term culture and feed additions on recombinant antibody ...

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1

Investigating the influence of long-

term culture and feed additions on

recombinant antibody production

in Chinese hamster ovary cells

A thesis submitted to the University of Manchester for the

degree of Doctor of Philosophy in

The Faculty of Life Sciences

2010

Laura Anne Bailey

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CONTENTS

CONTENTS ..................................................................................................................... 2

LIST OF FIGURES ......................................................................................................... 8

LIST OF TABLES ......................................................................................................... 12

ABSTRACT ................................................................................................................... 13

DECLARATION ........................................................................................................... 14

COPYRIGHT ................................................................................................................. 14

ACKNOWLEDGEMENTS ........................................................................................... 15

DEDEDICATIONS ....................................................................................................... 15

ABBREVIATIONS ....................................................................................................... 16

CHAPTER 1. INTRODUCTION ............................................................................... 21

1.1 INTRODUCTORY REMARKS .............................................................................. 22

1.2 EXPRESSION SYSTEMS ...................................................................................... 24

1.2.1 Bacterial and yeast systems ............................................................................. 24

1.2.2 Mammalian systems ........................................................................................ 24

1.2.2.1 PER.C6® cells ........................................................................................ 25

1.2.2.2 NS0 myeloma cells ................................................................................ 25

1.2.2.3 CHO cells ............................................................................................... 25

1.2.2.4 The DHFR vector system for recombinant protein synthesis ................ 26

1.2.3.5 The GS vector system for recombinant protein synthesis ...................... 27

1.3 MONOCLONAL ANTIBODIES AS THERAPEUTICS ........................................ 28

1.4 CELL BIOMASS AS A POTENTIAL DETERMINANT

OF RECOMBINANT PROTEIN PRODUCTION .................................................. 32

1.4.1 Cell cycle progression ..................................................................................... 32

1.4.2 Cell cycle regulators ........................................................................................ 33

1.5 METABOLIC ACTIVITY AS A POTENTIAL DETERMINANT


1.6 TRANSCRIPTION AS A POTENTIAL DETERMINANT


1.7 TRANSLATION AS A POTENTIAL DETERMINANT


1.7.1 Translational initiation .................................................................................... 39

1.7.2 RNA interference ........................................................................................... 40

1.8 PROTEIN FOLDING AS A POTENTIAL DETERMINANT


1.8.1 N-linked glycosylation .................................................................................... 43

1.8.2 Calnexin/calreticulin (CNX/CRT) cycle ......................................................... 44

1.8.3 The Unfolded Protein Response (UPR) .......................................................... 45

1.8.3.1 IRE-1 ...................................................................................................... 46

1.8.3.2 ATF6 ...................................................................................................... 47

1.8.3.3 PERK .................................................................................................... 47

1.8.3.3.1 ATF4 ....................................................................................... 49

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1.8.3.3.2 GADD153 ............................................................................... 49

1.8.3.3.3 GADD34 ................................................................................. 50

1.8.4 ER associated-degradation (ERAD) and ER stress-associated apoptosis . 50

1.8.4.1 ERAD ......................................................................................... 50

1.8.4.2 Macroautophagy ......................................................................... 51

1.8.4.3 ER stress-associated apoptosis ................................................... 51

1.9 PROTEIN SECRETION AS A POTENTIAL DETERMINANT

OF RECOMBINANT PROTEIN PRODUCTION ................................................. 53

1.10 IMPROVING PROTEIN PRODUCTION

BY FEED AND CHEMICAL ADDITIONS ......................................................... 54

1.11 INVESTIGATING INSTABILITY IN RECOMBINANT CHO CULTURES .... 55

1.12 SUMMARY AND PROJECT AIMS .................................................................... 56

CHAPTER 2. MATERIALS AND EQUIPMENT .................................................... 59

2.1 GENERAL MATERIALS ....................................................................................... 60

2.1.1 Sources of chemicals and reagents .................................................................. 60

2.1.2 Preparation and sterilisation of solutions ........................................................ 60

2.1.3 pH measurements ........................................................................................... 60

2.1.4 Mammalian cell lines and culture medium ..................................................... 60

2.2 GENERATION AND PURIFICATION OF PLASMIDS

IN BACTERIAL CELLS ......................................................................................... 61

2.2.1 Bacterial growth medium ................................................................................ 61

2.2.2 Generation of competent bacterial cells .......................................................... 61

2.2.3 Transformation of competent DH5α E.Coli cells .......................................... 61

2.2.4 Midi-preparation of plasmid DNA .................................................................. 61

2.2.5 Determination of nucleic acid concentration and purity ................................. 62

2.2.6 Restriction enzyme digestion .......................................................................... 62

2.3 CELL CULTURE .................................................................................................... 62

2.3.1 Maintenance of CHO cells .............................................................................. 62

2.3.2 Generation of batch cultures .......................................................................... 63

2.3.3 Determination of cell number, viability and diameter .................................... 63

2.3.4 Cryopreservation of cells ................................................................................ 64

2.3.5 Revival of cells from liquid nitrogen .............................................................. 64

2.3.6 Medium osmolality determination .................................................................. 64

2.3.7 Mycoplasma detection .................................................................................... 65

2.4 FLOW CYTOMETRY............................................................................................. 65

2.4.1 Cell cycle phase analysis ................................................................................. 65

2.4.2 Quantification of intracellular antibody .......................................................... 65

2.5 PROTEIN ANALYSIS ............................................................................................ 66

2.5.1 Detection of antibody by ELISA..................................................................... 66

2.5.2 Determination of total protein synthesis ......................................................... 67

2.5.3 Western blot analysis ...................................................................................... 68

2.5.3.1 Protein extraction ................................................................................... 68

2.5.3.2 SDS-PAGE ............................................................................................. 68

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2.5.3.3 Protein transfer ....................................................................................... 69

2.5.3.4 Stripping nitrocellulose membranes ....................................................... 69

2.5.3.5 Densitometric analysis ........................................................................... 70

2.5.4 N- linked glycan analyses ............................................................................... 70

2.5.4.1 Antibody purification ............................................................................. 70

2.5.4.2 Deglycosylation of purified recombinant antibody................................ 70

2.5.4.3 Precipitation and lyophilisation of glycans ............................................ 71

2.5.4.4 Desalting using Graphite ........................................................................ 71

2.5.4.5 MALDI-ToF analysis ............................................................................. 71

2.6 DETERMINATION OF COPY NUMBER ............................................................ 72

2.6.1 Southern blot analysis ..................................................................................... 72

2.6.1.1 DNA extraction, phenol extraction and ethanol precipitation ............... 72

2.6.1.2 Determination of genomic DNA per cell ............................................... 72

2.6.1.3 Preparation of plasmid standards and genomic DNA

for Southern analysis .............................................................................. 73

2.6.1.4 Agarose gel electrophoresis of DNA samples ....................................... 73

2.6.1.5 Capillary blot transfer of DNA to nylon membrane .............................. 74

2.6.1.6 Isolation of DNA probes for Southern analysis ..................................... 74

2.6.1.7 Radioactive labelling of probes .............................................................. 75

2.6.1.8 Pre-hybridisation ................................................................................... 76

2.6.1.9 Hybridisation and washing ..................................................................... 76

2.6.1.10 Autoradiography .................................................................................. 76

2.6.1.11 Membrane stripping ............................................................................ 77

2.6.2 Quantitative PCR (q-PCR) .............................................................................. 77

2.6.2.1 Preparation of standard curve................................................................. 77

2.6.2.2 Preparation of samples ........................................................................... 77

2.6.2.3 Real-time q-PCR reaction ...................................................................... 77

2.6.2.4 Analysis of q-PCR results ...................................................................... 78

2.7 DETERMINATION OF mRNA ............................................................................. 79

2.7.1 Quantitative reverse transcriptase PCR (q-RTPCR) ....................................... 79

2.7.1.1 RNA isolation ........................................................................................ 79

2.7.1.2 DNase treatment of RNA ....................................................................... 79

2.7.1.3 cDNA synthesis from RNA ................................................................... 79

2.7.1.4 Preparation of samples and „check‟ sample ........................................... 80

2.7.1.5 Quantitation of mRNA .......................................................................... 80

2.7.2 Polymerase Chain Reaction (PCR) ................................................................ 81

2.8 POLYSOME PROFILING ...................................................................................... 82

2.8.1 Sucrose gradient preparation ........................................................................... 82

2.8.2 Extract preparation for polysome analysis ..................................................... 82

2.8.3 Sedimentation of extracts ................................................................................ 83

2.9 MICROSOPY ANALYSES .................................................................................... 83

2.9.1 Preparation of metaphase spreads ................................................................... 83

2.9.2 Metaphase staining .......................................................................................... 83

2.9.3 Image acquisition ............................................................................................ 84

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2.9.4 Immunofluorescence ....................................................................................... 84

2.10 METABOLITE ANALYSES ................................................................................ 85

2.10.1 Glucose assay ................................................................................................ 85

2.10.2 Lactate assay ................................................................................................. 85

2.10.3 Gas chromatography-mass spectrometry (GC-MS) ...................................... 85

2.10.3.1 Sample derivatization .......................................................................... 85

2.10.3.2 GC-MS analysis ................................................................................... 86

2.10.4 Intracellular metabolite extraction ............................................................... 86

2.10.5 ATP assay ..................................................................................................... 87

2.10.6 NAD+/NADH assay ...................................................................................... 87

2.11 CALCULATIONS ................................................................................................. 87

2.11.1 Calculation of cell doubling time (dt) ........................................................... 87

2.11.2 Calculation of specific productivity (Qp)

and rates of metabolite production and utilisation ........................................ 88

2.11.3 Statistical methods ........................................................................................ 88

CHAPTER 3. CHARACTERISATION OF CELL LINE 3.90

IN DETERMINATION OF CELL LINE STABILITY .................. 90

3.1 INTRODUCTORY REMARKS .............................................................................. 91

3.2 ANALYSIS OF GROWTH CHARACTERISTICS

AND PRODUCTIVITY OF CELL LINE 3.90 ....................................................... 91

3.3 MOLECULAR INVESTIGATION OF ANTIBODY TITRE LOSS

DURING LTC OF CELL LINE 3.90 .................................................................... 101

3.3.1 Analysis of genomic stability during LTC .................................................... 101

3.3.2 Analysis of recombinant gene mRNA expression during LTC .................... 103

3.3.3 Investigating polysome profile characteristics during culture ...................... 103

3.3.4 Analysis of protein synthesis and secretion during LTC .............................. 104

3.4 THE REGULATION OF UPR MARKERS DURING CULTURE ...................... 116

3.5 METABOLIC ANALYSIS OF CELL LINE 3.90 ............................................... 127

3.6 DISCUSSION ........................................................................................................ 137

3.7 SUMMARY ........................................................................................................... 143

CHAPTER 4. CHARACTERISATION OF CELL LINE 3.90

IN REPSONSE TO FEED ADDITION .......................................... 150

4.1 INTRODUCTORY REMARKS ............................................................................ 151

4.2 ANALYSIS OF GROWTH CHARACTERISTICS AND PRODUCTIVITY

OF CELL LINE 3.90 IN RESPONSE TO FEED ADDITION ............................. 151

4.3 MOLECULAR INVESTIGATIONS OF RECOMBINANT CELL LINE 3.90

IN RESPONSE TO FEED ADDITION ................................................................. 163

4.3.1 Analysis of recombinant gene mRNA expression from cultures

with feed addition .......................................................................................... 163

4.3.2 Investigating characteristics of polysome profiles

in response to feed addition .......................................................................... 164

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4.3.3 Analysis of intracellular recombinant protein

in response to feed addition .......................................................................... 165

4.4 DETERMINING THE REGULATION OF UPR MARKERS

IN RESPONSE TO FEED ADDITION ................................................................ 175

4.5 METABOLIC ANALYSIS OF CELL LINE 3.90

IN RESPONSE TO FEED ADDITION ................................................................. 183

4.6 DISCUSSION ........................................................................................................ 193

4.7 SUMMARY ........................................................................................................... 196

CHAPTER 5. CHARACTERISATION OF CELL LINE 3.90 IN RESPONSE TO

DIMETHYL SULFOXIDE (DMSO) ADDITION ......................... 202


5.2 ANALYSIS OF GROWTH CHARACTERISTICS AND PRODUCTIVITY

OF CELL LINE 3.90 IN RESPONSE TO DMSO ................................................ 204


IN RESPONSE TO DMSO ADDITION ............................................................... 214

5.3.1 Effect of DMSO addition on recombinant gene mRNA expression ............ 214

5.3.2 Effect of DMSO addition on polysome profiles .......................................... 214

5.3.3 Effect of DMSO addition on intracellular recombinant protein ................... 215

5.4 THE UPR STATUS OF CULTURES AFTER DMSO ADDITION .................... 219

5.5 FUNCTIONALITY OF THE SECRETED ANTIBODY ..................................... 226

5.6 METABOLISM OF 3.90 CULTURES

IN RESPONSE TO DMSO ADDITION ............................................................... 228

5.6.1 Effects of DMSO addition on the production of metabolites ....................... 228

5.6.2 Effects of DMSO addition on rates of glucose and lactate utilisation .......... 229

5.7 DISCUSSION ........................................................................................................ 237

5.8 SUMMARY ........................................................................................................... 240

CHAPTER 6. CELL LINE 51.69 HAS CHARACTERISTICS SIMILAR

TO THOSE OF CELL LINE 3.90 ................................................... 245


6.2 ANALYISIS OF CELL LINE 51.69 IN RESPONSE TO LTC ............................ 247

6.2.1 Final antibody titres and viable cell densities were lower as a result of LTC247

6.2.2 Antibody titre loss was not at the level of

recombinant mRNA expression .................................................................. 248

6.2.3 Late generation 51.69 cultures had greater rates of lactate utilisation .......... 249

6.3 ANALYSIS OF CELL LINE 51.69 IN RESPONSE TO FEED ADDITION ....... 257

6.3.1 Feed addition increased recombinant protein production ............................. 257

6.3.2 Feed addition significantly lowered GADD153

mRNA and protein expression ...................................................................... 258

6.3.3 Metabolic profiles were altered for 51.69 in response to feed addition ........ 258

6.4 ANALYSIS OF CELL LINE 51.69 IN RESPONSE TO DMSO ADDITION ..... 265

6.4.1 Cell line 51.69 encountered growth arrest in response to DMSO addition .. 265

6.4.2 GADD153 mRNA and protein expression was significantly lowered

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in response to DMSO addition ..................................................................... 266

6.4.3 DMSO addition increased the rates of glucose utilisation

for 51.69 cultures .......................................................................................... 267

6.5 DISCUSSION ........................................................................................................ 281

6.5.1 How does 51.69 compare to 3.90 in response to LTC? ................................ 281

6.5.2 How does 51.69 compare to 3.90 in response to feed addition? ................... 282

6.5.3 How does 51.69 compare to 3.90 in response to DMSO addition? .............. 283

6.6 SUMMARY ........................................................................................................... 284

CHAPTER 7. OVERALL DISCUSSION ................................................................ 288

7.1 IS INSTABILITY CONNECTED TO A SPECIFIC CELLULAR EVENT? ....... 290

7.2 HOW IS RECOMBINANT PROTEIN PRODUCTION INCREASED

IN RESPONSE TO FEED ADDITION? ............................................................... 293

7.3 HOW IS RECOMBINANT PROTEIN PRODUCTION INCREASED

IN RESPONSE TO DMSO ADDITION? ............................................................. 295

7.4 ARE THERE MARKERS TO PREDICT THE LIKELIHOOD

OF INSTABILITY IN RECOMBINANT PROTEIN PRODUCTION? ............... 296

7.5 FUTURE WORK ................................................................................................... 297

REFERENCES ............................................................................................................. 299

APPENDICES ............................................................................................................. 329

APPENDIX1 – MATERIALS, CHEMICALS AND SPECIAL EQUIPMENT ......... 330

APPENDIX 2 – RELATIVE CONCENTRATION OF AMINO ACIDS ................... 338

APPENDIX 3 – OSMOLALITY MEASURMENTS .................................................. 341

APPENDIX 4 – INVESTIGATING CHEMICAL ADDITIONS ............................... 342

APPENDIX 5 – INVESTIGATING EXPRESSION OF UPR MARKERS

FOR THE PARENTAL CELL LINE ............................................... 345

APPENDIX 6 - MYCOPLASMA TESTING .............................................................. 349

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LIST OF FIGURES

Figure 1.1 Cellular events which control secreted protein production .......................... 23

Figure 1.2 Reaction catalysed by DHFR........................................................................ 27

Figure 1.3 Reaction catalysed by GS ............................................................................. 28

Figure 1.4 Schematic of an antibody.............................................................................. 30

Figure 1.5 The cell cycle ................................................................................................ 33

Figure 1.6 Metabolic production of ATP, NAD+ and NADH ....................................... 35

Figure 1.7 ATP is critical at multiple sites of protein expression .................................. 37

Figure 1.8 Diagram of translation initiation ................................................................... 40

Figure 1.9 The role of PDI in disulphide bond formation ............................................. 42

Figure 1.10 Common N-linked glycan structures .......................................................... 44

Figure 1.11 The CNX/CRT cycle .................................................................................. 45

Figure 1.12 Activation of the UPR ................................................................................ 48

Figure 1.13 ER stress-mediated degradation and cell death pathways .......................... 52

Figure 2.1 Restriction sites for DNA probes for Southern blot analysis ....................... 75

Figure 3.1 Analysis of recombinant antibody titre, viable cell densities,

and cell viability for 3.90 cultures................................................................. 94

Figure 3.2 Effect of LTC on viable cell growth and CCT ............................................ 96

Figure 3.3 Analysis of cell cycle distribution in response to LTC................................. 97

Figure 3.4 Effect of culture generation time on specific productivity (Qp)................... 99

Figure 3.5 Effect of culture generation time on cell size ............................................. 100

Figure 3.6 Effect of culture generation time on chromosome number ........................ 102

Figure 3.7 Analysis of heavy chain gene and light chain gene copy number

for early and late generation cultures .......................................................... 106

Figure 3.8 Effect of culture generation time on recombinant mRNA expression ....... 108

Figure 3.9 Analysis of polysome profiles during culture ............................................. 110

Figure 3.10 Effects of culture on the relative area of monosome

and polysome peaks ................................................................................. 112

Figure 3.11 Measuring global protein synthesis and secretion

for early and late generation cultures ....................................................... 114

Figure 3.12 Analysis of intracellular heavy chain and light chain

protein during culture ............................................................................... 115

Figure 3.13 Effects of culture generation time on the mRNA expression

of UPR markers ........................................................................................ 119

Figure 3.14 Analysis of ATF4 and GADD153 protein expression


Figure 3.15 Analysis of BiP protein expression


Figure 3.16 Analysis of PDI protein expression


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Figure 3.17 Analysis of XBP-1(s) mRNA during culture............................................ 125

Figure 3.18 Analysis of amino acid utilisation during culture ..................................... 129

Figure 3.19 Effects of culture generation time on metabolite accumulation ............... 131

Figure 3.20 Analysis of glucose and lactate concentrations during culture ................. 134

Figure 3.21 Investigating rates of glucose and lactate utilisation during culture......... 135

Figure 3.22 Correlation between antibody titre and proportion of cells in G0/G1 ...... 144

Figure 3.23 Potential metabolic changes in response to LTC ...................................... 146

Figure 3.24 Investigating ATP, NAD+ and NADH

concentrations for 3.90 cultures ............................................................... 147

Figure 3.25 Alterations to nutrient utilisation, UPR stress markers, cell biomass

and antibody titre in response to LTC ...................................................... 149

Figure 4.1 Effect of feed addition on recombinant antibody titre,

viable cell densities and cell viability during batch culture ....................... 154

Figure 4.2 Effect of feed addition on CCT ................................................................. 156

Figure 4.3 Effect of feed addition on cell cycle phase distribution

during batch culture.................................................................................... 157

Figure 4.4 The percentage of cells in G0/G1 cell cycle phase for culture

with and without feed addition .................................................................... 159

Figure 4.5 Effect of feed addition on specific productivity ......................................... 160

Figure 4.6 Effect of feed addition on cell diameter ..................................................... 162

Figure 4.7 Effect of feed addition on recombinant mRNA expression ....................... 166

Figure 4.8 Analysis of recombinant mRNA expression between cultures

with and without feed addition .................................................................. 168

Figure 4.9 Investigating characteristics of polysome profiles

in response to feed addition ....................................................................... 170

Figure 4.10 Quantification of monosome and polysome peak areas ........................... 172

Figure 4.11 Analysis of intracellular heavy chain and light chain protein

after feed addition ................................................................................... 174

Figure 4.12 Effect of feed addition on the mRNA expression

of ATF4, GADD34, and GADD153 ........................................................ 177

Figure 4.13 Effects of LTC on the mRNA expression of UPR markers

from cultures supplemented with feed addition ....................................... 179

Figure 4.14 Analysis of ATF4 and GADD153 protein in response to feed addition .. 181

Figure 4.15 Analysis of XBP-1 mRNA splicing in response to feed addition ............ 182

Figure 4.16 Effects of feed addition on amino acid concentrations ............................. 186

Figure 4.17 Increased metabolites in response to feed addition .................................. 188

Figure 4.18 Effects of feed addition on glucose and lactate concentrations ................ 191

Figure 4.19 Alterations to 3.90 cultures in response to feed addition.......................... 198

Figure 4.20 Concentrations of ATP, NAD and NADH

in response to feed addition ..................................................................... 199

Figure 4.21 Time-line of changes to late generation cultures with feed addition ........ 201

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Figure 5.1 Effect of DMSO addition on antibody titre, viable cell growth and CCT

for early generation cultures ....................................................................... 206


for late generation cultures .......................................................................... 208

Figure 5.3 Effect of DMSO on specific productivity (Qp) .......................................... 211

Figure 5.4 Effect of DMSO addition on G0/G1 cell cycle phase transition ................ 212

Figure 5.5 Cell size comparisons for cultures with and without DMSO addition ....... 213

Figure 5.6 Effect of DMSO addition on recombinant mRNA expression ................. 216

Figure 5.7 Quantification of monosome and polysome peaks

in response to DMSO addition ................................................................... 217

Figure 5.8 Analysis of intracellular recombinant protein

in response to DMSO addition ................................................................... 218

Figure 5.9 Effect of DMSO addition on GADD153 mRNA expression ..................... 221

Figure 5.10 Effect of DMSO addition on expression

of PDI, ATF4 and GADD153 protein ..................................................... 222

Figure 5.11 Analysis of XBP-1(s) mRNA in response to DMSO addition ................. 224

Figure 5.12 Effects of culture conditions on secreted glycan profiles ....................... 227

Figure 5.13 Analysis of glycerol, glycine, alanine and lactate accumulation

from early generation cultures in the presence of DMSO........................ 231

Figure 5.14 Analysis of glycerol, glycine, alanine and lactate accumulation

from late generation cultures in the presence of DMSO .......................... 233

Figure 5.15 Investigating glucose utilisation rates in response to DMSO addition ..... 235

Figure 5.16 Investigating lactate production rates in response to DMSO addition ..... 236

Figure 5.17 Effect of DMSO addition on global protein synthesis ............................. 241

Figure 5.18 Correlation between antibody titre and rates of glucose utilisation ......... 242

Figure 5.19 Alterations to 3.90 cultures in response to DMSO addition ..................... 244

Figure 6.1 Analysis of recombinant antibody titre, viable cell densities,

CCT and Qp for cell line 51.69 ................................................................... 250

Figure 6.2 The percentage of cells in G0/G1 was lower for

late generation 51.69 cultures ..................................................................... 252

Figure 6.3 Expression of recombinant mRNA was not altered in response to LTC .... 254

Figure 6.4 ATF4 and GADD153 mRNA increased during batch culture.................... 255

Figure 6.5 Late generation cultures had greater rates of lactate utilisation ................. 256

Figure 6.6 Feed addition increased final antibody titres for cell line 51.69................. 259

Figure 6.7 Feed addition enhanced specific productivity (Qp) for cell line 51.69 ...... 260

Figure 6.8 The mRNA expression of ATF4, GADD34 and GADD153 were lower

for cultures with feed addition ................................................................... 261

Figure 6.9 GADD153 protein was significantly lowered in response to feed addition 262

Figure 6.10 XBP-1(s) mRNA was less after feed addition .......................................... 263

Figure 6.11 Rates of glucose utilisation and lactate production were increased

in response to feed addition ..................................................................... 264

Figure 6.12 DMSO addition to for early generation 51.69 cultures

did not enhance antibody titres ................................................................ 268

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Figure 6.13 Antibody titres were enhanced for late generation 51.69 cultures

in the presence of feed and DMSO .......................................................... 270

Figure 6.14 Qp values were increased in response to DMSO addition ....................... 272

Figure 6.15 Recombinant mRNA expression was not altered in response to DMSO . 273

Figure 6.16 DMSO addition to 51.69 cultures lowered GADD153 expression ......... 275

Figure 6.17 XBP-1(s) mRNA was lowered in response to DMSO.............................. 277

Figure 6.18 Rates of glucose utilisation were increased for 51.69 cultures

in the presence of DMSO ......................................................................... 279

Figure 6.19 Alterations to nutrient utilisation, ER stress markers and antibody titre

for cell line 51.69 in response to LTC...................................................... 285

Figure 6.20 Alterations to 51.69 cultures in response to feed addition........................ 286

Figure 6.21 Alterations to 51.69 cultures in response to DMSO addition ................... 287

Figure 7.1 A pathway linking mitochondrial Ca2+

and ATP concentrations

to mis/unfolded proteins ............................................................................. 291

Figure A2.1 The relative concentrations of amino acids during batch culture ........... 338

Figure A4.2 Preliminary investigation of different chemical additions

to improve recombinant protein production ............................................ 342

Figure A5.1 Parental cells have lower GADD153 and XBP-1(s) mRNA

than recombinant CHO cultures ............................................................... 345

Figure A5.2 ATF4 is significantly lower for the parental cell line .............................. 346

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LIST OF TABLES

Table 1.1 MAbs on the market ...................................................................................... 31

Table 2.1 Details of antibodies used for western blot analysis ...................................... 70

Table 2.2 Primers used in real-time q-PCR ................................................................... 78

Table 2.3 Primers used in real-time q-RTPCR .............................................................. 81

Table 2.4 Details of PCR primers .................................................................................. 81

Table A3.1 Osmolality measurements in response to feed and DMSO addition......... 341

Table A4.1 Analysis of different DMSO additions on cell growth and final antibody

titres ........................................................................................................... 344

Table A6.1 Mycoplasma is not detected during batch culture ..................................... 349

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ABSTRACT

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production

of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability

to perform correct post-translational modifications. A major issue for use of CHO cells

lines for the production of recombinant proteins is the selection of cell lines that do not

retain stable protein expression during long-term culture (LTC). Instability of

expression impairs process yields, effective usage of time and money, and regulatory

approval.

Protein production is complex and is influenced by cell growth, transcription,

translation, protein folding and post-translational processing and secretory events,

which may interact to determine stability of expression during prolonged culture. This

thesis aims to identify features associated with stability/instability of recombinant

protein expression and methods to improve protein production, with the addition of

chemically defined (CD) feed and chemicals.

Two exemplar CHO cell lines, which secrete the same recombinant antibody were

characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and

51.69) exhibited unstable protein production over LTC, with a loss in final antibody

titres and specific productivity (Qp). The instability observed within the exemplar cell

lines was not due to decreased recombinant gene copy numbers or mRNA expression

but was associated with lower viable cell densities, increased ER stress (GADD153 and

spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during

the decline phase of batch culture). Improvement of recombinant protein expression in

response to feed or DMSO addition was associated with lower expression of ER stress

markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein

level) and alterations to the metabolic activity of the cultures (prevention of alanine and

lactate re-utilisation, and greater glucose utilisation between the stationary and decline

phase of batch culture).

Although feed or DMSO addition improved recombinant protein production, these

additions did not reverse the appearance or progression of instability for cells after LTC.

ER stress expression was not abolished as a consequence of feed or DMSO addition.

Expression of stress markers at earlier time points may be the factor that limits antibody

production and secretion. The consequences of the presence of feed and DMSO addition

on ER stress markers and antibody production serves to highlight approaches that may

be utilised for engineering more productive or stable protein production phenotypes in

parental cell lines.

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DECLARATION

No portion of this work referred to in the thesis has been submitted in support of an

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institute of learning.

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15

policy on presentation of Theses from the Dean of the Faculty of Life Sciences, for

Faculty of Life Sciences' candidates.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my supervisors, Alan Dickson and Diane

Hatton. I am sincerely grateful to them for their advice, enthusiasm and patience over

the course of my PhD.

I would like to thank my advisor, Neil Bullied, for his guidance and for the PDI

antibody, kindly donated. I would also like to thank my colleagues at MedImmune

Cambridge, in particular Tori Crook, Wyn Forrest-Owen, Alison Mason, and Ray Field,

for cell line creations and feed developments, and for general help and advice

throughout my studies. Many thanks also to Chris Sellick for assistance with GC-MS

analysis and Eleanor Taylor for her help in the development of the polysome protocol.

I wish to thank all the members of Lab B2075, and Suzanne Hunt, Hayley Campbell

and Verity Nancollis, for making my time as a postgraduate a wonderful experience.

Thanks also to Alexandra Croxford, whose advice and support throughout my entire

PhD will never be forgotten.

I would also like to acknowledge the BBSRC and MedImmune Cambridge for funding

my PhD. I am extremely grateful.

DEDICATIONS

This thesis is dedicated to my family, for their unconditional love and support.

Especially dedicated to my mum, Joy, for being my inspiration, and to my husband,

Andrew, for keeping things simple and making me smile.

16

ABBREVIATIONS

A - absorbance

AARE - amino acid response element

ACE - artificial chromosome expression

ACN - acetronitrile

ADCC - antibody-dependent cytotoxicity

ADP - adenosine diphosphate

AMDIS - automated mass spectral deconvolution and identification

software

APC - allophycocyanin

ASK1 - apoptosis signal-regulating kinase 1

ATF - activating transcription factor

ATP - adenosine triphosphate

BHK - Baby Hamster Kidney

BiP - heavy chain binding protein

BSA - Bovine serum albumin

CBF - CCAAT-binding factor

CCT - cumulative cell time

CD - chemically-defined

CDC - complement-dependent cytotoxicity

CDI - cyclin-dependent kinase inhibitors

CDK - cyclin-dependent kinases

cDNA - complementary DNA

C/EBP - CCAAT/enhancer binding protein (C/EBP)

CHO - Chinese Hamster Ovary

CIRP - cold-inducible RNA binding protein

CNX - calnexin

CRT - calreticulin

CV - coefficient of variation

DAbs - domain antibodies

DAPI - 4‟6-diamino-2-phenylindole

ddH2O - double distilled water

17

DEPC - diethylpyrocarbonate

DHB - 2,5-dihydroxybenzoic acid

DHFR - dyhydrofolate reductase

Dicer - ribonuclease III-like enzyme

DMSO - dimethyl sulfoxide

DNA - deoxyribonucleic acid

dt - doubling time

DTT - dithiothreitol

ECL - enhanced chemiluminescence

E. coli - Escherichia coli

EDTA - ethylenediaminotetra acetic acid

eIF - eukaryotic initiation factor

ELISA - enzyme-linked immunosorbent assay

ER - endoplasmic reticulum

ERAD - ER-associated degradation

ERdj - ER-resident J-domain co-chaperones

ERO - ER-resident oxidoreductases

ERSE - ER stress element

Fab - antibody binding region

FAD - flavin adenine dinucleotide

FDA - food and drug administration

FITC - fluorescein isothiocyanate

Fuc - fucose

GADD - growth arrest and DNA damage genes

Gal - galactose

GC - gas chromatography

GDP - guanosine diphosphate

Glc - glucose

GlcNAc - N-acetylglucosamine

GRP 78 - glucose-regulated protein 78

GS - glutamine synthetase

GTP - guanosine triphosphate

hr - hour

HAT - histone acetyltransferase

18

HEK-293 - Human embryonic kidney-293

hCMV - human cytomegalovirus promoter

HDAC - histone deacetylase

HRP - horseradish peroxidase

IFN - interferon

Ig - immunoglobin

IRE-1 - inositol requiring protein-1

IRS - Integrated Stress Response

JDP - jun dimerization protein 2

JIK - c-Jun NH2-terminal inhibitory kinase

JNK - c-Jun NH2-terminal kinase

kb - kilobase pair

L - litre

LB - Luria Bertani

LCR - locus control region

LDH - lactate dehydrogenase

LTC - long-term culture

M - Molar

MAbs - monoclonal antibodies

Man - mannose

MAPK - mitogen activated protein kinase

MDH - malate dehydrogenase

Met - Methionine

min - minute

miRNA - microRNA

mg - milligram

ml - millilitre

mM - milliMolar

mRNA - messenger RNA

MS - mass spectrometry

MSTFA - trimethylsilyltrifluoroacetamide

MSX - methionine sulphoximine

mTOR - mammalian target of rapamycin

MTX - Methotrexate

19

NAD+/ NADH - nicotinamide adenine dinucleotide/reduced NAD

+

ng - nanogram

NS0 - non-secreting, clone 0

OD - optical density

PAGE - polyacrylamide gel electrophoresis

PBA - 4-phenylbutyric acid

PBS - phosphate buffered saline

PCR - polymerase chain reaction

PDI - protein disulfide isomerise

PERK - protein kinase RNA (PKR)-like ER kinase

pg - pictogram

PI - propidium iodide

PI3K - phosphatidylinositol-3‟-kinase

pM - picoMolar

PMT - photomultiplier tube

QC - quality control

Qp - specific productivity

q-PCR - quantitative PCR

q-RTPCR - quantitative reverse transcription PCR

Rb - retinoblastoma

REDD1 - regulated in development and DNA damage responses

RIPA - Radio Immunoprecipitation buffer

RISC - RNA-interference silencing complex

RNA - ribonucleic acid

RNAi - RNA interferance

RNase - ribonuclease

rpm - revolutions per minute

rRNA - ribosomal RNA

RT - reverse transcription

SD - standard deviation

SDS - sodium dodecyl sulfate

sec - seconds

SEM - standard error of mean

siRNA - small interfering RNA

http://en.wikipedia.org/wiki/Sodium_dodecyl_sulfate

20

S/MAR - scaffold/matrix attachment regions

SP - site protease

SSC - standard saline citrate

SV40 - simian virus 40 promoter

TBE - tris, borate, EDTA

TCA - trichloroacetic acid

TCA cycle - tricarboxylic acid cycle

TE - tris, EDTA

TEMED - N, N, N‟, N‟-tetramethylethylenediamine

TEN - tris, EDTA, and NaCl

TMAO - tri-methylamine-N-oxide

TMB - 3, 3‟, 5, 5‟-tetramethylethylenediamine

TMCS - trimethylchorosilane

TRAF2 - TNF receptor-associated factor 2

Tris - tris (hydroxymethyl) aminomethane

tRNA - transfer RNA

Tween - polyethylene glycol sorbitan monolaurate

UCOE - ubiquitous chromatin opening element

UDP - uridine diphosphate

UGGT - α-glucosidase II and UDP-glucose:glycoprotein glucosyl

transferase

uORF - upstream open reading frame

UPR - unfolded protein response

UPRE - UPR response element

UTR - untranslated region

UV - ultraviolet

v/v - volume per volume

w/v - weight per volume

WCS - working cell stock

XBP-1 - X-box binding protein 1

XBP-1(s) - spliced XBP-1

µg - microgram

µl - microlitre

4E-BP1 - 4E binding protein1

21

1

CHAPTER 1

INTRODUCTION

22

1. INTRODUCTION

1.1 INTRODUCTORY REMARKS

In 1986 human tissue plasminogen activator (tPA, Activase; Genentech, S. San

Francisco, CA, USA) became the first therapeutic protein generated from recombinant

mammalian cells to obtain market approval. Since then the recombinant

biopharmaceutical market has increased dramatically with approximately 200 approved

peptide and protein pharmaceuticals on the food and drug administration (FDA) list

(Demain & Vaishnav, 2009). These protein pharmaceuticals include recombinant

hormones, cytokines, blood-related products (such as coagulation factors), vaccines,

therapeutic enzymes and monoclonal antibodies (MAbs, Section 1.3).

The production of recombinant proteins within an industrial environment must follow

strict procedures, including that mammalian-derived cell lines should be clonal and

remain stable over long-term culture (LTC) in accordance with the ICH guidelines

(ICH, 1996). Stable cell lines are regarded as cells that retain constant protein

production for extensive periods of culture, generally throughout a period of at least 60

generations beyond the production of a Manufacturers Working Cell Bank (Birch, 1993;

Brown, 1992). A failure of the recombinant cell line to maintain stability during LTC

can result in problems for process yields, protein quality, effective usage of time and

money, and regulatory approval (Barnes et al, 2003).

There are many cellular events that regulate overall recombinant protein expression and

long-term stability of recombinant cell lines (highlighted in Figure 1.1). As the ideal

characteristics of cell line development is to achieve high, stable recombinant protein

production within this Introduction I will discuss the intracellular events controlling

protein expression at the level of transcription (Section 1.6), translation (Section 1.7),

protein folding (Section 1.8) and protein secretion (Section 1.9). I will also discuss the

influence of cell biomass (Section 1.4) and metabolic control (Section 1.5) on protein

production.

23

Figure 1.1 Cellular events which control secreted protein production

The production of proteins from DNA involves several steps including transcription,

translation, protein folding and protein secretion. These events are also influenced by

the cell biomass, including the rates of cell division and cell death, and the metabolic

activity of the cell.

My research involves the Chinese hamster ovary (CHO) cell line and although this cell

line is mainly focused upon within this Introduction there are other expression systems

used in the production of recombinant proteins, including insect cell lines (Chai et al,

1996; Davis et al, 1993; Kost & Condreay, 1999; Wickham et al, 1992), fungal cell

lines (Gerngross, 2004; Keränen & Penttilä, 1995), transgenic animals (Houdebine,

2009; Lonberg, 2005) and transgenic plants (Daniell et al, 2001; Larrick & Thomas,

2001). The most commonly used expression systems for recombinant protein

production are bacterial, yeast and mammalian cells. These expression systems are

discussed in more detail in the next Section.

Transcription

Translation

Protein

Folding

Protein

Secretion

Nucleus

ER

24

1.2 EXPRESSION SYSTEMS

1.2.1 Bacterial and yeast systems

Bacterial and yeast species offer the advantages of rapid cell growth and high yields

with relatively low production costs (Baneyx, 1999; Demain & Vaishnav, 2009; Swartz,

2001). However, there are limitations with these expression systems. High cell densities

of cultures can result in toxicity due to acetate formation, and excessive recombinant

protein in the cytoplasm often becomes misfolded and segregates into soluble

aggregates, known as inclusion bodies (Baneyx, 1999; Demain & Vaishnav, 2009).

Enhanced secretion of recombinant proteins within bacteria can be achieved by

manipulating the cytoplasmic folding environment by increasing molecular folding

chaperones and foldases (Baneyx & Mujacic, 2004; Thomas et al, 1997), or by the

addition of specific signal sequences to the recombinant peptides (Choi & Lee, 2004). A

major disadvantage with using bacterial strains arises from the limited ability to perform

N- and O-linked glycosylation, essential for the function of many human therapeutic

proteins (Jenkins, 2007; Jenkins et al, 1996). Although yeast species have the advantage

of providing an environment capable of performing post-translational modifications

(Verma et al, 1998), the number and type of glycans observed for normal human

proteins differs from that provided by yeast (Cereghino et al, 2002). Approaches to

overcome this problem have included removal of yeast-specific glycosylation sites

(Asami et al, 2000), and genetic engineering yeast strains to perform human

glycosylation at high fidelity. The outcome from this engineering technology is a library

of engineered yeast strains with different glycosylation capabilities (Hamilton et al,

2006; Li et al, 2006a).

1.2.2 Mammalian systems

Although mammalian cells exhibit some disadvantages when compared to bacteria or

yeast systems, for example the expense of complex media and lower cell biomass, these

cells are still extensively used industrially due to their ability to perform correct post-

translational modifications (Andersen & Krummen, 2002; Werner et al, 1998). Post-

translational modifications dictate the pharmacokinetic and pharmocodynamic

properties of recombinant proteins and hence their biological activity (Chirino & Mire-

Sluis, 2004). Mammalian cell lines used for recombinant protein expression include

25

CHO (Section 1.2.2.3), NS0 myeloma (Section 1.2.2.2) and human PER.C6®

cells

(Section 1.2.2.1).

1.2.2.1 PER.C6® cells

PER.C6® cells are derived from primary culture of human fetal retinoblast immortalized

upon transfection with an E1 minigene of adenovirus type 5 (Fallaux et al, 1998). The

cells can be grown in suspension to high cell densities in serum-free medium (Pau et al,

2001). The PER.C6® cell line offers a reliable, safe and scalable solution for the

production of recombinant therapeutic proteins, with the advantage of human-type

glycosylation (Crucell, 2010).

1.2.2.2 NS0 myeloma cells

NS0 myeloma cells were derived in the 1960s from a plasmacytoma induced in a mouse

via peritoneal injection of mineral oils, myeloma cells from the tumour were then

cloned and selected until secretion of antibodies ceased, hence the name NS0 (non-

secreting, Galfre & Milstein, 1981; Potter & Boyce, 1962). Stable protein expression

has been shown for GS-NS0 cells over periods of extended batch culture (Barnes et al,

2001).

1.2.2.3 CHO cells

The CHO progenitor cell line was originally derived from partially inbred female adult

Chinese hamsters (Cricetulus griseus, Puck et al, 1958). CHO cells were considered

useful models in radiation cytogenetics, due to the low chromosome number of Chinese

hamsters (2n=22). It was soon found that these cells grew readily in vitro, with short

doubling times. These features of CHO cells and their ability to gain regulatory

approval has led to approximately 70% of all recombinant proteins being derived from

CHO cells (Jayapal et al, 2007).

The development of the CHO cell line has resulted in several CHO sub-clones, these

include sub-clones that require proline for growth (Kao & Puck, 1967), more commonly

known as CHO-K1. CHO-K1 cells have been used extensively for industrial purposes

http://www.crucell.com/glossary/item/cell+line

http://www.crucell.com/glossary/item/recombinant

26

and several further sub-clones have been generated from these cells including

dihydrofolate reductase (DHFR)-deficient mutant cell lines (DXB11 and DG44, Urlaub

& Chasin, 1980; Urlaub et al, 1983). These cells contain no DHFR enzyme so allow for

the transfected DHFR gene to be used as a selectable amplifiable marker for

heterologous gene expression (Section 1.2.2.4).

1.2.2.4 The DHFR vector system for recombinant protein synthesis

DHFR is an enzyme essential for the formation of folate for use in purine and

pyrimidine biosynthesis. Therefore introduction of a heterologous gene into a cell

deficient in DHFR can be selected for by co-transfection with a functional copy of the

heterologous DHFR gene. Clonal selection is achieved by growing cells in medium

without glycine, hypoxanthine and thymine, so only cells which have stably integrated

the DHFR transgene survive to form colonies (Kaufman, 1990). An advantage of using

DHFR as a selectable marker is that amplification of the heterologous DHFR gene,

along with associated transgenes, can be achieved by using methotrexate (MTX), a folic

acid analogue that competitively inhibits DHFR (Figure 1.2). By treating cells with

increasing concentrations of MTX (MTX amplification) the surviving cells can contain

several hundred to a few thousand copies of the integrated plasmid (Wurm et al, 1986).

Most 'amplified' cells produce more recombinant protein than the unamplified cells

(Jiang et al, 2006; Yoshikawa et al, 2000). Although MTX amplification results in the

isolation of very high producing cell lines, the process can be long and laborious, often

taking over 6 months to isolate and screen for high-producing cell lines (Page, 1988).

27

Figure 1.2 Reaction catalysed by DHFR

Dihydrofolate reductase (DHFR) catalyses the conversion of dihydrofolate to

tetrahydrolate, which is important in nucleoside and amino acid biosynthesis.

Methotrexate (MTX) inhibits the activity of DHFR.

1.2.3.5 The GS vector system for recombinant protein synthesis

The glutamine synthetase (GS) gene is another commonly used selection marker. The

incorporation of a GS gene in a plasmid vector provides a source of glutamine for the

cells and therefore allows for selection of cells in glutamine-free medium (Bebbington

et al, 1992). Glutamine is an essential amino acid, necessary for protein synthesis,

purine and pyrimidine biosynthesis, ammonia formation and the biosynthesis of amino

acids (Meister, 1976). NS0 cells, unlike CHO cells, are GS-deficient, and require

exogenous glutamine. This phenotype can allow for selection of successful transfectants

using the GS system. CHO cells, however, require methionine sulphoximine (MSX), an

inhibitor of GS activity to effectively identify transfected clones (Brown, 1992). The

reaction catalysed by GS is shown in Figure 1.3.

The GS vector system has been successfully used by over 85 biotechnology and

pharmaceutical companies worldwide (Lonza, 2010). Many of these companies have

utilised the GS selection technology in the production of the MAbs (Section 1.3).

Dihydrofolate + NADPH + H+ Tetrahydrofolate + NADP+

DHFR

MTX

28

Figure 1.3 Reaction catalysed by GS

Glutamine synthetase (GS) catalyses the conversion of glutamate and ammonia to

glutamine in the presence of magnesium. Glutamine is important in purine and

pyrimidine biosynthesis. Methionine sulphoximine (MSX) is used as an inhibitor of GS

activity.

1.3 MONOCLONAL ANTIBODIES AS THERAPEUTICS

Five major classes of human antibody (immunoglobulin, Ig) are defined as IgM, IgG,

IgA, IgD and IgE (Jefferis, 2009a). IgE is associated with allergy, IgM, is known to

protect against bacterial and fungal infection, whereas the function of IgD remains less

clear (Woof & Burton, 2004). IgA has been shown to provide a critical role in mucosal

protection (Underdown & Schiff, 1986; Woof & Kerr, 2004), whilst IgG provides the

majority of antibody-based immunity against invading pathogens (Karupiah &

Chaudhri, 2004). Four subclasses of human IgG are defined according to their relative

concentrations in normal serum: IgG1, IgG2, IgG3 and IgG4, which respectively

account for approximately 60%, 25%, 10% and 5% of serum IgG (Jefferis, 2009b). The

choice of IgG subclass is a crucial decision when developing recombinant MAb

therapeutics (Jefferis, 2007). For example, in oncology the IgG1 subclass is the isotype

of choice as it has maximal potential to eliminate targeted cancer cells by inducing

antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent

cytotoxicity (CDC, Jefferis, 2009b). The IgG isotype will be dependent on its intended

therapeutic action.

Since the first generation of mouse, chimeric and humanised IgG1 antibodies reached

the market in the late 1990s, the variety of antibody structures has greatly increased

Glutamate + Ammonia Glutamine

GS

MSX

Mg2+

29

(Beck et al, 2010), including human antibodies of other IgG isotypes (IgG2 and IgG4)

(Lonberg, 2008), and IgG-related products such as domain antibodies (DAbs, Nelson &

Reichert, 2009).

In its simplest form an individual IgG molecule is composed of two identical light

chains and two identical heavy chains, linked by disulphide bonds (Steinmeyer &

McCormick, 2008). The light and heavy Ig chains are arranged to form two antigen-

binding (Fab) regions that are linked to an Fc region to form the tertiary structure of the

antibody, comprised of non-covalently paired heavy chain domains and covalently

linked inter-heavy chain disulphide-bonded hinge regions (Jefferis, 2009b; Woof &

Burton, 2004). Antibodies are thought to be connected by 16-28 disulphide bonds,

depending on their isotype (Borth et al, 2005). A generic antibody structure is shown in

Figure 1.4.

As discussed in Section 1.2.2 the major advantage of utilising mammalian cells for

therapeutic protein production is their capacity to perform the correct post-translational

modifications, including glycosylation. N-linked glycosylation (Section 1.8.1) is found

on the constant heavy chain regions of antibodies (Jefferis, 2009b; Rudd et al, 2001),

and on the antibody binding region of polyclonal human IgG antibodies (Holland et al,

2006). The oligosaccharides found on the antigen binding regions are attached to the

variable region of the kappa or lambda light chains or to the variable heavy chain

regions (Jefferis, 2009b). CHO cells hold a major advantage in recombinant protein

production as they can glycosylate variable heavy chain and light chain regions in a

manner similar to that observed for normal human IgG (Lim et al, 2008).

30

Figure 1.4 Schematic of an antibody

An antibody contains both heavy chain and light chain proteins linked by disulphide

bonds. Variable regions are found at the amino acid terminal ends of the heavy and

light chain proteins which confer antigen binding specificity. The Fc domain modulates

effector functions (adapted from Steinmeyer & McCormick, 2008).

Key for Figure 1.4

Therapeutic murine MAbs entered clinical studies in the early 1980s, but problems

arose due to lack of efficacy, and the rapid clearance of the murine MAbs due to the

patient‟s production of human anti-mouse antibodies (the HAMA response). These

issues became the driving forces for the evolution of MAb production technology

(Reichert et al, 2005) and ultimately the development of MAbs, and related proteins.

MAbs have been used successfully in a variety of disease therapies including several

forms of cancer, multiple sclerosis and immunological disorders (Table 1.1, Jefferis,

2009a; Shukla & Thömmes, 2010).

Heavy chainHeavy chain

Fc domain

Variable region

Constant region

Hinge region

Disulphide bonds

31

Table 1.1 MAbs on the market

Note: these products may not be approved for use in all countries. * MAbs derived from

recombinant CHO cells, a Synagis is also known as palivizumab (adapted from Shukla

& Thömmes, 2010).

Recently approved antibodies include Arzerra (ofatumumab) and Prolia (denosumab).

Arzerra, a MAb specific for CD20, was approved by the US FDA in October 2009 for

the treatment of chronic lymphocytic leukemia, and is under regulatory review in

Europe for the same indication (Keating et al, 2010). Prolia (denosumab), a

breakthrough fully-human MAb , approved by the FDA in 2010 for use in the treatment

and prevention of bone loss in hormone-treated prostate and breast cancer patients, has

also been fast-tracked by the FDA for treatment and prevention of postmenopausal

osteoporosis (Ellis et al, 2008; McClung et al, 2006).

Although MAbs have great therapeutic potential they can be constrained by their large

molecular size (Reichert et al, 2005). Domantis has pioneered DAbs, based on the

Name Target Indication Company Year

Orthoclone OKT3 CD3 Acute kidney transplant rejection Ortho Biotech 1986

ReoPro Platelet GP Blot clot prevention Centocor 1994

Panorex 17-1A Colorectal cancer Genentech/Biogen-Idec 1995

Rituxan * CD20 Non-Hodgkin's Lymphoma GlaxoSmithKline 1997

Zenapax * IL2R (CD25) Acute kidney transplant rejection Hoffman-LaRoche 1997

Simulect IL2R Prophylaxis of acute organ rejection Novartis 1998

Synagis *a

RSV Respiratory Synctial Virus MedImmune 1998

Remicade TNFα Rheumatoid arthritis Centocor 1998

Herceptin * Her2 Metastatic breast cancer Genentech 1998

Mylotarg * CD33 Acute mylogenous lymphoma Wyeth-Ayerst 2000

Campath * CD52 B cell chronic lymphocytic leukemia Takeda 2001

Zevalin * CD20 Non-Hodgkin's Lymphoma Biogen-Idec 2002

Humira * TNFα Rheumatoid arthritis Abbott 2002

Bexxar CD20 Non-Hodgkin's Lymphoma Corixa/GlaxoSmithKline 2003

Xolair * IgE Allergy Genentech/Novartis 2003

Eritux EGFR/Her1 Colorectal cancer Bristol-Myers 2004

Squibb/Imclone (Eli Lilly)

Avastin * VEGF Colorectal cancer Genentech 2004

Raptiva CD11 Psoriasis Genentech/Xoma 2004

Tysabri * A4 integrin Multiple sclerosis Biogen-Idec/Elan 2004

Vectibix * EGFR/Her1 Colorectal cancer Amgen 2006

Soliris * C5 complement Paroxysmal nocturnal hemoglobinuria Alexion 2007

Stelara IL12 and IL23 Psoriasis Centocor 2008

Simponi TNFα Rheumatoid arthritis Centocor 2008

Actemra IL6 Rheumatoid arthritis Roche 2009

Arzerra CD20 Non-Hodgkin's Lymphoma Genmab 2009

Prolia * RANK ligand Prevention of bone loss in cancer patients Amgen 2010

32

smallest functional binding units of human antibodies. DAbs contain either the variable

domain of an antibody light chain or heavy chain, ranging in size from 11 kDa to 15

kDa. DAbs are bioactive as monomers but they can also be formatted into larger

molecules to create drugs with prolonged serum half-lives or other pharmacological

activities (Holt et al, 2003).

1.4 CELL BIOMASS AS A POTENTIAL DETERMINANT OF RECOMBINANT

PROTEIN PRODUCTION

1.4.1 Cell cycle progression

Cell cycle progression and division in mammalian cells is controlled by a network of

checkpoints that are regulated by a complex network of kinases, inhibitors and signalling

molecules. The cycle itself is divided into two distinct phases interphase and M phase,

with each having further subphases (Figure 1.5). The genetic material of the cell is

replicated during S phase, a subphase of interphase. Two gap phases of interphase,

GAP1 (G1) and GAP2 (G2), occur before and after S phase and allow the cell to grow

and prepare for either the replication of nuclear material (S phase) or the separation into

two progenic cells (M phase). M phase, which consists of mitosis and cytokinesis,

follows immediately after G2 resulting in daughter cells, allowing the cell cycle to re-

initiate. Non-proliferating cells that are arrested during G1 phase may also enter the

quiescent G0 phase. These cells are maintained in G0 through the phosphorylation of

key cell cycle regulators (Section 1.4.2, Sunley & Butler, 2010). Previous investigations

have given differing reports with regards to how productive certain cell cycle phases are.

Maximum protein expression has been related to G1 phase (Al-Rubeai & Emery, 1990;

Dutton et al, 2006; Kromenaker & Srienc, 1991), S phase (Banik et al, 1996; Gu et al,

1996; Kubbies & Stockinger, 1990) and G2/M phase (Aggeler et al, 1982).

1.4.2 Cell cycle regulators

Progression through each phase of the cell cycle is tightly correlated with the expression

and rapid degradation of cyclin and cyclin-dependent kinase (CDK) complexes. From

early Gl the D-type cyclins form complexes with CDKs 4 or 6 (Bates et al, 1994;

33

Meyerson & Harlow, 1994), which initiates hyperphosphorylation of retinoblastoma

(Rb, Kitagawa et al, 1996). Hyperphosphorylation of Rb releases transcription factor

E2F1, and allows for the transcription of genes required for G1/S transition, such as

cyclin A, cyclin D and cyclin E (Dyson, 1998). Cyclin E-CDK2 is required to initiate S

phase, cyclin A-CDK2 is expressed during DNA synthesis in the cell, whilst cyclin B-

CDK1 is needed to drive entry into mitosis (Hochegger et al, 2008; Nurse, 2000)

Figure 1.5 The cell cycle

Cell cycle phases and the key cyclin-CDK complexes involved in the cell cycle.

Key for Figure 1.5

Various techniques have been used to regulate the proliferation of mammalian cells, for

example, via activation of cyclin-dependent kinase inhibitors (CKIs) and via

temperature control.

CKIs are divided into two major families, the INK4 (inhibitor of CDK4) family, which

specifically inhibit cyclin D-associated kinases (CDKs 4 and 6), and the Cip/Kip (kinase

inhibitor protein) family, consisting of p21cip1/waf1

, p27kip1

and p57kip2

, which inhibit

most CDKs (Dai & Grant, 2003). Enhancement of p21cip1

in a recombinant GS-CHO

cell line increased both productivity and final titre by arresting cells in the G1 phase of

MG0

G2

G1

S

Cyclin A-CDK2

Cyclin B-CDK1

Cyclin E-CDK2

Interphase

Mitosis

34

the cell cycle (Bi et al, 2004), whilst overexpression of p27kip1

also resulted in growth-

arrest and greater recombinant protein production from recombinant CHO cultures

(Mazur et al, 1998). Although growth arrest has been shown to improve protein

production from CHO cells it can occur with higher cellular energy expenditure. For

example, p27kip1

mediated CHO cell growth arrest resulted in increased rates of oxygen,

glutamine and glucose consumption, with greater production of lactate and ammonia

(Carvalhal et al, 2003).

Marchant et al, have also shown that CHO cell growth can also be regulated by

controlling culture temperature. A decrease in culture temperature from 37°C to 27°C

resulted in G1 cell cycle arrest and lower maximal cell densities. Although total

antibody titres were not enhanced, increased specific protein production rate per cell

(specific productivity [Qp]) was observed (Marchant et al, 2008). An associated

increase in Qp during mild-hypothermic conditions has also been observed for other

recombinant CHO cultures (Nam et al, 2008; Rodriguez et al, 2005; Yoon et al, 2006;

Yoon et al, 2003). Exposure of mammalian cells to low temperatures has resulted in the

determination of cold-stress genes (Al-Fageeh & Smales, 2006), such as the cold-

inducible RNA-binding protein (CIRP), which is highly expressed during mild-

hypothermic conditions but not physiological temperatures (Nishiyama et al, 1997).

Overexpression of CIRP has been shown to increase both recombinant CHO protein

titre and Qp values at 37°C, without affecting viable cell densities (Tan et al, 2008).

1.5 METABOLIC ACTIVITY AS A POTENTIAL DETERMINANT OF

RECOMBINANT PROTEIN PRODUCTION

Assessment of cell growth and metabolic activities are essential to the success in the

control and improvement of a cell culture processes (Tsao et al, 2005). A proliferating

cell must replicate all of its cellular contents. This imposes a large requirement for

nucleotides, lipid, amino acids and carbon sources for effective replication (Van der

Heiden et al, 2009).

Glucose serves as both a main carbon source and an important energy intermediate in

most medium formulations. Entry of glucose into the glycolytic pathway leads to the

35

formation of pyruvate. In mammalian cells, pyruvate can either be shuttled into the

tricarboxylic acid (TCA) cycle or be converted into lactate (Tsao et al, 2005). The

oxidation of glycolytic pyruvate in the TCA cycle produces NADH, needed to

maximize ATP production via oxidative phosphorylation (shown in Figure 1.6, Van der

Heiden et al, 2009). Metabolic production of ATP is necessary for many cellular

processes, including transcription, translation, protein folding, secretion and degradation

(highlighted in Figure 1.7).

Figure 1.6 Metabolic production of ATP, NAD+ and NADH

ATP, NAD+ and NADH production via metabolic pathways involved in glycolysis, the

TCA cycle, oxidative phosphorylation and the utilisation of lactate and alanine.

The consumption rate of glucose and the accumulation rate of lactate can reflect the

metabolic activities and cell growth of the cultures. For example, CHO cell lines with

low rates of glucose utilisation and lactate production had low rates of cell growth

(Marchant et al, 2008), whilst cultures exposed to limited glucose concentrations also

Glucose Glucose-6-phosphate

Acetyl CoA

Oxaloacetate TCA Cycle

Lactate

Glycolysis

Succinyl

CoA

Oxidative

Phosphorylation

NADH

NAD+

O2

ADP

ATP

NADH

NAD+

ATP

ADP

NADH

Mitochondria

Cytosol

α-Ketoglutarate

Malate

NAD+

NADH

NADH

NAD+

AlanineAlanine

NAD+

NADH

Lactate

Glyceraldehyde 3-phosphate

1,3-Diphosphoglycerate

3-Phosphoglycerate

ADP

ATP

NAD+

NADH

2-Phosphoglycerate

Phosphoenolpyruvate

ADP

ATP

Pyruvate

NAD+


Acetyl CoA

Oxaloacetate TCA Cycle

Lactate

Glycolysis

Succinyl

CoA

Oxidative

Phosphorylation

NADH

NAD+

O2

ADP

ATP

NADH

NAD+

O2

ADP

ATP

NADH

NAD+

ATP

ADP

NADH

Mitochondria

Cytosol

α-Ketoglutarate

Malate

NAD+

NADH

NADH

NAD+

AlanineAlanine

NAD+

NADH

Lactate

Glyceraldehyde 3-phosphate

1,3-Diphosphoglycerate

3-Phosphoglycerate

ADP

ATP

NAD+

NADH

2-Phosphoglycerate

Phosphoenolpyruvate

ADP

ATP

Pyruvate

NAD+

36

had decreased viable cell densities (Altamirano et al, 2006; Lu et al, 2005), less

intracellular ATP, and increased utilisation of amino acids (Lu et al, 2005)

Metabolic engineering has also been employed to increase recombinant protein

production. Many strategies have involved lowering the expression of lactate

dehydrogenase (LDH), the enzyme involved in the conversion of glucose-derived

pyruvate to lactate. Less LDH within CHO cultures resulted in lower lactate

concentrations with greater intracellular ATP and recombinant antibody production

(Chaya et al, 2008; Jeong et al, 2006; Jeong et al, 2004; Kim & Lee, 2007).

Components of the TCA cycle have also been targeted to improve productivity of

recombinant CHO cells. Overexpression of malate dehydrgoenase II (MDH II), which

converts malate to oxaloacetate in the mitochondria as part of the TCA cycle, resulted in

lower lactate secretion, a three- to four-fold increase in ATP and NADH, and enhanced

MAb titre (Chong et al, 2010). A recombinant yeast pyruvate carboxylase expressed in

baby hamster kidney (BHK-21) cells has also been shown to affect the metabolic

activity of mammalian cultures, increasing the flux of glucose into the TCA

consequently resulting in a higher intracellular ATP content and greater recombinant

protein production (Irani et al, 2002).

37

Figure 1.7 ATP is critical at multiple sites of protein expression

ATP is an important energy source used at various stages of recombinant protein

production. ATP is also needed for cell survival as cells deficient in ATP often undergo

apoptosis (Izyumov et al, 2004).

1.6 TRANSCRIPTION AS A POTENTIAL DETERMINANT OF


The variability of transgene expression is often attributed to the number of gene copies

integrated, and to the particular site of integration within the host chromatin structure

(Davies & James, 2009). Chromatin is a DNA-protein complex whose basic repeating

unit is the nucleosome (Kornberg & Lorch, 1999). The nucleosome contains a tripartite

core of eight histones (two molecules of each histone H2A, H2B, H3 and H4), around

which is wrapped 146 bp DNA (Luger et al, 1997). Each core histone has two domains,

a histone fold domain, which is involved in histone-histone interaction and in wrapping

DNA around the nucleosome, and an amino-terminal tail domain that lies on the outside

Ribosome

mRNA

Transcription

Polypeptide

Amino-acylated

tRNAs

Amino acids

free

tRNAs

ADPATP

ATPADP

GTP GDP

ChaperoneUnfolded

polypeptide

ATP

ADPFolded

polypeptide

ER associated-

degradation

ADPATP

Ubiquitination

& proteasomal degradation

UbUb

Ub

Protein

Folding

Translation

Degradation

38

of the nucleosome allowing interactions with other regulatory proteins and DNA, as

well as providing a site for post-translational modifications (Jones & Wolffe, 1999).

Chromatin can be subdivided in two forms, condensed heterochromatin, which is

generally accepted to exist in a transcriptionally silent state, and the transcriptionally

active decondensed euchromatin (Davies & James, 2009). It has been observed that

transcriptionally active genes are enriched in acetylated histones (Kouzarides, 2000).

Histone acetylation is a lysine amidation reaction catalyzed by acetyltransferases, which

acts to neutralize the positive charge of the histone tails thereby lowering their affinity

for DNA (Hong et al, 1993). Histone acetylases (HATs) and histone deacetylases

(HDACs) act as transcription coactivators and corepressors, respectively (Pazin &

Kadonaga, 1997; Struhl, 1998). Sodium butyrate, an inhibitor of HDAC, has been

shown to increase the specific productivity of recombinant proteins in CHO cells by

enhancing gene accessibility to transcription factors (Jiang & Sharfstein, 2008; Zhou &

Sharfstein, 2008).

Improvement of gene expression can also occur by site-specific integration or by

flanking the transgene with genomic DNA elements that promote high transcriptional

activity (Davies & James, 2009). There are many transgene flanking DNA elements

used to improve transgene expression and stability, these include locus control regions

(LCRs), boundary and insulator elements, scaffold/matrix attachment regions (S/MAR)

and ubiquitous chromatin opening elements (UCOEs). S/MAR elements have been used

to improve both stable gene expression (Kim et al, 2004) and antibody production from

recombinant CHO cells (Zahn-Zabal et al, 2001), whilst vectors containing an UCOE

element have been reported to provide a higher proportion of positive CHO clones with

greater recombinant protein expression (Benton et al, 2002). Gene-targeting vectors

have also been used to target desired genes within the CHO cell genome to obtain high

recombinant proteins producers (Huang et al, 2007; Kito et al, 2002).

39

1.7 TRANSLATION AS A POTENTIAL DETERMINANT OF RECOMBINANT

PROTEIN PRODUCTION

1.7.1 Translational initiation

The majority of translational control is exerted on the initiation stage of protein

synthesis, during which the ribosomes bind the mRNA and locate the start codon (AUG,

Gingras et al, 1999; Sonenberg & Hinnebusch, 2009). The eukaryotic translation

initiation factor (eIF) 4E plays a key role by binding to the cap structure at the 5'-end of

the mRNA and recruiting initiation factors to the mRNA (Proud, 2002a). To initiate

translation, the 40S ribosomal subunit, stabilised by association with the large

multisubunit initiation factor eIF3, binds to Met-tRNAi and the mRNA. The Met-tRNAi

is brought to the 40S ribosomal subunit as part of an eIF2–GTP complex (eIF2-GTP-

Met-tRNAi), and together with other initiation factors, eIF3, eIF1, eIF1A and eIF5,

forms the 43S preinitiation complex. On most mRNAs, 48S complexes form by a

„scanning‟ mechanism, whereby the 43S preinitiation complex attaches to the capped 5‟

proximal region of mRNA‟s 5‟ terminal secondary structure by eIF4A, eIF4B and

eIF4F. After initiation codon recognition the 48S complex formation, eIF5 and eIF5B

promote the hydrolysis of eIF2-bound GTP, the displacement of eIFs and the joining of

a 60S subunit. (Figure 1.8, Fraser & Doudna, 2007; Sonenberg & Hinnebusch, 2009).

Under stressed conditions eIF4F assembly is blocked by 4E-binding protein (4E-BP)

binding to eIF4E via the mammalian target of rapamycin (mTOR) pathway (Carrera,

2004; Fingar et al, 2004; Kimball & Jefferson, 2004).

Two main mechanisms control initiation through reversible phosphorylation. The first

phosphorylation of eIF4E binding proteins stimulates the binding of eIF4E to the

mRNA allowing translation initiation. The second phosphorylation of eIF2α halts

translation by interfering with binding of initiator methionyl-tRNA to the 40S ribosomal

subunit (Kaufman et al, 2002). Translation attenuation by eIF2α phosphorylation will be

discussed in more detail Section 1.8.3.2.

Engineering at the mRNA translation initiation step via transient expression of non-

phosphorylatable eIF2α has been shown to improve reporter activity within CHO cells

by improving rates of protein synthesis (Underhill et al, 2003).

40

Figure 1.8 Diagram of translation initiation

Translation of mRNA into protein begins after assembly of initiator tRNA, mRNA and

both ribosomal subunits. The pre-initiation complex eIF2-GTP-Met-tRNAi together with

eIF3, eIF1, eIF1A and eIF5 binds to the mRNA at the 5' terminal cap structure with

help of the eIF4F protein complex and proceeds to scan the mRNA until it encounters

the initiation codon. GTP hydrolysis allows the joining of the 60S subunit and recycling

of the initiation factors.

1.7.2 RNA interference (RNAi)

RNAi can also regulate gene expression at the level of translation. RNAi has been

shown to occur specifically in the presence of double-stranded (ds) RNA with a

sequence complementary to the mRNA being targeted (Tuschl et al, 1999; Wianny &

Zernicka-Goetz, 2000). Studies of transgene-induced and virus-induced gene silencing

in plants identified the presence of small interfering RNAs (siRNAs), approximately 22

nucleotides in length (Hamilton & Baulcombe, 1999). Ribonuclease III-like enzyme

(Dicer) is the enzyme responsible for processing dsRNA into siRNAs (Bernstein et al,

2001). The antisense strand of the siRNA is used by RNA-interference silencing

complex (RISC, Hammond et al, 2000) to guide mRNA cleavage and mRNA

degradation (McManus & Sharp, 2002). siRNAs can also function as microRNAs

eIF2-GTP

Met-tRNAi

eIF2-GTP-Met-tRNAi

43S preinitiation complex

eIF3

eIF1

eIF1A

eIF5

40S

eIF4F

60S

Ribosome Scanning

GTP hydrolysis

eIF5B-GTP

80S initiation complex

eIF2-GDP + Pi

eIF2B

AUG

GTP

GDP

mRNA

mRNA

41

(miRNA, Doench et al, 2003). miRNAs, are non-coding RNAs predicated to pair with

30% of mammalian protein coding genes, possibly to direct translational repression or

mRNA degradation (Baek et al, 2008; Bartel, 2004; Bartel, 2009; Filipowicz et al,

2008). The upregulation of growth inhibitory miRNAs, miR-21 and miR-24 has been

shown during CHO-K1 batch culture (Gammell et al, 2007).

In addition to the natural actions of siRNAs, RNAi technology has been exploited by

the industry to increase recombinant CHO cell productivity. For example, Lim et al,

used siRNA to knock down pro-apoptotic genes Bak and Bax, resulting in greater

viability of CHO batch cultures with improved interferon (IFN) product titre (Lim et al,

2006).

1.8 PROTEIN FOLDING AS A POTENTIAL DETERMINANT OF


Once effectively translated, proteins destined for secretion are directed to the ER

through a predominantly hydrophobic signal sequence and either co- or post-

translationally traverse the ER membrane through an aqueous channel, the Sec61

complex (Rutkowski et al, 2003). The ER plays a crucial role in the folding, assembly

and glycosylation of newly synthesised proteins. As many secretory proteins contain

disulphide bonds, a central role is played by ER-resident oxidoreductases (ERO),

including protein disulphide isomerase (PDI), and protein chaperones such as BiP

(Ellgaard & Helenius, 2003).

BiP (also known as glucose-regulated protein [GRP] 78) is an ER homologue of

HSP70, containing both an peptide-binding domain and ATPase domain (Bole et al,

1986; Haas & Wabl, 1983; Hendershot et al, 1987; Hendershot, 2004; Lee, 2001)). BiP

interacts with heavy chain proteins (in the absence of light chain proteins, Vanhove et

al, 2001), and hydrophobic residues exposed on unfolded proteins (Gregory et al, 1991).

When in its open conformation BiP is bound to ATP. ATP hydrolysis allows BiP to

bind to mis/unfolded proteins (Mayer et al, 2000), a process stimulated by ER-resident

J-domain (ERdj) co-chaperones, including ERdj3, ERdj4, ERdj5 and ERdj6 (p58IPK

,

Otero et al, 2009). BiP also assists PDI binding to mis/unfolded proteins, allowing it to

access the incorrectly folded protein (Mayer et al, 2000).

42

PDI aid protein re-folding by catalysing disulphide bond formation, with the assistance

of ERO1 (Bulleid & Freedman, 1988; Roth & Pierce, 1987). ERO1 is oxidized by

molecular oxygen and in turn acts as a specific oxidant of reduced PDI, whilst at the

same time reducing its cofactor flavin adenine dinucleotide (FAD) to FADH2 (Figure

1.11, Frand & Kaiser, 1999; Tu et al, 2000; Tu & Weissman, 2004). In mammalian cells

the capacity of oxidative protein folding machinery depends on two conserved resident

oxidases ERO1α and ERO1β. ERO1α is expressed in most cell types, whilst ERO1β is

induced by ER stress (Cabibbo et al, 2000; Mezghrani et al, 2001; Pagani et al, 2000).

Increased ERO1 expression in mammalian cells has been shown to increase the rate of

PDI-dependent immunoglobulin oxidation (Mezghrani et al, 2001).

Figure 1.9 The role of PDI in disulphide bond formation

Protein disulphide isomerase (PDI) operates in both an oxidised and reduced form to

assist in disulphide bond formation with the cooperation of ER-resident oxidoreductase

(ERO1).

Studies involving upregulation of ER chaperone proteins have given differing accounts

relating to the improvement of recombinant protein production from CHO cultures.

Borth et al, found that enhanced BiP expression in CHO cells resulted in lower antibody

productivity due to increased accumulation of intracellular heavy chain proteins (Borth

et al, 2005), whilst Hayes et al, found that overexpression of PDI had no effect on

recombinant CHO IgG4 MAb titres (Hayes et al, 2010). However, other investigations

have shown increased Qp values from recombinant CHO cell lines with the

upregulation of PDI expression alone (Borth et al, 2005; Chaya et al, 2007; Mohan et al,

H2O

O2

FAD

FADH2

ERO1 reduced

ERO1 oxidised

SH

PDI oxidised

PDI reduced

SHSH

SH

SS

SS

SS

SHSH

43

2007), or in combination with ERO1 (Mohan & Lee, 2010). These contradictory

findings highlight the cell line specific nature of the CHO cell line.

Protein folding in the case of MAbs also requires the coordinated expression of both

heavy chain and light chain proteins (Dinnis & James, 2005). It has been previously

suggested that excess copies of light chain proteins are necessary for optimal rates of

MAb assembly in mammalian cells (Smales et al, 2004), whilst heavy chain mRNA

translation was found to exert most control on a recombinant IgG4 antibody producing

CHO cell line (Schlatter et al, 2005). Again the dependency of heavy chain or light

chain expression on optimal MAb formation may be cell line specific.

1.8.1 N-linked glycosylation

As mentioned in Section 1.3 N-linked glycosylation is common to recombinant

antibodies, and is also important for protein folding (Hammond et al, 1994). When a

nascent protein enters the ER lumen through the Sec61 translocon complex, their

sequence is scanned by the luminal oligosaccharyltransferase (OST) for asparagine in

consensus Asn-X-Ser/Thr motifs. These are modified covalently by the addition of

preassembled, tri-antennary core glycan composed of two N-acetylglucosamine

(GlcNAc), nine mannose (Man) and three glucose (Glc) residues (Glc3Man9GlcNAc2,

Ruddock & Molinari, 2006). The glycoprotein is then monitored within the ER by the

calnexin (CNX)/calreticulin (CRT) cycle (Section 1.8.2) to ensure it has been correctly

folded. Once the glycoprotein has correctly folded it travels to the Golgi and becomes

characterised by the addition of new oligosaccharides including GlcNAc, galactose

(Gal), fucose (Fuc) and sialic acid (Helenius & Aebi, 2001; Jefferis, 2005; Jenkins et al,

1996). It has been reported that the sialylated profiles of antibodies produced from CHO

and human IgGs differ. However, the amount of sialylated oligosaccharides in both

human-and CHO-derived antibodies is so low that the difference in sialylation patterns

is not considered disadvantageous to recombinant CHO cell protein production (Raju et

al, 2000).

After terminal glycosylation only Man3GlcNAc2 from the original core is present. A

shorthand system of nomenclature for oligosaccharides uses G0, G1 and G2 for those

glycans bearing zero, one or two galactose residues, respectively. An F is added after

44

the oligosaccharide number to indicate the presence of fucose (Jefferis, 2009b). The

common N-linked glycan structures are shown in Figure 1.10

Figure 1.10 Common N-linked glycan structures

The shorthand system of nomenclature for the oligosaccharide structures commonly

found on antibodies.

The glycan profile of the therapeutic protein is extremely important as it can modulate

the immunogenic potential of the glycoprotein by defining all or part of an epitope

(Cumming, 1991). It is also important for maintaining quality control (QC) within the

ER, ensuring ER homeostasis.

1.8.2 Calnexin/calreticulin (CNX/CRT) cycle

The CNX/CRT cycle (Figure 1.11) acts as part of the QC mechanism in the ER to

monitor protein conformations and dictate whether a molecule is exported or targeted

for degradation. CNX and CRT are homologous lectins resident to the ER (Hammond et

al, 1994). As previously mentioned (in Section 1.8.1) N-linked glycosylation occurs

through the transfer of Glc3Man9GlcNAc2 to the nascent polypeptide chain as it enters

the ER lumen. Soon after transfer trimming of the core oligosaccharide occurs by the

successive action of ER glucosidases I and II to produce Glc1Man9GlcNAc2. The

monoglucosylated form interacts with CRT and CNX, shuttling through cycles of de-

G0

G1

G2

Non-Fucosylated Fucosylated (F)

GlcNAc

GlcNAc

Man

Man

Man GlcNAc GlcNAc

GlcNAc

GlcNAc

Man

Man

Man GlcNAc GlcNAc

Fuc

GlcNAc

GlcNAc

Man

Man

Man GlcNAc GlcNAc

GlcNAc

GlcNAc

Man

Man

Man GlcNAc GlcNAc

Fuc

Gal Gal

GlcNAc

GlcNAc

Man

Man

Man GlcNAc GlcNAc

GlcNAc

GlcNAc

Man

Man

Man GlcNAc GlcNAc

Fuc

Gal Gal

GalGal

45

and re-glucosylation by α-glucosidase II and uridine diphosphate (UDP)-

glucose:glycoprotein glucosyl transferase (UGGT, Ellgaard & Helenius, 2003). UGGT

preferentially recognizes and glucosylates partially mis/unfolded glycoproteins.

Mis/unfolded proteins are then retained within the CNX/CRT cycle, via interactions

with ERp57 (a thiol-disulphide oxidoreductase) until the protein is folded correctly or

targeted for degradation (Section 1.8.4, Ellgaard & Helenius, 2001; Frickel et al, 2002).

Figure 1.11 The CNX/CRT cycle

The calnexin (CNX) and calreticulin (CRT) cycle is a quality control mechanism within

the ER to ensure only correctly folded glycoproteins are secreted.

CNX and CRT can also act by binding to BiP if N-linked glycosylation is blocked, this

process results in the activation of an unfolded protein response (UPR, Section 1.8.3,

Balow et al, 1995; Zhang et al, 1997).

1.8.3 The Unfolded Protein Response (UPR)

As a major post-translational protein processing organelle, the ER monitors, and

controls via feedback mechanisms, the protein production load and ensures the QC of

proteins within the exocytic pathway (Ellgaard & Helenius, 2003; Rutkowski &

Kaufman, 2004). Ectopic expression of recombinant proteins can compromise the ER

protein folding capacity and result in accumulation of unfolded proteins within the ER

ER

Glucosidase I and II

CRT

Glc1Man9GlcNAc2

ERp57

Unfolded protein

Man9GlcNAc2

Glucosidase II

Man9GlcNAc2

Folded protein

Glc1Man9GlcNAc2

UGGT

Ribosome/sec61 complex

Glc1Man9GlcNAc2

ERp57

CNX

46

(Cudna & Dickson, 2003). Increased demands on the secretory pathway can activate the

UPR, which consists of four main processes (Brewer & Hendershot, 2005). These

processes include (i) transcriptional induction of molecular chaperones and folding

enzymes (Dorner et al, 1992), (ii) attenuation of global protein synthesis (Harding et al,

1999), (iii) ER-associated degradation (ERAD) of mis/unfolded proteins (Yoshida et al,

2003), and (iv) cellular apoptosis (Kim et al, 2006). Stress sensors, inositol requiring

protein-1 (IRE-1), activating transcription factor 6 (ATF6) and protein kinase RNA

(PKR)-like ER kinase (PERK) are activated upon the dissociation of BiP. These stress

sensors are discussed in more detail below.

1.8.3.1 IRE-1

Genetic screens in yeast identified IRE-1, a type I transmembrane protein with

serine/threonine protein kinase activity, as the ER stress receptor (Cox et al, 1993). The

accumulation of mis/unfolded proteins and dissociation of BiP activates IRE-1,

allowing it to homodimerize and transphosphorylate (Welihinda & Kaufman, 1996). In

yeast the endonuclease activity in the C-terminal domain of IRE-1 catalyses splicing

and activation of HAC1 mRNA (Cox & Walter, 1996; Sidrauski & Walter, 1997) to

allow induction of ER stress genes to aid in protein folding, as well as ER and Golgi

transport (Mori et al, 2000; Rüegsegger et al, 2001).

The mammalian IRE-1 pathway is similar to yeast, although there are two mammalian

IRE-1 genes that have been cloned, IRE1α (Tirasophon et al, 1998) and IRE1β (Wang

et al, 1998). Once activated the cytosolic endonuclease component of IRE-1 splices a

26-bp intron from X-box-binding protein (XBP)-1 mRNA (Bertolotti & Ron, 2001;

Yoshida et al, 2001), the mammalian homolog of HAC1 (Calfon et al, 2002; Tirasophon

et al, 1998). Activated and spliced XBP-1 (XBP-1[s]) migrates to the nucleus where it

binds with the general transcription factor, nuclear factor (NF)-γ, and the ER stress-

response element (ERSE), a promoter sequence encoding for XBP-1, BiP and P58IPK

(Calfon et al, 2002; Lee et al, 2003).

The cytosolic domain of IRE1α has also been shown to interact with the apoptosis-

associated proteins Bax and Bak, an interaction which increases during ER stress (Hetz

et al, 2006). The regulation of ER stress-induced apoptosis is discussed in Section

1.8.4.3.

47

1.8.3.2 ATF6

Two isoforms of ATF6 exist in mammalian cells, ATF6α and ATF6β (Haze et al, 1999;

Yoshida et al, 1998). The accumulation of mis/unfolded protein causes BiP dissociation

from ATF6 revealing two Golgi localisation signals. The localisation signals allow

ATF6 to enter COPII vesicles and translocate to the Golgi (Shen et al, 2002), where it is

proteolytically processed by two Golgi resident enzymes: site-1 protease (S1P) and site-

2 protease (S2P, Shen & Prywes, 2004). The cleavage yields a free cytoplasmic domain,

a 50-60kD cytosolic bZIP-containing fragment that migrates to the nucleus, and in the

presence of the CCAAT-binding factor (CBF), binds exclusively to the ERSE (Shen et

al, 2001). CHO cells defective in S2P are unable to proteolytically process ATF6 and

effectively respond to ER stress (Nadanaka et al, 2006).

ATF6 and IRE-1 differ in their timing but interact to induce XBP-1 transcription and

mRNA splicing (Yoshida et al, 2003). This splicing event creates a translational

frameshift in XBP1 mRNA allowing production of an active transcription factor, co-

inducing the UPR-response elements (UPRE, Calfon et al, 2002; Lee et al, 2002; Shen

et al, 2001; Yoshida et al, 2001). Since XBP-1(s) binds and activates both ERSE- and

UPRE-containing promoters, it has been considered a global regulator for sustained

stress recovery across the entire endomembrane/endocytic systems (Tigges &

Fussenegger, 2006).

1.8.3.3 PERK

PERK is type I ER-resident protein that senses ER stress through its luminal domain

(Harding et al, 2000a; Harding et al, 2000b; Harding et al, 1999). BiP dissociation

causes autophosphorylation and dimerisation of PERK (Bertolotti et al, 2000). Once

activated PERK phosphorylates serine-51 of eIF2α (Harding et al, 2000a; Harding et al,

2000b; Shi Y, 1998). Phosphorylated eIF2α prevents the exchange of eIF2-GDP to

eIF2-GTP, which is needed to bind to Met-tRNAi and assemble the 43S preinitiation

complex (Section 1.7.1). This process results in a transient decrease in protein synthesis

(Brostrom & Brostrom, 1998; Prostko et al, 1993). Although global protein synthesis is

inhibited, translation of ATF4 mRNA is preferentially initiated due to the position of

two upstream open reading frames (uORFs) allowing re-initiation at 5‟ sites (Harding et

48

al, 2000a; Lu et al, 2004). Activated PERK also blocks the translation of cyclin D1

mRNA, resulting in the loss of cyclin D-dependent kinase activity. This loss, in turn,

leads to inhibition of cyclin E- and A-dependent CDK2 leading to G1 cell cycle arrest

(Brewer & Diehl, 2000; Brewer et al, 1999). Cell cycle progression is discussed in

Section 1.4.

The stress sensor pathways involved in the UPR are shown in Figure 1.12. The proteins

affected by the PERK pathway are discussed in more detail below.

Figure 1.12 Activation of the UPR

The UPR pathway is mediated by three resident proteins, PERK, ATF6 and IRE-1

which act as mis/unfolded protein sensors. Upon activation the sensors initiate

responses that lower ER protein load by inhibiting translation and by enhancing

expression of UPR genes, such as ER chaperones.

Ribosome/Sec61 complex

BiP

PDI

P

IRE-1

Golgi

ATF6 (p50)

ATF6 (p90)P P

eIF2α

ATF4

GADD34

Cyclin D1translation

Cell cycle arrest

XBP-1splicing

PERK

P

P

ER

XBP-1(s)

GADD153

Global Translation

Attenuation

Induction of UPR genes

49

1.8.3.3.1 ATF4

ATF4 belongs to the ATF/CREB family of basic region-leucine zipper transcription

factors and is induced by many stress signals including ER stress, amino acid

deprivation, oxidative stress and anoxia/hypoxia. As mentioned earlier ATF4 mRNA is

translationally upregulated by ER stress via the PERK pathway (Harding et al, 2000a;

Lu et al, 2004) and is also transcriptionally upregulated by nutrient deprivation, through

the activation of GCN2 by the direct binding of uncharged tRNAs that accumulate

during amino acid limitation (Siu et al, 2002, Hinnebusch, 2005). The activation of

GCN2 also results in the eIF2α phosphorylation, which acts to limit protein synthesis,

ensuring amino acids are not completely depleted (Harding et al 2000a).

The downstream targets of ATF4 include genes involved in amino acid import, redox

regulation and cell growth (Harding et al, 2003), such as growth arrest and DNA–

damage–inducible genes (GADD34 & GADD153, Fawcett et al, 1999; Ma &

Hendershot, 2003).

1.8.3.3.2 GADD153

GADD153 is a bZIP-containing transcription factor that was identified as a member of

the CCAAT/enhancer binding protein (C/EBP) family (Ron & Habener, 1992), hence

also referred to as C/EBP homologous transcription factor (CHOP). Ma et al,

demonstrated that the C/EBP ATF composite site was essential for stimulation of

GADD153 promoter activity under ER stress (Ma et al, 2002).

The regulation of GADD153 is also dependent on the nutrient status of the cell, and is

induced during glucose (Carlson et al, 1993) and leucine (Bruhat et al, 2000; Bruhat et

al, 1997) deprivation. Induction of GADD153 expression by amino acid starvation

requires both ATF4 and phosphorylated ATF2 (Averous et al, 2004; Bruhat et al, 2007;

Bruhat et al, 2000). Bruhat et al, reported that in vivo binding of phosphorylated ATF2

and ATF4 to the GADD153 amino acid response element (AARE) is associated with

the acetylation of histones H4 and H2B in response to amino acid starvation, which

promotes the modification of the chromatin structure to enhance the transcription of

amino acid-regulated genes (Bruhat et al, 2007).

50

GADD153 activation affects a series of target genes including TRB3, Bcl-2, ERO1, and

GADD34 (Section 1.8.3.3.3). GADD153 acts to downregulate the expression of Bcl-2,

an anti-apoptotic protein (McCullough et al, 2001), whilst TRB3, a tribbles ortholog,

associates with GADD153 to block its proapoptotic activity whilst inhibiting its own

induction (Ohoka et al, 2005).

1.8.3.3.3 GADD34

GADD34 is a regulatory unit of phosphatase PP1 that binds to the catalytic subunit

(PP1c) resulting in dephosphorylation of eIF2α (Novoa et al, 2001). This negative

feedback loop acts to restore protein synthesis and allow translation of stress-induced

transcripts (Brush et al, 2003). GADD34 is not only transcriptionally induced but also

translationally regulated to ensure maximal expression during eIF2α phosphorylation.

Findings suggest that GADD34 translation is regulated by an unique 5′UTR uORF

mechanism, similar to that of ATF4, to ensure increased GADD34 expression during

eIF2α phosphorylation (Lee et al, 2009b). Overexpression of GADD34 resulted in

enhanced recombinant protein production from CHO cultures, possibly due to increased

rates of recombinant protein translation (Omasa et al, 2008).

Although UPR mechanisms act to ensure only correctly-folded proteins are secreted

from the ER persistent UPR would be undesirable in a stable production cell line as it

would decrease protein synthesis and ultimately lead to ERAD and cell death (Dinnis &

James, 2005).

1.8.4 ER associated-degradation (ERAD) and ER stress-associated apoptosis

1.8.4.1 ERAD

Mannose trimming by the ER α1,2-mannosidase I occurs when proteins are unable to

fold correctly and leads to their association with ER degradation-enhancing 1,2-

mannosidase-like protein (EDEM, Hosokawa et al, 2001; Olivari et al, 2006).

Transcriptional induction of EDEM is mediated by mis/unfolded proteins via the IRE1-

XBP1 pathway (Yoshida et al, 2003). It is thought that EDEMs deliver ERAD

substrates to the retro-translocation channel (Kanehara et al, 2007). Ubiquitylation of

51

the substrates on the cytoplasmic side of the membrane is essential for their retro-

translocation to the cytoplasm. There are various ubiquitin-protein ligases (E3s), such as

HRD1 and gp78 which have been shown to mediate the ubiquitylation of ER-derived

proteins (Fang et al, 2001; Schulze et al, 2005). Upon their ubiquitylation substrate

proteins are recognized by a protein complex consisting of p97, a cytosolic ATPase

responsible for removal of substrates for degradation by the cytosolic 26S proteasome

in an ATP-dependent process (Oberdorf et al, 2001).

1.8.4.2 Macroautophagy

Macroautophagy is another intracellular degradation system in which cells can remove

mis/unfolded proteins. Distinct classes of phosphatidylinositol-3‟-kinases (PI3Ks) are

involved in the control of macroautophagy in mammalian cells (Petiot et al, 2000). The

initial nucleation and assembly of the primary autophagosomal membrane requires a

kinase complex that consists of class III PI3K, p150 myristylated protein kinase and

beclin 1. The autophagosomes ultimately fuse with lysosomes leading to the degradation

of the sequestered material (Li et al, 2008). This degradation event is activated by the

IRE-1 pathway in order to prevent cell death in response to ER stress (Ding et al, 2007)

and nutrient deprivation (Hwang & Lee, 2008). The anti-apoptotic properties of

macroautophagy may be due to the clearance of mitochondria, thereby lowering the

amount of cytochrome c release and subsequent cell death (Ravikumar et al, 2006).

1.8.4.3 ER-stress induced apoptosis

Studies have linked ER stress with apoptosis mediated by mitochondrial pathways

involving Bcl-2, Bax and Bak. During ER stress Bax and Bak undergo conformational

changes allowing the release of Ca2+

from the ER to the cytoplasm (Scorrano et al,

2003; Zong et al, 2003). Increased Ca2+

in the cytosol activates m-calpain, which

subsequently cleaves and activates procaspase 12, initiating a caspase cascade

(Nakagawa & Yuan, 2000; Morishima et al, 2002). The caspase family is broadly

divided into two groups: initiator caspases (caspase-8, -9, and -12) and effector caspases

(caspase-3, -6, and -7). Initiator caspases undergo autoprocessing for activation in

response to apoptotic stimuli. Active initiator caspases in turn process precursors of the

effector caspases responsible for dismantling cellular structures (Morishima et al, 2002).

52

IRE-1α has also been found to induce apoptosis following ER stress, via recruitment of

the adaptor protein TNF receptor-associated factor 2 (TRAF2, Wang et al, 1998), c-Jun

NH2-terminal inhibitory kinase (JIK, Yoneda et al, 2001) and apoptosis signal-

regulating kinase 1 (ASK1, Nishitoh et al, 1998). The c-Jun NH2-terminal kinase

(JNK), which is activated by the IRE1α/JIK/TRAF2/ASK1 pathway, can phosphorylate

Bcl-2 (Yamamoto et al, 1999). Bcl-2 in a phosphorylated state is unable to bind and

prevent activation of proapoptotic members, resulting in changes to the concentration of

cytosolic Ca2+

, again causing cytochrome c release (Bassik et al, 2004). The ER stress-

mediated degradation and cell death pathways are summarised in Figure 1.13.

Figure 1.13 ER stress-mediated degradation and cell death pathways

ER associated-degradation (ERAD) aims to ensure ER homeostasis by degrading

mis/unfolded proteins or removing material which may cause cell death. If the protein

balance within the ER cannot be controlled and ER stress becomes severe ER-stress

associated apoptosis occurs.

Ribosome/Sec61 complex

BiP

PDI

P

IRE-1P P

PERK

P

ER

JIKTRAF2ASK

JNK

Bcl-2

MtCytochrom c

Bax/Bak

Ca2 +

m-calpain

Casp 12

Casp 9

Casp 3

Apoptosome

P

-

ERAD

ER-stress mediated

cell death pathways

lysosome

Proteasomaldegradation

EDEMUb

Ub

UbHRD1

gp78

p97

Autophagosome

MtCytochrome c

Apoptosome

53

Genetic engineering strategies to directly maintain or extend viable cell density to

improve protein production have concentrated on the inhibition of apoptotic cell death

(Singh & Anand, 1994). Overexpression of the anti-apoptotic proteins Bcl-2 and Bcl-xL

have been shown to increase maximal viable cell densities and suppress apoptosis in

CHO cultures (Mastrangelo et al, 2000; Tey et al, 2000; Kim et al, 2009, Kim & Lee,

2000), with the improvement of recombinant protein titres (Meents et al, 2002; Kim &

Lee, 2000). Recently upregulation of myeloid cell leukemia (Mcl)-1, a member of the

Bcl-2 family protein, has also been shown to increase viable cell densities and antibody

production in recombinant CHO cell cultures (Majors et al, 2009).

Recent strategies for improving viable cell densities, and recombinant protein

production, have also involved the mRNA knockdown of pro-apoptotic proteins. CHO

cells lacking Bax and Bak were able to grow but failed to activate caspases in response

to apoptotic stimuli. Antibody production was increased for these cultures (Cost et al,

2009). Direct inhibition of caspases by siRNA has also been shown to improve

recombinant protein titres and Qp values from CHO cultures (Wong et al, 2006).

1.9 PROTEIN SECRETION AS A POTENTIAL DETERMINANT OF


Protein secretion is the final part of the cellular recombinant protein production process.

Factors that regulate flow through the secretory pathway include regulation of ER and

Golgi transport, glycosylation and other post-translational modifications and the

segregation of proteins depending upon their intended destination (Barnes et al, 2003).

Any disruption to these factors could ultimately limit protein secretion and lower

protein expression for the recombinant cell line.

Transport through the secretory pathway involves vesicle intermediates, which bud

from the donor compartments and fuse with the acceptor compartments. Vesicle

budding is mediated by coat protein complexes (COPI, COPII and clathrin, Peng &

Fussenegger, 2009a), while vesicle fusion is catalyzed by SNARE and Sec1/Munc18

proteins (Wickner & Schekman, 2008).

54

Secretion engineering is a potential strategy to increase the production of

biopharmaceuticals. The ER and the Golgi of transgenic CHO-K1-derived cell lines

have been expanded through the ectopic expression of human-derived XBP-1,

increasing overall production capacity of these cells (Tigges & Fussenegger, 2006). Ku

et al, also suggested that transient expression of XBP-1(s) increased recombinant CHO

cell productivity by improving the secretory capacity of the cell (Ku et al, 2008).

However, other studies have shown no improvement in recombinant protein titres with

enhanced expression of XBP-1(s) in CHO cells (Ohya et al, 2008). It was suggested that

protein titres were not increased for this cell line as a secretory bottleneck was not a

limiting factor.

Recently overexpression of Munc18b has been shown to enhance the secretory capacity

of HeLa and HEK-293, observed with improved protein titres (Peng & Fussenegger,

2009b; Peng et al, 2010). It may be possible to utilise this technology to improve

secretion, and recombinant protein production, from CHO cultures.

1.10 IMPROVING PROTEIN PRODUCTION BY FEED AND CHEMICAL

ADDITIONS

Over the last twenty years improvements in cell line process control and media

formulation have increased product titres by 100-fold (Wurm, 2004). Although

chemically-defined (CD) media are commercially available, they may not adequately

meet the specific nutrient requirements of individual cell lines. Companies meet feeding

requirements by adding concentrated solutions of commercial media or standard amino

acids plus glucose and/or glutamine to cells usually during mid-culture stage (Huang et

al, 2004). Ultimately, optimisation of a fed-batch process involves interplay between

variables including feed solution constituents, concentration, timing and duration of

feed (Lavric et al, 2005; Wong et al, 2005). Many feed regimes act to increase

recombinant protein production in CHO cultures by enhancing viable cell numbers

(Choi et al, 2007; Kuwae et al, 2005; Wong et al, 2005), whilst decreasing rates of

ammonia and lactate production (Wong et al, 2005) and glucose utilisation (Kuwae et

al, 2005). There is limited information regarding the impact of feed additions on gene

55

transcription or protein translation. However, it is likely that feeding affects many

cellular events involved in recombinant protein production and secretion.

It has been suggested that a series of low-molecular-weight compounds like dimethyl

sulfoxide (DMSO), glycerol, trimethylamine-N-oxide (TMAO), 4-phenylbutyric acid

(PBA) can alleviate defective or impaired protein folding by stabilising proteins in their

native conformation (Perlmutter, 2002; Römisch, 2004). Both Glycerol (1% [v/v], Liu

& Chen, 2007a) and DMSO (1% [v/v], Liu & Chen, 2007b) addition to recombinant

CHO cultures has been shown to significantly enhance Qp values. Although chemical

additives, such as glycerol and DMSO, can improve recombinant Qp values overall titre

values were not necessarily increased due to induced growth arrest (Li et al, 2006c;

Rodriguez et al, 2005).

Understanding regulation of CD feeds and chemical additions in improving

recombinant protein titres and Qp values may provide valuable information for optimal

cell line development.

1.11 INVESTIGATING INSTABILITY IN RECOMBINANT CHO CULTURES

As mentioned in Section 1.1 cell line stability in terms of stable protein expression

during LTC is needed from a recombinant cell line in order to gain regulatory approval.

Transcription, translation, protein folding and secretion are events critical to

recombinant protein production. Any limitations to these cellular events could

potentially affect protein titres and cell line stability.

Loss of productivity from DHFR-CHO cell lines in response to LTC has been shown to

be dependent on transgene chromosomal location (Kim & Lee, 1999) and loss of

recombinant gene copies. Kim et al, showed that Qp was approximately 80% lower

after 8 weeks of culture due to a loss of amplified gene copies (Kim et al, 1998). As

discussed in Section 1.2.3.4 the amplification of the DHFR-CHO selection system in the

presence of MTX can increase recombinant cell line production. However, DHFR-CHO

instability has been shown to occur upon removal of the MTX selection (Fann et al,

2000; Kim et al, 1998; Yoshikawa et al, 2000).

56

Instability in terms of a loss in protein production has also been shown to act beyond

gene copy number or the site of gene integration (Jun et al, 2006). Decreased MAb

production in recombinant CHO cultures after 36 passages has been associated with a

decrease in heavy and light chain mRNA expression (Chusainow et al, 2009). Current

investigations have involved systems which allow for targeted transfection of single or

multiple genes. The artificial chromosome expression (ACE) system has been

developed based on pre-engineered artificial chromosomes with multiple recombination

acceptor sites and allowed for stable MAb expression during 70 days of culture, with

good Qp values (approximately 40 pg/cell/day, Kennard et al, 2009).

Stable protein expression is achievable, witnessed with the high number of therapeutic

proteins derived from recombinant CHO cultures. Recently large-scale antibody

productions using a DHFR-CHO cell line found this cell line to be stable in terms of

recombinant protein production during the scale-up period, which accounted to 53 days

in culture. However, Qp was found to decrease during the scale-down period (days 53

to 109 of culture). The 40% decrease in Qp was not due to genomic changes but was

associated with increased cell doubling time (dt, Kennard et al, 2009).

Although protein production instability is a published issue for recombinant CHO cells

the exact molecular mechanisms leading to instability are not yet fully understood

(Chusainow et al, 2009; Derouazi et al, 2006). Concerns regarding instability within

CHO, and other industrial mammalian cell lines, are often left unpublished due to

confidentiality. Many publications make speculations for the reason behind decreased

protein expression in response to LTC but fully understanding the precise mechanisms

for CHO cell instability is vital in producing consistently stable cell lines.

1.12 SUMMARY AND PROJECT AIMS

Recombinant protein expression is a complex task, there are many cellular events that

can effect production including transcriptional and translational regulation, protein

folding and protein secretion. All these events are also regulated by the metabolic state

of the cell and ATP availability. Although from an industrial perspective high

production titres are important it is crucial that the secreted protein is functional. Protein

57

events within the ER ensure only correctly-folded proteins are secreted. Mis/unfolded

proteins alter the ER homeostasis and result in an UPR, if mis/unfolded proteins cannot

be folded to the correct conformation by ER chaperones, such as BiP or PDI, or by the

CNX/CRT cycle, the protein is targeted for degradation or the cell undergoes apoptosis.

Alterations to these cellular events involved in protein production can increase

productivity. The insertion of S/MAR elements, overexpression of PDI, Bcl-2 and Bcl-

xL, and downregulation of Bax, Bak and certain caspases all enhanced recombinant

protein production from recombinant CHO cultures. Production of CHO cultures can

also be improved with feed and chemical additions, or under hypothermic conditions.

Feed addition mainly increased antibody titres by enhancing viable cell numbers, whilst

chemical and low temperatures arrested cell growth so although Qp was increased final

protein titres were often not.

Although improving productivity is advantageous when developing cell lines, ensuring

cell lines remain stable during LTC is vital in gaining regulatory approval. CHO cell

line instability during LTC has been shown to occur with a loss of gene copies,

chromosomal alterations and decreased recombinant mRNA expression. However, the

cause of instability is not always determined.

The overall aim of my project is to gain an improved understanding of regulatory events

that determine productivity and overall stability of an industrially-relevant CHO cell

line (engineered with the GS vector system to secrete a recombinant MAb). A greater

understanding may potentially offer approaches to optimise cell line selection and

culture, and the potential to engineer cells so product titre is optimum.

Therefore the main aims of my project are to:

Determine whether recombinant protein production is maintained at constant

levels over LTC (100 generations).

Characterise the exemplar cell line (3.90) to determine if antibody production is

linked to changes in cell growth, mRNA expression, translation or secretion.

Determine if protein titres can be enhanced by feed and chemical (DMSO)

additions.

58

Investigate the effects of feed and DMSO addition on intracellular factors that

control protein production.

To determine if the effects seen during LTC, and with feed and chemical

additions, exhibit cell line-specific profiles.

The results presented in this thesis fall into four discrete chapters. Chapter 3

characterises the main cell line (cell line 3.90) in determination of cell line stability

during a period of LTC (100 generations). Chapter 4 investigates the effects of feed

addition on protein production and stability, and Chapter 5 examines the effects of

DMSO addition to cell line 3.90. The fourth chapter (Chapter 6) aims to determine if the

effects of LTC, feed and DMSO addition are cell line specific.

All Chapters are collated to present an overall discussion. Chapter 7 aims to consider

the following questions:

Is instability connected to a specific cellular event?

How is recombinant protein production increased in response to feed addition?

How is recombinant protein production increased in response to DMSO

addition?

Are there any markers to predict the likelihood of recombinant protein

production?

What further work is required to answer these questions?

59

2

CHAPTER 2

3

MATERIALS AND METHODS

60

2. MATERIALS AND EQUIPMENT

2.1 GENERAL MATERIALS

2.1.1 Sources of chemicals and reagents

All chemical reagents were of the highest grade and obtained from standard sources.

The materials used, and their suppliers, are listed in Appendix 1.

2.1.2 Preparation and sterilisation of solutions

All solutions were prepared in milliQ water (ddH2O) unless otherwise stated. All

solutions used in the processing of RNA were made in ddH2O that had initially been

treated for 12-16 hr with 0.05% (v/v) diethylpyrocarbonate (DEPC), a ribonuclease

inhibitor. Phosphate buffered saline (PBS) solution was created with PBS tablets.

Solutions were sterilised by autoclaving in a LTE Scientific Series 250 autoclave or by

filtration through a 0.2µm filter where autoclaving was not viable. Solutions were stored

at room temperature, unless otherwise stated.

2.1.3 pH measurements

Measurements of pH were made using a digital Corning pH meter 120 with a glass

electrode. The pH was adjusted using hydrochloric acid or sodium hydroxide, as

appropriate, unless otherwise stated.

2.1.4 Mammalian cell lines and culture medium

The CHO cell lines 3.90 and 51.69 had been engineered to produce a recombinant IgG

antibody using the glutamine synthetase expression system, which is proprietary to

Lonza Biologics. The recombinant CHO cells, and untransfected parental cell line, were

cultured in CD-CHO medium. For the parental cells lacking the glutamine synthetase

gene the medium was supplemented with 6mM L-glutamine. Transfected recombinant

cells were grown in the medium supplemented with 25µM L-methionine sulphoximine

(MSX). All cell culture media were pre-warmed to 37oC, prior to use.

61

2.2 GENERATION AND PURIFICATION OF PLASMIDS IN BACTERIAL

CELLS

2.2.1 Bacterial growth medium

Luria Bertani (LB) broth was used as bacterial growth medium, unless otherwise stated,

and comprised of 1% (w/v) tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) sodium

chloride and supplemented with ampicillin (50µg/ml), where appropriate. Solid medium

contained the above constituents with the addition of 1.5% (w/v) agar. Ampicillin was

added to the LB agar once it was below a temperature of 55oC.

2.2.2 Generation of competent bacterial cells

DH5α E.Coli cells (100µl) were added to 10ml LB medium and grown overnight at

37oC at 250rpm. 2ml of the overnight culture were added to 40ml LB medium and

grown for about another 2 hr, or until the absorbance at 550nm was approximately 0.3.

Bacterial cells were harvested by centrifugation at 7,000g for 4 min and cells were

resuspended in 20ml ice-cold sterile 50mM calcium chloride, and incubated on ice for

20 min. Cells were stored with 20% (v/v) glycerol at -80oC.

2.2.3 Transformation of competent DH5α E.Coli cells

10µl of the recombinant IgG plasmid DNA (1ng/µl) was added to 100µl of competent

cells (Section 2.2.2), swirled to mix, and incubated on ice for 30 min. The cells were

then heat-shocked at 42oC for 2.5 min before addition of 1ml of room temperature LB

medium. The cells were incubated at 37oC with shaking at 250rpm for 1 hr. The cells

were diluted with LB medium, spread onto agar plates and incubated at 37oC overnight.

Colonies were then observed and counted.

2.2.4 Midi-preparation of plasmid DNA

Midi-preparation was used when relatively large quantities of pure plasmid were

required for transfection of cells or for further plasmid manipulation. Plasmid was

prepared from 200ml bacterial culture using the Qiagen® Plasmid Midi Kit. The

62

protocol for low-copy number plasmids was followed from the Qiagen plasmid

purification handbook.

2.2.5 Determination of nucleic acid concentration and purity

Concentrations were estimated using the NanoDrop® UV/Vis spectrophotometer,

according to manufacturer‟s instructions. The purity was assessed by using the

A260nm/A280nm ratio, where a ratio of 1.6-2.0 was considered pure.

2.2.6 Restriction enzyme digestion

Restriction enzyme digestion was performed to verify plasmid identity. Digestions were

performed in a final volume of 15µl using 1µg DNA, 5-10 units (U) of appropriate

restriction enzyme and the appropriate restriction enzyme buffer (final concentration

1x). After brief vortexing and pulse-centrifugation at 14,000g at room temperature, the

digests were incubated at 37oC for 1-3 hr, for plasmid samples, or overnight, for

genomic DNA samples. To evaluate if the restriction digest had reached completion,

and to investigate the pattern of cleaved DNA, the samples were analysed on agarose

gels (Section 2.6.4.1).

2.3 CELL CULTURE

2.3.1 Maintenance of CHO cells

Suspension cell lines were cultured routinely in 125ml and 250ml Erlenmeyer flasks

(referred to as shake flasks) at 36.5oC and 140 rpm with gassing 5% CO2 (v/v) in air.

Cells were sub-cultured every 4 days into a final volume of 30-50ml. At each sub-

culture cell density and viability were estimated by light microscopy and trypan blue

exclusion, respectively (Section 2.3.3), and an appropriate volume of each culture was

diluted in the relevant medium to give a cell density of 0.2x106 viable cells/ml. All

solutions and equipment used for mammalian cell culture were either sterilised by the

manufacturer prior to receipt, or as stated in Section 2.1.2. All cell culture procedures

63

were performed within sterile laminar flow cabinets. Upon reaching the correct

generation time batch cultures were created (Section 2.3.2).

2.3.2 Generation of batch cultures

Batch cultures were created at time of subculture, once cells had reached the correct

generation time, generation 20, 40, 60, 80 and 100. Generation identification began

from the working cell stock (WCS, also known as generation 0), and calculated from

the dt of the cell (Section 2.11.1). Each culture doubling was equivalent to one

generation. Due to the dt of the cell, and day of subculture, the production of batch

cultures at each specific generation (generations 20, 40, 60, 80 and 100) may be created

at the specific generation, ±4 generations. For example, a generation 100 batch culture

could be created at generations 96, 97, 98 etc to 104.

CD-CHO media was used to dilute the cells to initial densities of 0.2x106 viable cells/ml

using 250ml and 500ml shake flasks. The 250ml and 500ml shake flasks contained

culture volumes of 50ml and 100ml, respectively. Where appropriate a CD feed and/or

DMSO was added. A 2% (v/v) CD feed addition, together with 0.25% (v/v) sodium

bicarbonate, was added on days 3, 4, 5, 6 and 7 of batch culture, whilst a 2% (v/v)

DMSO addition was added on day 5 of batch culture, to the relevant cultures. Cell

number and viability counts were made every 48 hrs from day 0 of batch culture

(Section 2.3.3), with samples taken to allow determination of recombinant protein

production. Samples were centrifuged at 10,000g for 30 sec at room temperature. The

supernatant was then stored at -80°C until required for Enzyme-Linked Immunosorbent

Assay (ELISA, Section 2.5.1), N-linked glycan analysis (Section 2.5.4) or metabolite

analyses (Section 2.10). Exhaustion batch cultures were continued until cell viability

reached ≤ 30%.

2.3.3 Determination of cell number, viability and diameter

The cell densities and cell viability of cultures were determined by light microscopy and

trypan blue dye exclusion, respectively. Cell samples were diluted with 0.5% (w/v)

trypan blue dye in PBS (with a further dilution in PBS where appropriate) and counted

using an Improved Neubauer haemocytometer. Viable cells excluded the blue dye as

64

they had an intact membrane. Dead cells, however, appeared blue due to the uptake of

the trypan blue dye through damaged plasma membranes. The average of four

haemocytometer fields of view (0.1 mm3 each) was used to calculate both total and

viable cell number. Cells on the haemocytometer were also measured for cell diameter

using a Widefield Axiovision microscope and Axiovision software.

2.3.4 Cryopreservation of cells

Exponentially-growing CHO cells were centrifuged at 100g for 5 min at room

temperature. The supernatant was removed and the cell pellet was gently resuspended in

a mixture 92.5% (v/v) CD-CHO media and 7.5% (v/v) DMSO to give a concentration

of 1.5x107 cells/ml. 1ml cell suspension was distributed into 1.8ml cryovials, and then

placed at -80oC overnight in a polystyrene box. Frozen cells where then transferred to

liquid nitrogen for long-term storage.

2.3.5 Revival of cells from liquid nitrogen

After removal from liquid nitrogen, cells (1ml aliquot) were thawed and transferred into

30ml of medium, pre-warmed to 37oC, and centrifuged at 100g for 5 min. The

supernatant was discarded and cell pellet was resuspended gently with 10ml of pre-

warmed medium. A sample was taken for cell counting (Section 2.3.3) and diluted in

the appropriate medium to a density of 0.3x106 viable cells/ml, until first subculture

after 3 days. Subsequent subcultures were every 4 days (Section 2.3.1).

2.3.6 Medium osmolality determination

The osmolality of cell cultures was measured using an automatic micro-osmometer,

which measures the freezing point of aqueous solutions. Cell culture samples were

centrifuged at 10,000g for 30 sec at room temperature. The freezing point was

determined from 100µl of supernatant. Water (zero mOsmole) and a 300 mOsmole

standard solution (Minimum Essential Medium, containing Earle‟s salts and L-

glutamine) were used to calibrate for the measurement of samples.

65

2.3.7 Mycoplasma detection

Cell cultures were routinely tested for the presence of mycoplasma during LTC.

Following the instructions of the MycoAlert® Detection Kit, the presence of

mycoplasma could be determined by lysing the viable mycoplasma and allowing the

reaction of mycoplasmal enzymes with the MycoAlert® Substrate, which results in

elevated ATP levels. The emitted light intensity (linearly related to the ATP

concentration) was measured using a luminometer.

2.4 FLOW CYTOMETRY

2.4.1 Cell cycle phase analysis

1x106 cells, taken during batch culture (Section 2.3.2) were resuspended in 200µl cold

PBS, 2ml ice-cold 70% (v/v) ethanol, the solution was vortexed and left on ice for 30

min. The cells were fixed in this state at 4oC for a maximum of one week. When needed

the cells were centrifuged at 100g for 5 min, the ethanol was removed and the cells were

resuspended in 400µl PBS. Together with 50µl RNase A (1mg/ml) and 50µl Propidium

Iodide (PI, 400µg/mL) the cells were incubated for 30 min at 37oC. The cells were

analysed by a CyAn ADP flow cytometer, using the 488nm excitation laser, according

to manufacturer‟s instructions. The emission was measured by a 613/20 nm bandpass

filter, the voltage applied to the photomultiplier (PMT) tube was adjusted to ensure the

histogram plots obtained were within range. The data was gated to select single cells

using a plot of the height of the PI signal against the area of the PI signal. The data was

analysed by Summit 4.3 and ModFit LT software.

2.4.2 Quantification of intracellular antibody

1x107 cells, removed on days 4 and 9 of batch culture (Section 2.3.2), were centrifuged

at 100g for 5 min. The cell pellet was washed twice with PBS at room temperature prior

to cell fixation by resuspension in 5ml ice-cold 70% (v/v) methanol. The samples were

then stored at -20oC until required.

66

2 x 106 cells (1ml of fixed cells) were removed from the previously fixed cells and

washed with pre-chilled 3ml PBS containing 1% (w/v) bovine serum albumin (BSA).

The cells were centrifuged at 100g for 5 min, the supernatant was removed, and the

cells were further washed with 5ml PBS, 1% (w/v) BSA, and again centrifuged at 100g

for 5 min. After removal of the supernatant the pellet was re-suspended 2ml PBS, 1%

(w/v) BSA supplemented with 10µg goat anti-human IgG, Fcγ-APC and 6µg goat anti-

human lambda light chain-FITC. The cells were incubated in the dark for 30 min at 4oC.

After incubation the stained cells were washed twice with pre-chilled 3ml PBS, 1%

(w/v) BSA. Unstained and stained parental cells were also required for use with the

CyAn-ADP flow cytometer.

The samples were then analysed by a CyAn ADP flow cytometer, using the 488nm and

infra-red excitation lasers to excite the FITC and APC conjugates. The unstained cells

and the stained parental cells were used to set the initial parameters. The voltage applied

to the PMT tube was adjusted to ensure the histogram plots obtained were within range.

The data was gated to select single cells and analysed by Summit 4.3 software.

2.5 PROTEIN ANALYSIS

2.5.1 Detection of antibody by ELISA

Nunc 96 well immunoassay plates were coated with monoclonal goat anti-human IgG

antibody at a final concentration of 3.25µg/ml, diluted in sterile PBS (100µl per well),

and incubated at 4oC overnight. The following day the coating solution was discarded

and plates were washed three times by filling each well with 250µl wash buffer (0.1%

[v/v] Tween-20 in sterile PBS). After the final wash the plates were blotted dry. Plates

were then blocked by addition of 150µl blocking solution per well (2% [w/v] milk in

sterile PBS) and incubated at room temperature for 1 hr. Supernatant samples (taken

routinely during batch culture, Section 2.3.2) were diluted in blocking solution at

1:1600 or 1:6400 (dependent on day of batch culture). 100µl diluted samples or

standards (highest concentration of standard 0.625µg/ml) were added (in duplicate) to

the well and combined with 100µl of blocking solution. The samples were serially

diluted directly on the plate using blocking solution. The plates, containing 100µl of

67

diluted sample per well, were incubated at room temperature for 1 hr.

Samples/standards were then discarded and the plate was washed three times with wash

buffer and blotted dry. 100µl of detection antibody (sheep anti-human lambda

peroxidise conjugate, 0.4µg/ml) was added to each well and incubated at room

temperature for a further 1 hr. Development solution was prepared by dissolving two

TMB (3,3‟,5,5‟ tetramethyl benzidine chromogen) tablets and 5µl 30% hydrogen

peroxide in 12ml TMB substrate solution (10mM sodium acetate and 10mM sodium

citrate, pH 5.5). After the plate was washed and dried, 100µl development solution was

added per well and incubated for 6 min at room temperature in darkness. The reaction

was stopped by addition of 100µl 0.2M sulphuric acid to each well. Absorbance in wells

was read at 450 nm.

2.5.2 Determination of total protein synthesis

The rate of protein synthesis was measured as the rate of L-[4,5-3H] leucine

incorporation into trichloroacetic acid (TCA) precipitable-material. Incubations were

carried out in 24 well plates containing 500µl cell suspensions (cell suspensions were

taken from cultures on day 7 of batch culture [Section 2.3.2]). 10µl of L-[4,5-3H]

leucine (final specific radioactivity of 14µCI/µmole [1:100 dilution]) was added to the

cell suspensions and incubated over a 48 hr time period. At 24 hr time intervals samples

were mixed with equal volumes of ice-cold 10% (w/v) TCA. After 30 min at 4oC the

sample was centrifuged for 2 min at 12,000g. The pelleted precipitate was washed three

times by resuspension in 500µl of 5% (w/v) TCA, followed by recentrifugation at

12,000g for 2 min. The pelleted precipitate was solubilised using 50µl of NCS tissue

solubiliser for 1 hr at room temperature, before addition to 1ml of Ecoscint scintillation

fluid. The remaining supernatant was washed with resuspension in 500µl of 5% (w/v)

TCA, with 10mg/ml BSA, followed by centrifugation at 12,000g for 2 min. The

supernatant precipitate was then solubilised using 50µl of NCS tissue solubiliser for 1 hr

at room temperature, before addition to 1ml of Ecoscint scintillation fluid. The

radioactivity was measured using a scintillation analyser.

68

2.5.3 Western blot analysis

2.5.3.1 Protein extraction

Cellular protein was extracted routinely during batch culture (Section 2.3.2). 1x107 cells

were centrifuged at 100g for 5 min, the pellet was washed with 10ml PBS and

recentrifuged at 100g for 5 min. The pellet was resuspended in 500µl Radio

Immunoprecipitation (RIPA) buffer (1% [w/v] triton X-100, 0.2% [w/v] SDS, 125mM

sodium chloride, 10mM trisodium phosphate, 0.5% [w/v] sodium deoxycholate, 10mM

sodium orthovanadate, 25mM HEPES and 10mM sodium fluoride) supplemented on

day of use with the protease inhibitors (0.1% [w/v] leupeptin, 0.1% [w/v] aprotinin and

1% [w/v] phenyl-methyl sulfonyl fluoride). To ensure the pellet was fully resuspended

the extract was syringed through a needle several times, after which the extract was left

on ice for 30 min. Protein concentration was determined by the Bradford Protein Assay

(Bio-Rad) using BSA as a standard. 60g of the protein extract was resolved by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Section 2.5.3.2).

2.5.3.2 SDS-PAGE

The Bio-Rad mini gel II slab system was used for SDS-PAGE. The system consisted of

a 12% (w/v) separating gel overlaid by a 4% (w/v) stacking gel. The separating gel was

prepared by mixing 3.6ml Protogel solution (30% [w/v] acrylamide), 3.75ml separating

buffer (1.5M Tris, 14mM SDS, pH 8.8) and 5.05ml ddH2O. Stacking gel was prepared

by mixing 1.6ml Protogel solution, 2.5ml stacking buffer (0.5M Tris, 14mM SDS, pH

6.8) and 6ml ddH2O. Polymerisation was initiated by the addition of ammonium

persulphate and TEMED to a final concentration of 0.2µg/ml and 0.2% (v/v),

respectively.

Extracted protein samples (Section 2.5.3.1) were mixed 1:1 with sample buffer (20%

[v/v] glycerol, 7mM SDS, 6.2M Tris, 0.025% (w/v) bromophenol blue). Immediately

before use 2-mercaptoethanol was added to the sample buffer to a final concentration of

1.75% (v/v). The samples were heated at 100oC for 5-10 min and cooled before use.

Electrophoresis was performed in electrode buffer (10mM Tris, 80mM glycine, 1.4mM



69

SDS) and the samples, together with a protein marker, were electrophoresed at 75V

until the bromophenol blue front had moved into the separating gel and then at 200V

until the dye had reached the bottom of the gel.

2.5.3.3 Protein transfer

Separated proteins from SDS-PAGE were transferred onto a nitrocellulose membrane,

pre-soaked along with thick filter paper in the blotting buffer (25mM Tris, 190mM

glycine, 20% [v/v] methanol, pH 7.4) for 2-5 min prior to use. The transfer was set up

on a Bio-Rad Semi-Dry electroblotter at 15V for 60 min. To assess the efficiency of the

transfer, the membrane was stained with Ponceau-S (0.5% [w/v] in 1% [v/v] glacial

acetic acid). In order to minimise non-specific binding of antibodies the membrane was

incubated in blocking buffer (3% [w/v] milk in PBS) for 1 hr shaking at room

temperature, or overnight shaking at 4oC. The primary antibodies, detailed in Table 2.1,

were diluted in blocking buffer and incubated with the membrane for 1 hr shaking at

room temperature, or overnight shaking at 4oC. The secondary antibody-HRP conjugate

was also diluted in the blocking buffer (as stated in Table 2.1). The secondary antibody-

HRP conjugates were added to the membrane for 30-60 min, whilst shaking at room

temperature. In-between each incubation the membrane was washed with 1% (v/v)

Tween-20 in PBS, three times for 5-10 min. Protein bands were detected using an

enhanced chemiluminescence (ECL) system according to manufacturer‟s instructions.

The membrane was exposed to Kodak film. Band density was assessed by densitometric

analysis (Section 2.5.3.5)

2.5.3.4 Stripping nitrocellulose membranes

Membranes were stripped using stripping buffer (0.1M glycine, pH 2.5) for 1 hr whilst

shaking at room temperature. The membranes were then ready to be blocked, and

incubated with ERK antibody (dilutions stated in Table 2.1), before detection with ECL

and exposure.

ERK antibody was used as a standard for protein loading. All antibodies were then

compared to the densitometric analysis of ERK (Section 2.5.3.5).

70

2.5.3.5 Densitometric analysis

The densities of bands were analysed using Image J software, to determine the intensity

of the protein of interest relative to ERK.

Table 2.1: Details of antibodies used for western blot analysis

Primary

antibody Dilution

Secondary

antibody-HRP

conjugate

Dilution

pan ERK 1:5000 Mouse 1:2000

polyclonal ATF4

(CREB-2) 1:1000 Rabbit 1:1000

polyclonal

GADD153 1:1000 Rabbit 1:1000

polyclonal BiP

(GRP78) 1:1000 Goat 1:2000

polyclonal PDI 1:2000 Rabbit 1:1000

2.5.4 N-linked glycan analyses

2.5.4.1 Antibody purification

Antibody from the supernatant samples (taken from day 15 of batch culture, Section

2.3.2) was purified using Ab SpinTrap Protein A columns and concentrated using

MicroCon ultrafiltration units, both according to manufacturers‟ instructions.

2.5.4.2 Deglycosylation of purified recombinant antibody

Reaction mixes (50µl) containing 50-100µg antibody and a PNGase incubation buffer

(100mM sodium phosphate, pH 7.2, 25mM EDTA and 3U PNGase F) were incubated

71

overnight at 37oC. This enzyme is a native glycoaminidase cleaving the link between

asparagine and N-acetylglucosamines, allowing the study of N-linked carbohydrates.

2.5.4.3 Precipitation and lyophilisation of glycans

Proteins were removed by ethanol precipitation. 450µl 100 % ethanol was added to the

purified antibody prior to incubation at -20oC for 60 min. The samples were then

centrifuged at 15,000g for 10 min, the supernatant containing the glycans were

transferred to a fresh tube and lyophilised. .

2.5.4.4 Desalting using Graphite

Glycan samples were resuspended in 50µl of 0.05% (v/v) trifluoric acid (TFA). The

samples were desalted using the graphite tip „TopTip‟ columns according to the

manufacturer‟s instructions. Glycans were eluted with 20µl 60% (v/v) acetonitrile

(ACN), 0.05% (v/v) TFA.

2.5.4.5 MALDI-ToF analysis

The desalted glycan samples were mixed 1:1 with DHB (2,5-dihydroxybenzoic acid)

matrix, prepared in 60% (v/v) ACN, 0.05% (v/v) TFA. The sample was spotted onto the

MALDI target plate and analysed using a MALDI-ToF mass spectrometer (MS).

External calibration was used and the laser intensity was kept below 40% to prevent

fragmentation. The MALDI-ToF-MS spectrum was acquired in reflectron mode for

glycan profiling, and in MS/MS, CID LIFT™ mode, for glycan structural

determination.

72

2.6 DETERMINATION OF COPY NUMBER

2.6.1 Southern blot analysis

2.6.1.1 DNA extraction, phenol extraction and ethanol precipitation

An adapted version of the protocol detailed by Blin & Stafford (1976) was used to

extract genomic DNA from CHO cells. Approximately 2x107 viable cells (from time of

subculture [Section 2.3.2]) were harvested by centrifugation at 100g for 5 min at room

temperature. The pellet obtained was washed three times in PBS, with centrifugation as

above between each wash step. The final pellet was then resuspended in 200µl of PBS

before addition of 3ml of EDTA-sarcosine solution (0.1M EDTA, pH 8.0, containing

0.5% (w/v) N-lauroyl-sarcosine) which was added drop wise to the pellet. 60µl

Proteinase K (10mg/ml) and 10µl RNase A (10mg/ml) were then added. The mixture

was incubated at 55oC for 2 hr with inversion every 20 min.

After the 2 hr inversion an equal volume of phenol:chloroform:isoamyl alcohol

(25:24:1) was added to the DNA and mixed by rocking for 10 min at room temperature.

The solution was then centrifuged at 13,000g for 10 min and the upper aqueous layer

was removed into a fresh tube. The DNA was extracted via this method three times. 3M

sodium acetate (pH 5.5) was added to the upper aqueous layer to a final concentration of

0.3M 2.5 volumes of 100% ice cold ethanol were then added before mixing. The

mixture was centrifuged at 13,000g for 5 min at room temperature and the supernatant

was discarded. The pellet was washed with 1ml 70% (v/v) ethanol, and the final pellet

was air dried for approximately 5 min. The pellet was resuspended in a suitable volume

of ddH2O water (50µl–250µl) and DNA concentration and purity was measured by

NanoDrop® UV-Vis Spectrophotometer (Section 2.2.5).

2.6.1.2 Determination of genomic DNA per cell

1x107 viable cells (from time of subculture [Section 2.3.2]) were centrifuged at 100g for

10 min at room temperature. The pellet was resuspended in PBS and 0.5ml 0.5M

perchloric acid was added. The mixture was incubated on ice for 5 min and then

centrifuged at 200g for 10 min at room temperature. The pellet was then resuspended in

73

1ml 0.5M perchloric acid. A range of standards were created using calf thymus DNA

(diluted in 5mM sodium hydroxide). Standards contained between 0-100µg calf thymus

DNA diluted in 0.5M perchloric acid. Samples and standards were incubated at 80oC for

35 min and allowed to cool. 2ml of freshly made assay reagent (98% [v/v] glacial acetic

acid, 1.5% [v/v] concentrated sulphuric acid, 1.5% [w/v] diphenylamine, 0.5% [v/v]

acetaldehyde) was added to all samples and standards, which were then incubated for

12-16 hr at room temperature. Absorbance measurements were performed at 595nm

and, using the standard curve constructed from the calf thymus DNA samples and cell

numbers, DNA per cell was calculated.

2.6.1.3 Preparation of plasmid standards and genomic DNA for Southern

analysis

Genomic DNA was digested (Section 2.2.6) with HindIII and EcoRI, for copy number

analysis of light chain and heavy chain genes, respectively. Standards for copy number

analysis were generated by digestion of a known quantity of the recombinant plasmid,

and diluted using ddH2O to give a range of concentrations between 0-0.4ng of DNA.

Genomic DNA and plasmid standards were then separated on an agarose gel (Section

2.6.1.4).

2.6.1.4 Agarose gel electrophoresis of DNA samples

Agarose gel electrophoresis was used to separate DNA species on the basis of size.

Agarose gels were prepared by dissolving 1% (w/v) agarose in TBE buffer (0.09M Tris,

0.09M orthoboric acid and 0.2mM EDTA, pH8.0), by boiling in a microwave. Once the

gel had cooled to ≤ 55oC ethidium bromide was added to a final concentration of

0.1µg/ml. Gels were set and run in horizontal electrophoresis tanks with TBE as the

running buffer. DNA samples were mixed in a 5:1 ratio with the gel loading buffer

(1mM EDTA, pH 8.0, containing 50% [v/v] glycerol and 0.25% [w/v] bromophenol

blue) and loaded into wells. A voltage of 80V–100V was used to separate the DNA

based on size. Separated DNA species were then visualised by examination of the gel

on a UV transilluminator. Comparison of bands to a 10kb DNA ladder allowed

estimation of molecular size.

74

2.6.1.5 Capillary blot transfer of DNA to nylon membrane

After DNA separation, on the basis of size, by agarose gel electrophoresis (Section

2.6.1.4), capillary blot transfer was used to transfer DNA from agarose gels to nylon

membranes. The agarose gel was soaked three times in denaturing solution (1.5M

sodium chloride, 0.5M sodium hydroxide) for 15 min, ensuring the gel was covered

completely in all washes. The gel was then washed in ddH2O, and soaked a further three

times in neutralising solution (1M Tris, pH 7.4, containing 1.5M sodium chloride) for

10 min each time, again ensuring the gel was covered. The gel was again washed in

ddH2O, and then soaked in 10x SSC (1.5M sodium chloride, pH 7.0, containing 0.15M

sodium citrate) for 10 min. The nylon membrane was pre-soaked in ddH20 for 2 min

followed by soaking in 10x SSC for 2 min. The efficiency of the transfer to the nylon

membrane was checked by examination of both the membrane and the gel for DNA

species using a UV transilluminator. The membrane was air dried for 20 min, and the

DNA was linked using a UV cross-linker.

2.6.1.6 Isolation of DNA probes for Southern analysis

The probes for copy number for the light chain and heavy chain region were generated

by a restriction digest of the recombinant plasmid. Figure 2.1 shows a map of the

plasmid and indicates the position of all restriction digest sites that were used during

isolation of probes for Southern analysis. The probes for copy number analysis were

obtained by digesting with the recombinant plasmid with HindIII and EcoRI. The

fragment sizes were 900bp and 2150bp of light chain gene and heavy chain gene,

respectively. After digestion (Section 2.2.6) the products were separated on an agarose

gel (Section 2.6.1.4). Relevant bands were purified from the gel using a Qiagen®

Gel

Extraction Kit according to manufacturer‟s instruction.

The 18S probe was used a control, it was generated from plasmid p100-D9. This

plasmid contains a 220bp mouse 18S rRNA cDNA inserted into the PstI site of

pBR322. The entire plasmid was used for radioactive labelling (Section 2.6.1.7).

75

Figure 2.1 Restriction sites for DNA probes for Southern blot analysis

2.6.1.7 Radioactive labelling of probes

The probes were radioactively labelled using 32

P-αdATP, and a Random Primed Kit

according to the manufacturer‟s instructions (the reaction performed at 37oC was

stopped by the addition of 2µl of 0.2M EDTA [pH 8.0]). Unincorporated nucleotides

were then separated from DNA using a Sephadex G50 column. The mixture was added

to the column, with 100µl of TEN buffer (10mM Tris, pH 8.0, containing 0.1M sodium

chloride and 1mM EDTA) and the column was centrifuged at 800g for 30 sec at room

temperature until the column was packed to a volume of approximately 1ml. The

radioactive reaction mix was then added to the column and centrifuged as above.

The 18S probe was labelled using a Nick Translation Kit according to manufacturer‟s

instructions (the reaction performed at 15oC was stopped by the addition of 2µl of 0.2M

EDTA [pH 8.0]). The unincorporated nucleotides were separated from DNA as above.

HindIII

EcoRI

HindIII

EcoRI

76

2.6.1.8 Pre-hybridisation

The Southern analysis method used was adapted from that of Church and Gilbert

(1984). Nylon membranes, prepared as in Section 2.6.1.5, were incubated in a

hybridisation oven for 2-6 hr at 65oC in 15ml hybridisation solution (0.5M disodium

hydrogen phosphate, pH 7.2, containing 1mM EDTA, 7% [w/v] SDS and 1% [w/v]

BSA). The pH of all solutions used during pre-hybridisation, hybridisation and washing

were adjusted using 85% (v/v) orthophosphoric acid.

2.6.1.9 Hybridisation and washing

The solution used to pre-hybridise membranes was removed. The probes, generated as

detailed in Section 2.6.1.7, were added to 8ml fresh hybridisation solution and the

incubation with the pre-hybridised membrane was continued at 65oC for a further 12-16

hr.

After hybridisation, membranes were washed at 65oC for 15 min with 15ml initial wash

buffer (40mM disodium hydrogen phosphate, pH 7.2, containing 1mM EDTA, 5%

[w/v] SDS and 0.5% [w/v] BSA). A further two or three washes were performed at

65oC for 15 min each time using 15ml wash buffer (40mM disodium hydrogen

phosphate, pH 7.2, containing 1mM EDTA and 1% [w/v] SDS). The number of washes

was dependent on the level of background radioactivity present, detectable using a

hand-held giga monitor.

2.6.1.10 Autoradiography

The membrane was exposed to X-ray film in a cassette with intensifying screens.

Exposure was performed at -80oC for 3-7 days. The densities of bands on X-ray film

were determined by densitometric analysis on a Model GS-700 Imaging Densitometer

using Molecular Analyst® software.

77

2.6.1.11 Membrane stripping

Membranes were stripped by washing in boiling 0.05% (w/v) SDS with agitation.

Washing was continued until the solution had cooled to room temperature. The success

of the stripping was determined by exposing the membrane to a phosphoimager plate

for 12-16 hr.

2.6.2 Quantitative PCR (q-PCR)

The genomic DNA was extracted from cells as described in Section 2.6.1.1.

2.6.2.1 Preparation of standard curve

The recombinant plasmid vector was diluted to a final concentration of 1,000,000–1,347

copies per 5µl reaction, in a background of genomic DNA (10ng/µl) extracted from

parental cells. Background DNA was used to ensure the efficiency of the PCR reaction

was the same for all samples. Stock dilutions of the standards were made and stored in

aliquots at -80°C until required.

2.6.2.2 Preparation of samples

One sample was defined as a „check‟ sample that was run on all detection plates and

used to normalise the total DNA content in each well. The check sample was diluted to

final concentrations of 20, 10 and 5ng/µl, using ddH2O, and aliquots of these dilutions

were made and stored at -80°C, until required.

2.6.2.3 Real-time q-PCR reaction

The following reagents were added to each well of an MJ-white 96 well plate: 5µl of

genomic DNA (Section 2.6.1.1), 2.5µl of 10µM forward primer, 2.5µl of 10µM reverse

primer and 10µl of 2x SYBR®

Green I q-PCR MasterMix. Plates were sealed with clear

plastic caps and centrifuged at 900g. Primers used are detailed in Table 2.2. Samples

were analysed in triplicate. In addition, triplicate wells containing only 5µl ddH2O and

10ng/µl parental genomic DNA were prepared as negative controls for the reaction and

78

to check for any baseline expression from parental genomic DNA. Triplicate standards

were analysed concurrently. The PCR reaction was performed using an Chromo 4

thermal cycler with the following settings: 95°C for 10 min, followed by 35 cycles of

denaturation at 95°C for 10 sec, annealing at 57°C for 10 sec, elongation at 72°C for 20

sec and denaturation of any potential primer dimers at 76°C for 1 sec. A final elongation

step at 72°C for 10 min was performed, followed by a melting curve to check the

quality of the amplified product. Data was quantified using Opticon Monitor analysis

software.

Copy number data were normalised using a β-Actin real-time PCR reaction that was

performed as described. The β-Actin primers are detailed in Table 2.2. Due to

confidentiality concerns the recombinant PCR primers are not shown.

Table 2.2 Primers used in real-time q-PCR

Target gene Forward Reverse

β-Actin

(Genomic) 5‟-ACTGCTCTGGCTCCTAGCAC-3‟ 5‟-CATCGTACTCCTGCTTGCTG-3‟

All primers were designed using the primer 3 express software

(http://frodo.wi.mit.edu/).

2.6.2.4 Analysis of q-PCR results

Data was quantified using Opticon Monitor analysis software according to

manufacturer‟s protocol. Briefly, blanks and baseline fluorescence (as calculated as an

average over cycle range 1-10) were subtracted from fluorescent plots. The threshold

was raised manually to 0.05, and the cycle at which samples reached this fluorescence

were obtained (Ct value). A standard curve was obtained by plotting log [genomic

DNA] versus Ct values for the standards. Relative concentrations of genomic DNA,

compared to the standard sample, were extrapolated from the standard curve using the

Ct values. Total DNA content was normalised using a β-Actin q-PCR reaction using the

primers detailed in Table 2.2. The melting curve was checked to assess the quality of

the PCR product, where a pure product was indicated by a single peak at between 80°C

to 90°C.

http://frodo.wi.mit.edu/

79

2.7 DETERMINATION OF mRNA

2.7.1 Quantitative reverse transcriptase PCR (q-RTPCR)

2.7.1.1 RNA isolation

RNA was isolated from cells routinely taken during batch culture (Section 2.3.2). 5-10x

106 cells were pelleted by centrifugation at 400g for 4 min. Supernatant was removed

and RNA was isolated from the cell pellets using TRIzol® Reagent. Samples were

stored at -80oC until purification. Thawed samples were incubated at room temperature

for 5 min, 200µl of chloroform was added and the tubes were shaken vigorously for 15

sec. After incubation at room temperature for 5 min, samples were centrifuged at

13,000g for 15 min at 4oC. The upper aqueous phase was collected and the RNA was

precipitated by the addition of 500µl isopropanol at room temperature for 10 min.

Samples were centrifuged at 13,000g for 10 min at 4oC, supernatant was discarded and

the RNA pellets were washed with 1ml of 75 % (v/v) ethanol by vortexing. After

centrifugation at 5,000g for 5 min at 4oC, the pellets were air-dried for 5-10 min and

dissolved in 30-50µl of DEPC-treated ddH2O (Section 2.1.2). RNA was aliquoted and

stored at -80oC until needed.

2.7.1.2 DNase treatment of RNA

RNA samples (Section 2.7.1.1) were quantified using NanoDrop®

UV-Vis

Spectrophotometer (Section 2.2.5), and further diluted to 1μg/μl in DEPC-treated ddH2O

(Section 2.1.2). To remove any trace contamination of genomic DNA, RNA samples

were treated with DNase I, using a DNase I kit. DNase I treatment was performed by

mixing 1μl of RNA (1μg/μl), 7μl of 0.05% (v/v) DEPC-treated ddH2O, 1μl DNase 10x

reaction buffer and 1μl of DNase enzyme. The reaction was incubated at room

temperature for 20 min and then stopped by the addition of 1μl of 0.2mM EDTA.

2.7.1.3 cDNA synthesis from RNA

Reverse transcriptase (RT) production of cDNA was completed using a Bioline cDNA

Synthesis Kit. To the 11μl DNase I treated RNA (Section 2.7.1.2), the following

80

reagents were added: 4μl 5x cDNA synthesis buffer, 4μl dNTP mix, 1μl oligo dT and

1μl RT enzyme. The reaction was mixed and incubated at 42°C for 60 min. The reaction

was stopped by heating to 72°C for 10 min.

2.7.1.4 Preparation of samples and ‘check’ sample

One sample was again dedicated as a „check‟ sample that was run on all detection

plates. The cDNA reaction from the standard sample was diluted 1:5 in ddH2O, to give

the 100% standard. Serial dilutions of the 100% standard were then performed in

ddH2O to give 10%, and 1% final concentrations. All other samples were diluted once

at a ratio of 1:6 with ddH2O.

2.7.1.5 Quantitation of mRNA

The reaction, including concentrations and volumes of SYBR®

Green I MasterMix and

primers, is described in Section 2.6.2.3. The only alteration to the reaction mix is that

diluted cDNA (5µl per well, Section 2.7.1.3) was used instead of genomic DNA. The

primers were designed using the Primer 3 website (shown in Table 2.3). The PCR

reaction was performed using a Chromo 4 thermal cycler with the identical settings as

described in Section 2.8.3. Total mRNA content was normalised using a β-Actin real-

time q-RTPCR reaction using the primers detailed in Table 2.3. Again due to

confidentiality concerns the recombinant PCR primers are not shown.

Data was quantified using Opticon Monitor analysis software according to

manufacturer‟s protocol (Section 2.6.2.4).

81

Table 2.3 Primers used in real-time q-RTPCR

Target gene Forward Reverse

β-Actin

(mRNA) 5‟-TGTGACGTTGACATCCGTAAA-3‟ 5‟-CTCCCCTGTGTACAGCTTCAG-3‟

ATF4 5‟-CAGGTTGCCCCCTTTACATT-3‟ 5‟-CAGGCTTCCTGTCTCCTTCA-3‟

GADD34 5‟-CCTGGTCTGCAAAGTGCTGAT-3‟ 5‟CCAGCTCAGTCACTCCCTCTTC-3‟

GADD153 5‟-CACCACACCTGAAAGCAGAA-3‟ 5‟-ACCTCCTGCAGATCCTCATA-3



2.7.2 Polymerase Chain Reaction (PCR)

Reverse transcriptase (RT) production of cDNA was completed as described in Section

2.7.1.3. The PCR reaction was set up using the following reagents in a total volume of

49µl (made up with ddH20): 25ng of forward and reverse primers (Table 2.4), 0.2mM

each of dATP, dCTP, dTTP and dGTP, 2µl cDNA and 1x Taq polymerase buffer. The

PCR reaction was heated to 94ºC for 5 min prior to the addition of 5U of Taq DNA

polymerase, which was then followed by 35 reaction cycles consisting of denaturation

at 94ºC for 1 min, annealing at the appropriate temperature (Table 2.4) for 1 min and

elongation at 72ºC for 2 min. The overall reaction sequence was finished at 72ºC for 10

min. Products were checked by agarose gel electrophoresis (Section 2.6.1.4)

Table 2.4 Details of PCR primers

Primer

Name Forward Reverse

Annealing

Temp (oC)

XBP-1(s) 5‟CCTTGTGGTTGAG

AACCAGG-3‟

5‟AGAGGCTTGGTG

TATACATGGTC-3‟ 54

GAPDH 5‟GAGGACCAGGTT

GTCTCCTG-3‟

5‟CCCTGTTGCTGTA

GCCCGTAT-3‟ 57





82

2.8 POLYSOME PROFILING

Extracts were prepared (Section 2.8.2), layered onto 15-50% (w/v) sucrose gradient

(Section 2.8.1) and the gradients were analysed by spectophotometry (Section 2.8.3).

All solutions were prepared using DEPC-treated H2O (Section 2.1.2) to minimise

degradation of the polysomes. All disposables used were chilled to 4oC overnight prior

to preparation of polysome extracts to prevent polysome dissociation from mRNA.

2.8.1 Sucrose gradient preparation

Sucrose solutions of 50%, 42%, 33%, 24% and 15% (w/v) were prepared using DEPC-

treated water and polysome buffer (10mM Tris acetate [pH 7.4], 70mM ammonium

acetate, 4mM magnesium acetate). 2.25ml 50% (w/v) sucrose solutions was added to a

9/16 x 3½ inch polyallomer tube and frozen in liquid nitrogen. 2.25ml 42% (w/v)

sucrose solution was layer on top and then frozen, this process was repeated for the

remaining sucrose solutions. The gradients were stored at -80oC and thawed overnight

at -4oC before use.

2.8.2 Extract preparation for polysome analysis

Cells were grown until day 4 and 7 of batch culture (Section 2.3.2). 5x107

cells were

transferred into chilled tubes containing 500µl cycloheximide (10mg/ml). The tubes

were centrifuged at 8,000g for 5 min at 4oC and the cell pellet was resuspended in 25 ml

of chilled lysis buffer (20mM HEPES, pH 7.4, 2mM magnesium acetate, 100mM

potassium acetate, 100µg/ml cycloheximide, 0.1mM DTT). Cells were pelleted for

5 min at 8,000g at 4oC, and the pellet was resuspended in 800µl of lysis buffer, and

transferred to a chilled 1.5ml eppendorf tubes. Cells were repelleted and resuspended in

an equal volume of lysis buffer and glass acid washed beads. Each tube was vortexed

six times for 20 sec, at 40 sec intervals on ice. Lysates were cleared briefly by

centrifugation at 13,000g for 1 min, and then removed and re-centrifuged at 10,000g for

10 min. Both centrifugations were at 4oC. After the final centrifugation the A260nm was

measured and lysates were frozen in liquid nitrogen and stored at -80oC until required.

83

2.8.3 Sedimentation of extracts

2.5 A260 units of extracts (Section 2.8.2) were layered onto the sucrose gradients

(Section 2.8.1). The gradients were centrifuged in a SW41 rotor for 2.5 hr at

40,000 rpm, after which the gradients

were collected. The A254 was measured

continuously using an ISCO UA-6 UV/Vis detector to generate the traces. Monosome

and polysome peaks were quantified using Image J software. Monosome and polysome

peaks were quantified using Image J software using a straight baseline drawn manually

below to connect the monosome and polysome peak areas.

2.9 MICROSOPY ANALYSES

2.9.1 Preparation of metaphase spreads

CHO cells were grown in batch culture (Section 2.3.2) until mid-exponential phase of

their growth cycle before treatment with 130ng/ml (w/v) of colcemid for 20 hr. Cells

were centrifuged at 100g, supernatant was removed and cells were resuspended in

approximately 100μl of medium. To the resuspended cells, 10ml of hypotonic solution

(0.04M potassium chloride, 0.025M trisodium citrate) was added drop wise with gentle

mixing. Cells were centrifuged at 220g for 5 min, supernatant was removed and cells

were resuspended in 100μl of hypotonic solution. Added to the resuspended cells was

5ml of ice-cold methanol:acetic acid (3:1). The solution was centrifuged at 220g for 5

min and supernatant was removed. The process of ice-cold methanol:acetic acid

addition and centrifugation was repeated three times in total. After the final

centrifugation cells were resuspended in a 100μl of ice-cold methanol:acetic acid (3:1).

Approximately 10μl of this solution was dropped, from a height of approximately 40-

50cm, onto glass slides that had previously been wiped with acetic acid. Slides were left

overnight at room temperature.

2.9.2 Metaphase staining

Slides were stained with 20ng/ml (w/v) DAPI in PBS for 5 min at room temperature.

Slides were air dried, one drop of SlowFade® antifade reagent was applied, after which

84

they were covered with a coverslip and sealed. Images were acquired as described in

Section 2.9.3.

2.9.3 Image acquisition

Images were collected using an Olympus BX51 upright microscope using a coolsnap

ES camera through Metavue software. Slides were viewed using a 100x/1.30UPlanFLn

oil immersion objective.

2.9.4 Immunofluorescence

CHO cells were grown until day 9 of batch culture (Section 2.3.2). 1ml of cell

suspension was centrifuged at 100g for 10 min, and resuspended to give 3x106 cells in

200µl per coverslip. The cell suspension was air dried on the coverslip (pretreated with

20g/ml of poly-L-lysine) for 30 min at room temperature. After a 1 min wash in cold

PBS the cells were fixed on the coverslips using 4% (w/v) paraformaldehyde in PBS.

The cells were permeabilised by addition of using 0.5% (v/v) triton-X-100 in PBS for 5

min at room temperature, and then washed three times with PBS (each wash was for 5

min at room temperature). 200µl of the primary antibody (anti-ATF4, 1:100 dilution)

and 200µl of the fluorescent conjugated secondary antibody (Texas Red®

, anti-Rabbit,

1:100 dilution) were added to each coverslip and incubated separately for 30 min at

37C. Between each incubation the cells were washed, as described above. The

coverslips were then treated with DAPI (50ng/ml [w/v] diluted in PBS) for 1 min and

were mounted on slides using ProLong® antifade reagent. The coverslips were sealed

and stored at 4C until required.

Images were acquired at stated in Section 2.9.3, and analysed using Image J technology.

85

2.10 METABOLITE ANALYSES

2.10.1 Glucose assay

The glucose assay was based on the method developed by Trinder (1969). Glucose is

converted to glucoeimine in a two-step reaction catalysed by glucose oxidase and HRP.

Glucoeimine concentration is measured spectrophotometrically and correlates to initial

glucose concentration. To determine the glucose concentration 2µl from cell culture

supernatants (Section 2.3.2) were mixed with 200µl of assay buffer in 96 well plates.

The assay buffer (0.5M sodium phosphate, pH 7.5, 2U glucose oxidase/ml, 5U

peroxidise/ml, 10.6mM phenol, 1.5mM 4α-aminophenazone) was prepared on the day

of use (pH was adjusted using perchloric acid). The mixture of samples and assay buffer

was incubated at 37oC for 10 min before the absorbance at 505nm was measured.

Standards (0-20mM glucose) were analysed at the same time and used in determination

of glucose concentration.

2.10.2 Lactate assay

The concentration of lactate in samples was determined based on the catalysis to

pyruvate by LDH with the concomitant reduction of NAD+, which formed the L-isomer

of lactic acid (lactate at pH 7.0). To determine the amount of L-lactic acid 2.5µl from

cell culture supernatants (Section 2.3.2) was added to 1ml of assay buffer (2U lactate

dehydrogenase, 0.12mM hydrazine, 1mM NAD+, 0.1M glycine buffer, pH 9.0). The

mixture of samples and assay buffer was incubated at room temperature for 40 min

before the absorbance at 340nm was measured. Standards (0-25mM lactate) were

analysed at the same time and used in determination of lactate concentration.

2.10.3 Gas Chromatography (GC)-MS

2.10.3.1 Sample derivatization

20µl of cell culture supernatants (Section 2.3.2) for GC-MS analysis were spiked with

the internal standard (5µl of 3mg/ml myristic acid d 27) and lyophilised. To induce

volatility and thermal stability chemical derivatization was performed in two stages:

86

pellets were resuspended in methoxyamine hydrochloride in pyridine (40mg/ml; 10μl)

and incubated at 30°C for 90 min with gentle shaking. N-methyl-N-

trimethylsilyltrifluoroacetamide with 1% trimethylchorosilane (MSTFA + 1% TMCS)

(90μl) was then added and the samples were incubated at 37°C for 30 min. The samples

were then cooled to room temperature and transferred into silanized GC vials for GC-

MS analysis.

2.10.3.2 Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analysis was performed as detailed by Sellick et al, using a 7890A GC System

coupled to a 5975C Inert XL MSD with Triple-Axis Detector (using the manufacturer‟s

software, ChemStation, Sellick et al, 2010). Samples were injected onto a DB-

5MS + DG column using helium as the carrier gas. Components were separated by

isothermal chromatography for 1 min at 60°C, followed by an increase to 325°C at a

rate of 10°C/min then 10 min at 325°C. Mass spectra were scanned from 50 to 600 mass

units. Metabolite peaks in the raw chromatograms were identified using ChemStation

and automated mass spectral deconvolution and identification system software

(AMDIS). Metabolite identifications were based on retention times and fragmentation

patterns. The data were combined using an in-house Microsoft Excel macro and

normalized to the standard (myristic acid d 27).

2.10.4 Intracellular metabolite extraction

The cells were grown until day 5 and 9 of batch culture (Section 2.3.2). At the

appropriate time-points the cells were rapidly quenched by addition of 1x107 cells to 5

volumes of quenching solution (60% methanol with 0.85% [w/v] ammonium

bicarbonate [AMBIC, pH 7.4]) at -40°C. The cells in the quenching solution were

centrifuged at 1,000g for 1 min and the quenching solution was then removed. The

metabolites were extracted by resuspension of the cell pellet in 0.5ml of 100% methanol

followed by flash freezing in liquid nitrogen. After thawing on ice at 4°C samples were

vortexed for 30 sec, centrifuged at 800g and the supernatant removed. The pellet was

resuspended in 0.5ml of 100% methanol and the extraction procedure was again

repeated. The methanol extracts were pooled, centrifuged at 15,000g for 1 min, the

supernatant removed and the extracts were lyophilized. Dried metabolite extracts were

resuspended in 1ml of ddH20 prior to use.

87

2.10.5 ATP assay

ATP assays were performed using the Roche® ATP Bioluminescence Assay Kit CLS II

according to manufacturer‟s instructions. Intracellular metabolite extracts (50μl, Section

2.10.5) was mixed with an equal volume of luciferase reagent before measuring

luminescence in a luminometer according to manufacturer‟s instructions. ATP

concentration was confirmed using an ATP calibration curve. The ATP standard stock

was provided in the kit.

2.10.6 NAD+/NADH assay

NAD+/NADH assays were performed using the BioVision NAD

+/NADH Quantification

Kit in 96 well plates. NADH concentrations were determined by mixing intracellular

metabolite extracts (50μl, Section 2.10.5) with 50μl extraction buffer and decomposing

the NAD+ by incubation at 60°C for 1 hr after which 50μl was assayed. The assay was

performed by addition of 100μl of NAD cycling mix followed by incubation for 5 min

at room temperature, and then addition of 10μl of NADH developer. Plates were

incubated for 3 hr at room temperature before the absorbance at 450nm was measured

using a Multiskan Ascent plate reader.

2.11 CALCULATIONS

2.11.1 Calculation of cell doubling time (dt)

The cell dt was calculated as indicated below.

K = (1/ln) x ln (Nt/No)

Where K = the mean growth rate constant (generations/day)

No = the initial population number

Nt = the population at time (t)

88

2.11.2 Calculation of specific productivity (Qp) and rates of metabolite production

and utilisation

Qp or rates of production/utilisation = (P1-P0)/CCT

CCT = (VCC1 + VCC0/2)x(T1 –T0)

P0= antibody titre or metabolite concentration by first point of analysis

P1= antibody titre or metabolite concentration second point of analysis

VCC0= Viable cell density by first point of analysis

VCC1= Viable cell density by second point of analysis

T0= day of first point of analysis

T1= day of second point of analysis

2.11.3 Statistical methods

All data presented is represented as a mean ± standard deviation (SD), or mean ±

standard error of mean (SEM)

Standard Deviation (SD) = (√[∑{x-m}2]/{n-1})

x: observed value

m: mean of n observations

n-1: degrees of freedom

SEM = (SD/√n)

n: number of independent observations

The correlation coefficient (r value) was calculated for standard curves produced in

ELISAs (Section 2.5.1) and q-PCR assays (Section 2.8.4), using Microsoft Excel. Any

assays performed, where the standard curve had a correlation coefficient ≤ 0.98, were

disregarded.

Independent samples t-test was used to determine whether the difference between

samples was statistically significant. The independent samples t-test was performed

89

using SPSS (v14.0.2) software. Data was considered significant if p < 0.05 or p < 0.10.

The limits of p are stated where appropriate.

90

4

CHAPTER 3

5

CHARACTERISATION OF CELL

LINE 3.90 IN DETERMINATION

OF CELL LINE STABILITY

91

3. CHARACTERISATION OF CELL LINE 3.90 IN DETERMINATION OF

CELL LINE STABILITY


This Chapter focuses on the stability of recombinant antibody titre and specific

productivity from the recombinant CHO cell line 3.90 (3.90). I examined the cell

growth, antibody titre, and molecular characteristics of 3.90 in response to LTC.

Potential regulatory factors that might affect antibody secretion were also investigated,

these included analysis of proteins and chaperones involved in protein folding and

maintenance of ER homeostasis. Finally this Chapter addresses the metabolic status of

3.90 cultures created at early and late generations, to examine potential relationships

between the metabolic activity of the cell and the stability of recombinant gene

expression.

3.2 ANALYSIS OF GROWTH CHARACTERISTICS AND PRODUCTIVITY OF

CELL LINE 3.90

The growth characteristics and antibody titre of 3.90 batch cultures were examined at

generations 20, 40, 60, 80 and 100. Generation 20 and 40 cultures expressed final

antibody titres of approximately 1000 mg/L, whilst generation 60, 80 and 100 cultures

had final antibody titres of approximately 600 mg/L (Figure 3.1A). Therefore, 3.90 was

shown to be unstable, with a 40% decrease in final antibody titre values in response to

LTC. Intriguingly, antibody titre was similar for all generation cultures throughout

exponential stage of culture. It was only as cells moved beyond the exponential phase

that generation dependent alterations in antibody titre were observed.

The growth of batch cultures were analysed in parallel to determine if changes in viable

cell densities would account for the decrease in antibody titre. The growth pattern for all

cultures was similar. The cultures were in exponential phase until day 7 of batch culture.

After day 7 the cultures entered, and stayed in, stationary phase until day 11 of batch

culture. For all cultures viable cell densities were maximal on day 9 of batch culture

(Figure 3.1B). Viability declined after day 11 of batch culture, and for all cultures the

92

viability was measured as 30%, or below, on the final day of batch culture (day 15,

Figure 3.1C).

Although patterns of growth were similar for all generations, changes in viable cell

densities were apparent. Batch cultures created at late generations (generations 80 and

100) had lower cell densities in the stationary and decline phase of batch culture than

early generation cultures (generations 20 and 40, Figure 3.1B). Figure 3.2A highlights a

difference in viable cell densities between an early generation (generation 20) and a late

generation (generation 100) culture. Alterations to viable cell densities can also be

observed with changes to cumulative cell time (CCT). CCT values were significantly

lower on days 13 and 15 of batch culture for cultures created at generations 80 and 100

compared to CCT values for generation 20 cultures (Figure 3.2B).

The alterations in cell densities between early and late generation cultures were also

seen with variations to the cell cycle phase transition of cultures. At generations 20, 40,

60, 80 and 100 the cell cycle profile on day 3 of batch culture showed 50% of cells in

G0/G1 phase (Figure 3.3A), approximately 40% in S phase (Figure 3.3B), and 10% in

G2/M phase (Figure 3.3C). The percentage of cells in G2/M phase remained the same

as batch cultures progressed, whilst the percentage of cells in G0/G1 phase increased

and the percentage of cells in S phase decreased. The proportion of cells in G0/G1 phase

and in S phase during batch culture was dependent on the culture generation. On day 11

of batch culture early generation cultures (generations 20 and 40 cultures) had

approximately 75% of cells in G0/G1 phase, with approximately 15% of cells in S

phase, whilst late generation cultures (generations 60, 80 and 100 cultures) had fewer

cells in G0/G1 phase (65%) and more cells in S phase (25%). The cultures with a

greater percentage of cells in G0/G1 also had higher antibody titres. This is supported

by other publications, which had previously stated that CHO cells, and hybridoma cells,

were more productive in the G0/G1 phase of cell cycle distribution (Al-Rubeai &

Emery, 1990; Dutton et al, 2006; Kromenaker & Srienc, 1991).

As generation number influenced CCT the Qp was determined to quantitate antibody

production rate per cell. Qp was calculated from antibody titre and viable cell densities

during different stages of batch culture. Qp was determined for the entire batch culture,

using antibody titre values and cell densities measured on days 0 to 15 of culture, Qp

93

(d0-d15). Similarly, Qp was also determined for the early (exponential) phase of batch

culture, Qp (d0-d7), and for the end (decline) phase of batch culture, Qp (d9-d15). Qp

(d0-d15) decreased by approximately 30% between early generation cultures

(generations 20 and 40) and late generation cultures (generations 60, 80 and 100, Figure

3.4), again confirming instability of 3.90 in response to LTC. Qp was found to be

maximal during the early phase of batch culture. Qp (d0-d7) values were two-fold

greater than Qp (d0-d15) values, and three- to five-fold greater than Qp (d9-d15) values,

dependent on the generation time of culture.

Interpretation of Qp data shows that 3.90 instability (seen with a decrease in final

antibody titre) was not solely due to changes in viable cell densities, as Qp also

decreased as a result of LTC. However, it could be possible that other cellular

alterations, such as a change in cell size, were affecting Qp (Lloyd et al, 2000). The

average diameter for both early generation (≤ 40 generation) and late (≥ 60 generation)

generation cells were approximately 13µm (Figure 3.5A). This value is in agreement

with previously stated CHO cell diameters (Kuystermans & Al-Rubeai, 2009). The cells

analysed gave a range of diameters from 8µm to 22µm, similar for both early generation

and the late generation cells (Figure 3.5B). The decrease in Qp in response to LTC was

not a consequence of changes in cell size but could be the result of changes to

intracellular events that control the efficiency of cellular expression of the recombinant

gene.

94

Figure 3.1 Analysis of recombinant antibody titre, viable cell densities, and cell

viability for 3.90 cultures

3.90 was subject to LTC in suspension using MSX supplemented CD-CHO media. Batch

growth analysis was performed in shake flasks at generation numbers 20, 40, 60, 80

and 100, ± 4 generations (Section 2.3.2). Batch cultures were created at 0.2x106

cells/ml, and maintained at 37oC, 140 rpm and with a manual supply of 5% CO2 in air.

Cells were cultured under these conditions until viability was ≤ 30%. Antibody titres

(A), viable cell densities (B), and cell viabilities (C) are shown. Antibody titres were

measured by ELISA (Section 2.5.1) and viable cell densities and cell viabilities were

determined by light microscopy and trypan blue exclusion (Section 2.3.3) from samples

taken routinely during batch culture. Error bars represent SEM for three biological

replicates. Each biological replicate value is an average from duplicate technical

repeats. * indicates p<0.05, using independent samples t-test to compare cultures

created at generations 40, 60, 80 and 100 to generation 20 cultures (on the same day of

batch culture).

Annotation of the generation batch cultures in Figure 3.1

20

40

60

80

100

95

Figure 3.1 Analysis of recombinant antibody titre, viable cell densities, and cell

viability for 3.90 cultures

B.

C.

A.

0

200

400

600

800

1000

1200

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

0

2

4

6

8

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

20

40

60

80

100

0 5 10 15

Cell

via

bil

ity

(%)

Day

*** *** ***

96

Figure 3.2 Effect of LTC on viable cell growth and CCT

Viable cell densities were determined using light microscopy and trypan blue exclusion

(Section 2.3.3). A, highlights the change in viable cell densities between an early

generation batch culture (generation 20) and a late generation batch culture

(generation 100). The CCT was calculated from the growth of the batch cultures

(Figure 3.1B). B, shows the CCT during batch culture for cultures created at

generations 20, 40, 60, 80 and 100. For determination of CCT see Section 2.11.2. Error

bars represent SEM for three biological replicates. Each biological replicate value is

an average from duplicate technical repeats. * indicates p<0.05, using independent

samples t-test to compare cultures created at generations 40, 60, 80 and 100 to

generation 20 cultures (on the same day of batch culture).


B.

A.

0

2

4

6

8

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

10

20

30

40

50

60

70

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

l)

Day

20

40

60

80

100

** **

*

*

97

Figure 3.3 Analysis of cell cycle distribution in response to LTC

3.90 was cultured as previously described (Figure legend 3.1). 1x106 cells, taken on

days 3, 5, 7, 9, and 11 of batch culture, were analysed by flow cytometry using PI

excitation by a 488nm laser, and emission measured by a 613/20nm bandpass filter

(Section 2.4.1). The data was analysed by Summit 4.3 and ModFit LT software. The

percentage of cells in A, G0/G1 phase, B, S phase, and C, G2 phase are shown. Error

bars represent SEM for three biological replicates.


20

40

60

80

100

98

Figure 3.3 Analysis of cell cycle phase distribution in response to LTC

B.

C.

A.

0

20

40

60

80

100

3 5 7 9 11

G0

/G1

cell

cy

cle

ph

ase

(%)

Day

0

20

40

60

80

100

3 5 7 9 11

S c

ell

cy

cle

ph

ase

(%)

Day

0

20

40

60

80

100

3 5 7 9 11

G2

/M c

ell

cy

cle

ph

ase

(%)

Day

99

Figure 3.4 Effect of culture generation time on specific productivity (Qp)

Specific productivity (Qp) was determined from the antibody titre and cell density

values seen in Figure 3.1A and Figure 3.1B, respectively. Qp was calculated for the

entire batch culture, using antibody titre and cell density measurements from days 0 to

15 of culture, Qp (d0-d15). Qp was also calculated for the early (exponential) phase of

batch culture, using antibody titre and cell density measurements from days 0 to 7 of

culture, Qp (d0-d7), and for the end (decline) phase of batch culture, using antibody

titre and cell density measurements from days 9 to 15 of culture, Qp (d9-d15). For

determination of Qp see Section 2.11.2. Qp values are shown for cultures created at

generations 20, 40, 60, 80 and 100. Error bars represent SEM for three biological


repeats. * indicates p<0.05, and ♦ indicates p<0.10, using independent samples t-test to

compare the corresponding Qp values for cultures created at generations 40, 60, 80

and 100 to Qp values for generation 20 cultures.

Annotation of Qp values in Figure 3.4

0

5

10

15

20

25

30

35

20 40 60 80 100

Sp

ecif

ic p

ro

du

cti

vit

y (

Qp

)

(pg

/cell

/da

y)

Generation time

Qp (d0-d15)

Qp (d0-d7)

Qp (d9-d15)

* * *

* ♦ ♦

100

Figure 3.5 Effect of culture generation time on cell size

3.90 was cultured as previously described (Figure legend 3.1). On day 9 of batch

culture, early generation (≤ 40 generations) and late generation (≥ 60 generations)

cells were prepared for cell counting using trypan blue dye (as described in Section

2.3.3). 100 cells were counted and measured for cell diameter using a Widefield

Axiovision microscope, and analysed using the Axiovision software. A, shows the

average cell diameter for early and late generation cells, and B, shows the range of cell

diameter measurements for early and late generation cells. Error bars represent the SD

for 100 cells counted.

Annotation of Figure 3.5

A.

B.

0

5

10

15

20

25

30

35

40

Nu

mb

er o

f cell

s

(Freq

uen

cy

)

Cell diameter

(µm)

0

4

8

12

16

Early Late

Cell

dia

mete

r

(µm

)

Generation

Early generation cells

Late generation cells

101

3.3 MOLECULAR INVESTIGATION OF ANTIBODY TITRE LOSS DURING

LTC OF CELL LINE 3.90

Characterisation analysis of 3.90 showed this cell line to be unstable, with a 40%

decrease in final antibody titre and a 30% decrease in Qp (d0-d15) between early

generation (generations 20 and 40) and late generation (generations 60, 80 and 100)

cultures. In order to determine the reason for instability molecular investigations of 3.90

were performed. In the work described in Section 3.3, 3.90 cultures were examined for

genomic stability in response to LTC, by analysis of chromosomal spreads and plasmid

gene copy numbers. Antibody mRNA and polysome profiles were also examined to

determine changes at transcript level. Molecular investigations extended to the analysis

of protein synthesis and secretion for 3.90 cultures.

For the remainder of this Chapter batch cultures created at generations 20 and 40 are

referred to as early generation cultures and batch cultures created at generations 60, 80

and 100 are referred to as late generation cultures.

3.3.1 Analysis of genomic stability during LTC

CHO cells have been reported to exhibit genetic instability, and have a karyotype that

has undergone extensive chromosomal rearrangement (Bacsi & Wejksnora, 1986). To

determine if 3.90 was experiencing karotypic changes chromosomal spreads were

analysed. Chromosomal numbers observed in early generation metaphase spreads

(Figure 3.6A(i)) and late generation metaphase spreads (Figure 3.6A(ii)) were similar.

For all the cultures analysed 18-20 chromosomes were observed in each spread (Figure

3.6B). The average chromosome number determined for 3.90 cultures was similar to

reported literature for CHO cultures. For example, Kao and Puck found the model

chromosome number of CHO-K1 to be 21 (Kao & Puck, 1969), whilst Derouazi et al,

found CHO-DG44 had an average of 20 chromosomes per cell (Derouazi et al, 2006).

102

Figure 3.6 Effect of culture generation time on chromosome number

3.90 cells in exponential phase were fixed in metaphase using 130ng/ml (w/v) colcemid

(Section 2.9.1). Metaphase spreads were created from an early generation (≤ 40

generations), and late generation (≥ 60 generations) culture. Images were collected on

an Olympus BX51 upright microscope with a 100x/1.30UPlanFLn oil immersion

objective using a coolsnap ES camera, through Metavue software (Section 2.9.3).

Typical examples of a early generation spread (A(i)), and a late generation metaphase

spread (A(ii)) are shown. The chromosome number (determined from 40 early and late

generation metaphase spreads) is shown in B. White scale bars = 10μm.

Annotation of the batch cultures in Figure 3.6

0

5

10

15

20

25

30

18 19 20

Freq

uen

cy

Number of chromosomes

A.

B.

(i) (ii)

Early generation

Late generation

103

Previous literature has shown DHFR-mediated CHO cells to be genetically unstable as a

result of recombinant gene loss during culture (Kim et al, 1998). Copy number analysis

determined 3.90 cultures had approximately 10 copies of both the heavy chain (Figure

3.7A) and light chain (Figure 3.7B) recombinant genes per cell. The number of heavy

chain and light chain gene copies per cell were similar for both early and late generation

cultures. Southern blot analysis also confirmed there was no alteration in plasmid gene

copy number in response to LTC.

3.3.2 Analysis of recombinant gene mRNA expression during LTC

A decrease in final antibody titre (Figure 3.1A) and Qp (Figure 3.4) was witnessed in

response to LTC, however, the instability cannot be attributed to molecular changes in

gross chromosome number (Figure 3.6) or gene copy number (Figure 3.7). As Qp has

been associated with recombinant transcript levels in CHO cell lines (Lee et al, 2009a)

recombinant mRNA expression was investigated for 3.90. GS mRNA expression was

constant during batch culture, and in response to LTC (Figure 3.8A). Although heavy

chain (Figure 3.8B) and light chain (Figure 3.8C) mRNA increased during batch culture

there was no significant difference in recombinant mRNA between cultures created at

early generations (generations 20 and 40) and those created at late generations

(generations 60, 80 and 100).

3.3.3 Investigating polysome profile characteristics during culture

Even though the expression antibody mRNA was not dependent on generation time of

culture mRNA association with translational machinery may have altered in response to

LTC. Lower rates of translation between early and late generation cultures may be

responsible for the decrease in antibody titre. It is possible that alterations to the

translation efficiency of the cell can be identified by modifications to polysome

conformations (reviewed in Ross, 1995). From polysome profiles analysed an increase

in the 80S peak was observed from day 4 to day 7 of batch culture, whilst a

corresponding decrease was observed for the 60S and polysome peaks (Figure 3.9).

Although the monosome peak areas were similar for both the early and late generation

polysome profiles, the polysome peak was lower for late generation day 7 polysome

profiles (Figure 3.9B(ii)) than for early generation day 7 polysome profiles (Figure

104

3.9A(ii)). To determine if the change in polysome peak area was altered during LTC the

relative change in peak area was quantified using Image J software. The resultant data

are shown in Figure 3.10.

Quantitative analysis of the monosome and polysome peak areas found that from days 4

to 7 of batch culture the relative 40S peak area was unaltered (Figure 3.10A), the

relative 60S peak area decreased (Figure 3.10B), and the relative 80S peak area

increased (Figure 3.10C). The changes to the monosome peaks during batch culture

were similar for both early and late generation cultures, but the percentage decrease

observed for the polysome peak area during batch culture was dependent on the

generation time of culture. The relative polysome area from days 4 to 7 of batch culture

decreased by 40% and 75% for early generation cultures and late generation cultures,

respectively (Figure 3.10D), suggesting that late generation cultures had greater

polysome dissociation than early generation cultures. The change to the polysome

profiles during culture indicate alterations to molecular events relating both to

instability in response to LTC, and the difference in Qp between the early (exponential)

phase and the end (decline) phase of batch culture. Harding et al, reported that during

ER stress, polysomes dissociate and monosomes accumulate, resulting in translational

inhibition (Harding et al, 2000b). My findings may reflect stress during batch culture

and potentially lower translational efficiency on day 7 of batch culture, particularly for

late generation cultures.

3.3.4 Analysis of protein synthesis and secretion during LTC

A change at the overall polysome locus has a potential consequence for the general

protein translational capacity of the cultures. In order to investigate the possibility that

changes in translation occurred with LTC, global protein synthesis was analysed using

incorporation of tritiated leucine (L-[4,5-3H] leucine). L-[4,5-3H] leucine was added to

early generation and late generation cultures which had undergone 7 days of prior batch

culture and was followed into intracellular proteins (cell pellets) and extracellular

proteins (supernatant samples). The relative L-[4,5-3H] leucine incorporation in the

intracellular proteins increased approximately thirty-fold after 48 hrs, for both early and

late generation cultures (Figure 3.11A). Although there was no change in the

intracellular L-[4,5-3H] leucine incorporation between early and late generation cultures

105

the extracellular protein from early generation cell suspensions had greater L-[4,5-3H]

leucine incorporation than the extracellular protein from late generation cell suspensions

(after 48 hrs incubation, Figure 3.11B). The change in L-[4,5-3H] leucine incorporation

in extracellular protein mirrored the decrease in antibody titre data measured by ELISA

(Figure 3.1A). The L-[4,5-3H] leucine incorporation method analysed protein synthesis

and secretion on a global scale. To investigate intracellular changes to the recombinant

protein antibody APC- and FITC-conjugated dyes (which detect the heavy chain and

light chain proteins, respectively) were used. The mean APC and FITC fluorescence

intensity increased from days 4 to 9 of batch culture, but no difference in APC or FITC

mean fluorescence was observed between early and late generation cultures (Figure

3.12). This confirmed, at an intracellular level, the results seen with the L-[4,5-3H]

leucine incorporation assay.

A molecular investigation of 3.90 suggests decreased final antibody titres and lower Qp

values in response to LTC could be due to changes at the level of protein secretion. A

working hypothesis can now be created that late generation cells have limitations with

the folding and secretion of the antibody chains or complexes. Mis-folded and unfolded

proteins can be recognized in the ER and cause activation of UPR (Figure 1.12) and

ERAD pathways (shown in Figure 1.13), with consequences for protein synthesis and

cell growth (Ellgaard & Helenius, 2003; Rutkowski & Kaufman, 2004). It is possible to

monitor the activation of these pathways to determine changes in response to LTC.

106

Figure 3.7 Analysis of heavy chain gene and light chain gene copy number for

early and late generation cultures

3.90 was cultured as previously described (Figure legend 3.1). Batch cultures were

created at early generations (≤ 40 generations) and late generations (≥ 60 generations,

Section 2.3.2). Gene copy numbers were analysed from genomic DNA extracted during

the exponential phase of batch culture (Section 2.6.1.1). Plasmid copies per cell were

assessed, using the light chain and heavy chain primer sets, by q-PCR (Section 2.6.2).

Genomic DNA content was normalised using the β-Actin primer set. Copy number was

also assessed by Southern blot analysis (Section 2.6.1). For Southern analysis of gene

copy number the genomic DNA, and plasmid DNA for probe design, was digested with

HindIII and EcoR1 (Section 2.6.1.3). The membrane was probed with the radiolabelled

heavy or light chain region, prepared as detailed in Section 2.6.1.7. Standardisation of

the loading of DNA species was performed using a radiolabelled 18S RNA gene. Tables

detailing heavy chain gene copy number per cell (A) and light chain gene copy number

per cell (B) are shown. An example of a Southern blot analysed for light chain gene

copy number is also shown (C). Copy number values determined by q-PCR are the

average values from triplicate experiments ± SEM. The plasmid copies determined by

Southern blot are the result of one experiment after normalisation.

107

Figure 3.7 Analysis of heavy chain gene and light chain gene copy number for

early and late generation cultures

A.

B.

Heavy chain g ene Plasmid copies per cell

( q-PCR)

Plasmid copies per cell

(Southern Blot)

Early g eneration 10.9 ± 2.3 8.7

Late generation 10.9 ± 1.9 10.3

Light chain g ene Plasmid copies per cell

( q-PCR)

Plasmid copies per cell

(Southern Blot)

Early generation 9.5 ± 1.1 10.2

Late generation 7.4 ± 1.2 10.8

C.

Southern blot standard

Early Late

Generation

108

Figure 3.8 Effects of culture generation time on recombinant mRNA expression

3.90 was cultured as previously described (Figure legend 3.1). mRNA levels were

compared using q-RTPCR from samples taken on days 3, 5, 7 and 9 of batch culture (as

detailed in Section 2.7.1), using the mRNA specific primer sets for A, GS, B, heavy

chain, and C, light chain. Samples were normalised using mRNA β-Actin primers. Error



20

40

60

80

100

109

Figure 3.8 Effect of culture generation time on recombinant mRNA expression

A.

B.

C.

0

50

100

150

200

250

300

3 5 7 9

Lig

ht ch

ain

mR

NA

ex

press

ion

(% r

ela

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ard

an

d

β-A

cti

nm

RN

A e

xp

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ion

)

Day

0

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3 5 7 9

GS

mR

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ex

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(% r

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an

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β-A

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nm

RN

A e

xp

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ion

)

Day

0

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3 5 7 9

Hea

vy

ch

ain

mR

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ex

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(% r

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sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Day

110

Figure 3.9 Analysis of polysome profiles during culture



Section 2.3.2). Polysomes were extracted on day 4 and day 7 of batch culture (Section

2.8.2), and the extracts were analysed on a UV/Vis machine at 254nm after sucrose

gradient centrifugation, as described in Section 2.8.3. The peaks that contain the small

ribosomal subunit (40S), the large ribosomal subunit (60S), and both subunits (80S) are

indicated by arrows. The polysome peaks generated by 2, 3, 4 etc. 80S ribosomes on a

single mRNA are also indicated by an arrow. Figure 3.9A, represents a typical 3.90

early generation polysome profile, (i), shows a day 4 polysome profile, (ii), shows a day

7 polysome profile. Figure 3.9B, represents a typical 3.90 late generation polysome

profile, (i), shows a day 4 polysome profile, (ii) shows a day 7 polysome profile.

111

Figure 3.9 Analysis of polysome profiles during culture

60S

40S

80S

Polysomes40S

80S

60S

Polysomes

40S

80S

60S

Polysomes

60S

40S

80S

Polysomes

A.

B.

(i)(ii)

(i) (ii)

Early Generation Cultures

Late Generation Cultures

Day 4 Day 7

Day 4 Day 7

112

Figure 3.10 Effects of culture on the relative area of monosome and polysome

peaks

Polysomes were extracted and analysed (as described in Figure legend 3.9 and Section

2.8). The 40S, 60S, 80S and polysome peaks were analysed using Image J software to

provide a relative representation of peak area variation. The relative 40S peak area (A),

60S peak area (B), 80S peak area (C) and polysome peak area (D) are shown for early

and late generation day 4 and day 7 polysome profiles. Error bars represent SEM for

three biological replicates.


Early generation

Late generation

113

Figure 3.10 Effects of culture on the relative area of monosome and polysome

peaks

A.

B.

C.

0

10

20

30

40

50

4 7

Rela

tiv

e 4

0S

pea

k a

rea

Day

D.

0

20

40

60

80

100

120

4 7

Rela

tiv

e 6

0S

pea

k a

rea

Day

0

20

40

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80

100

120

4 7

Rela

tiv

e p

oly

som

e

pea

k a

rea

Day

0

50

100

150

200

4 7

Rela

tiv

e 8

0S

pea

k a

rea

Day

114

Figure 3.11 Measuring global protein synthesis and secretion for early and late

generation cultures

3.90 was cultured as previously described (Figure legend 3.1). Cell suspensions (500µl)

from early generation (≤ 40 generations) and late generation (≥ 60 generations) day 7

cultures were transferred to 24 well plates and incubated with L-[4,5-3H] leucine.

Protein synthesis and secretion was measured as the rate of incorporation of L-[4,5-

3H] leucine into trichloroacetic acid (TCA) precipitable-material over a 48 hr time

period (Section 2.5.2). The fold increase for intracellular protein (A) and extracellular

protein (B) was determined relative to early generation cultures at 0 hrs. Error bars

represent SEM for three biological replicates.

Annotation of time-points in Figure 3.11

A.

B.

(i) (ii)

0

10

20

30

40

Early Late

Fo

ld in

crea

se i

n

intr

acell

ula

r p

ro

tein

(rela

tiv

e to

ea

rly

gen

era

tio

n

va

lues

at

0 h

rs)

Generation

0

10

20

30

Early Late

Fo

ld in

crea

se i

n

ex

tra

cell

ula

r p

ro

tein

(rela

tiv

e t

o e

arly

gen

era

tio

n

va

lues

at

0 h

rs)

Generation

0 hrs

24 hrs

48 hrs

115

Figure 3.12 Analysis of intracellular heavy chain and light chain protein during

culture



Section 2.3.2). 2x106 fixed cells, from days 4 and 9 of batch culture, were washed,

blocked and incubated with 10µg goat anti-human IgG, Fcγ-APC and 6µg goat anti-

human lambda light chain-FITC. The samples were then analysed by a CyAn ADP flow

cytometer, using the 488nm and infra-red excitation lasers to excite the FITC and APC

conjugates (Section 2.4.2). Unstained 3.90 cells and stained parental cells were

required for setting initial parameters with the CyAn-ADP flow cytometer. The data was

gated to select single cells, and was analysed by Summit 4.3 software. Error bars


Annotation of antibody conjugates in Figure 3.12

0

20

40

60

80

4 9 4 9

Early Late

Rela

tiv

e m

ea

n f

luo

resc

en

ce i

nte

nsi

ty

(fo

ld in

crea

se c

om

pa

red

to

th

e

pa

ren

tal

cell

lin

e)

Day/Generation

APC conjugate antibody (for use in heavy chain protein detection)

FITC conjugate antibody (for use in light chain protein detection)

116

3.4 THE REGULATION OF UPR MARKERS DURING CULTURE

Instability of 3.90 was characterised by a 40% decrease in antibody titre and a 30%

decrease in Qp between early generation and late generation cultures (Section 3.2).

Molecular characterisation studies confirmed that early and late generation cultures had

similar copies of antibody genes per cell and similar expression of recombinant mRNA,

but highlighted a decrease in protein secretion in response to LTC (Section 3.3). If late

generation cultures were encountering problems in antibody secretion, possibly due to

an accumulation of mis/unfolded proteins and the ability to handle such proteins

appropriately, the UPR would be initiated. A greater UPR for late generation cultures

would prevent incorrectly folded proteins from being secreted, ultimately resulting in

lower antibody titres.

In the temporal changes in response to mis/unfolded proteins, the PERK pathway is the

first pathway activated during the UPR. As discussed in Section 1 PERK becomes

activated as unfolded proteins accumulate in the ER (Bertolotti et al, 2000; Liu et al,

2000). Activated PERK phosphorylates eIF2α (Harding et al, 1999; Prostko et al, 1992;

Shi Y, 1998) resulting in global protein synthesis inhibition, with the exception of ATF4

(Harding et al, 2000a; Lu et al, 2004). Increased translation of ATF4 upregulates

expression of ER stress target genes including GADD34 (Ma & Hendershot, 2003) and

GADD153 (Harding et al, 2000a). Pathways involved in the UPR are shown in Figure

1.12. Expression of ATF4, GADD34 and GADD153 mRNA were examined in response

to LTC.

The expression of ATF4 and GADD34 mRNA increased from days 3 to 9 of batch

culture, five-fold and three-fold, respectively (Figure 3.13A and 3.13B). Although the

expression of ATF4 mRNA was the same between early and late generation cultures,

GADD34 mRNA expression was slightly altered in response to LTC. On day 7 of batch

culture GADD34 mRNA was approximately 30% lower for late generation cultures than

early generation cultures. The expression of GADD153 mRNA was also dependent on

the culture generation time (Figure 3.13C). On day 7 of batch culture expression of

GADD153 mRNA was approximately three-fold greater for late generation cultures than

for early generation cultures. Increased GADD153 mRNA expression for late generation

117

cultures was interpreted to be an indication of an adaptation to increased cell stress as a

result of LTC.

In order to confirm the observations made at mRNA level, cellular ATF4 and

GADD153 protein were examined by western blot analysis. A 50% increase in ATF4

protein expression was detected as cells moved from the exponential to the stationary

phase of batch culture, similar for both early and late generation cultures (Figure

3.14A). A similar percentage increase was seen for ATF4 mRNA expression during

batch culture (Figure 3.13A). Expression of GADD153 protein was also upregulated

during batch culture (Figure 3.14B). From days 3 to 9 of batch culture there was an

approximate eight-fold increase in GADD153 protein expression, with no significant

difference in GADD153 protein intensity between early and late generation cultures. A

western blot for GADD153 protein shows the expression of GADD153 protein during

batch culture (Figure 3.14C), which peaks on day 9 of batch culture.

The increased expression of ATF4 and GADD153 mRNA and protein was interpreted

to indicate that the cells experienced a stress response during batch culture. To confirm

increased ER stress during batch culture ER chaperones, BiP and PDI, were also

examined. Increased BiP expression has been associated with activation of the UPR

(Kaufman, 2002; Wang et al, 1996). Western blot analysis of BiP showed a band at

approximately 75 kDa, corresponding to the MW observed by Lee (Lee, 1987). The up-

regulation of BiP protein is shown in the representative western blot in Figure 3.15B.

From days 3 to 11 of batch culture the expression of BiP protein increased five-fold for

early generation cultures, and four-fold for late generation cultures (Figure 3.15A).

Although BiP protein expression increased during batch culture there was no change in

BiP protein intensity between early and late generation cultures. Similar findings were

also seen for PDI protein expression. Western blot analysis of the ER chaperone PDI

showed a band of approximately 57 kDa, a MW similar to other published data. An

approximate three-fold increase in relative PDI protein expression was observed from

days 3 to 11 of batch culture, for both early and late generation cultures (Figure 3.16A).

PDI protein expression is shown in the representative western blot in Figure 3.16B.

So far, experiments have focused upon mRNA and protein expression at the first

response to ER stress, such as the increase in ER chaperones, BiP and PDI, and on

118

downstream factors from the PERK pathway, including ATF4, GADD34 and

GADD153. There are two other pathways involved in the UPR, which involve the

activation of ER transmembrane proteins, ATF6 and IRE-1. ATF6 and IRE-1 differ in

the timing of their response, but merge to induce XBP-1 transcription, and mRNA

splicing (Yoshida et al, 2003), shown in Figure 1.12. This splicing event creates a

translational frameshift in XBP-1 mRNA allowing production of an active transcription

factor, co-inducing UPRE (Calfon et al, 2002; Lee et al, 2002; Shen et al, 2001;

Yoshida et al, 2001). Treatment with tunicamycin, a known activator of ER stress,

resulted in spliced XBP-1 mRNA after a 24 hr incubation (Figure 3.17A). XBP-1

mRNA splicing was also shown for 3.90 cultures during batch culture (Figure 3.17B).

The ratio of spliced XBP-1 mRNA to total XBP-1 mRNA increased from days 3 to 9 of

batch culture, and by day 9 of batch culture the ratio was greater for late generation

cultures (Figure 3.17C). The extent of XBP-1 mRNA splicing gave another indication

that late generation cultures might be experiencing greater ER stress than early

generation cultures.

A UPR was detected for 3.90 during batch culture, shown by increased expression of

ATF4 and GADD153 mRNA and protein, and BiP and PDI protein. There was also

enhanced GADD153 mRNA and XBP-1(s) mRNA for late generation cultures. These

findings suggest that late generation cultures encountered more ER stress, potentially

due to a greater concentration of mis/unfolded proteins in the ER, or a failure of late

generation cultures to restore proteins to the correctly-folded conformation. A failure in

protein folding would result in enhanced protein degradation, which would have

detrimental consequences for antibody secretion and antibody titres.

119

Figure 3.13 Effects of culture generation time on the mRNA expression of UPR

markers

3.90 was cultured as previously described (Figure legend 3.1). mRNA levels were

compared using q-RTPCR from samples taken on days 3, 5, 7 and 9 of batch culture (as

detailed in Section 2.7.1), using the mRNA specific primer sets for A, ATF4, B,

GADD34, and C, GADD153. Samples were normalised using mRNA β-Actin primers.

Error bars represent SEM for three biological replicates. ♦ indicates p<0.10, using

independent samples t-test to compare cultures created at generations 40, 60, 80 and

100 to generation 20 cultures (on the same day of batch culture).


20

40

60

80

100

120

Figure 3.13 Effect of culture generation time on the mRNA expression of UPR

markers

A.

B.

C.

0

50

100

150

200

250

300

350

400

3 5 7 9

GA

DD

34

mR

NA

ex

press

ion

(% r

ela

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sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

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ion

)

Day

0

50

100

150

200

250

300

350

3 5 7 9

AT

F4

mR

NA

ex

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(% r

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β-A

cti

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RN

A e

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ion

)

Day

0

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100

150

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3 5 7 9

GA

DD

15

3 m

RN

A e

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(% r

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sta

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an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Day

♦ ♦

♦ ♦ ♦

♦ ♦

♦

121

Figure 3.14 Analysis of ATF4 and GADD153 protein expression for early and late

generation cultures



Section 2.3.2). Protein was extracted during batch culture (as detailed in Section

2.5.3.1). 60µg of protein was separated by SDS-PAGE (Section 2.5.3.2), transferred

(Section 2.5.3.3) and detected using anti-rabbit polyclonal ATF4 and GADD153

antibodies. Membranes were stripped and re-probed using an anti-mouse pan ERK

antibody (Section 2.5.3.4). Bands were examined using Image J software, and the

amount of ATF and GADD153 protein expression was calculated relative to ERK

expression (Section 2.5.3.5). A, shows the relative intensity of ATF4 expression, B,

shows the relative intensity of GADD153 expression, and C, provides a representative

western blot using the GADD153 antibody. Error bars represent SEM for three

biological replicates.


Early generation

Late generation

122

Figure 3.14 Analysis of ATF4 and GADD153 protein expression for early and late

generation cultures

A.

B.

C.

3 7 9 11 3 7 9 11

Early Late

GADD153

ERK

0

20

40

60

80

100

120

140

exponential stationary

AT

F4

pro

tein

in

ten

sity

( rela

tiv

e to

sta

nd

ard

ER

K)

Stage of culture

0

50

100

150

200

250

3 7 9 11

GA

DD

15

3 p

ro

tein

in

ten

sity

( rela

tiv

e to

sta

nd

ard

ER

K)

Day

Day

Generation

123

Figure 3.15 Analysis of BiP protein expression for early and late generation

cultures



Section 2.3.2). Protein was extracted on days 3, 7, 9 and 11 of batch culture (as detailed

in Section 2.5.3.1). 60µg of protein was separated by SDS-PAGE (Section 2.5.3.2),

transferred (Section 2.5.3.3) and then detected using an anti-goat polyclonal BiP

antibody. Membranes were stripped and re-probed using an anti-mouse pan ERK

antibody (Section 2.5.3.4). Bands were analysed using Image J software, and the

amount of BiP protein expression was calculated relative to ERK expression (Section

2.5.3.5). A, shows the relative intensity of BiP expression, and B, provides a

representative western blot. Error bars represent SEM for three biological replicates.


A.

B.

3 7 9 11 3 7 9 11

Early Late

BiP

ERK

0

100

200

300

400

500

3 7 9 11

BiP

pro

tein

in

ten

sity

(rela

tiv

e to

ER

K s

tan

da

rd

)

Day

Day

Generation

Early generation

Late generation

124

Figure 3.16 Analysis of PDI protein expression for early and late generation

cultures



Section 2.3.2). Protein was extracted on days 3, 7, 9 and 11 of batch culture (as detailed

in Section 2.5.3.1). 60µg of protein was separated by SDS-PAGE (Section 2.5.3.2),

transferred (Section 2.5.3.3) and then detected using an anti-rabbit polyclonal PDI

antibody. Membranes were stripped and re-probed using an anti-mouse pan ERK


amount of PDI protein expression was calculated relative to ERK expression (Section

2.5.3.5). A, shows the relative intensity of PDI expression, and B, provides a

representative western blot. Error bars represent SEM for three biological replicates.


A.

B.

0

50

100

150

200

250

300

3 7 9 11

PD

I p

ro

tein

in

ten

sity

(rela

tiv

e to

ER

K s

tan

da

rd

)

Day

3 7 9 11 3 7 9 11

Early Late

PDI

ERK

Day

Generation

Early generation

Late generation

125

Figure 3.17 Analysis of XBP-1(s) mRNA during culture



Section 2.3.2). cDNA was synthesised from RNA extracted on days 3 and 9, for early

and late generation cultures, and on day 3 for the positive control culture (Section

2.7.1.3). The positive control culture had been treated with tunicamycin (6µg/ml) 24 hrs

prior to RNA extraction. The PCR was performed using the newly synthesised cDNA

and the relevant primers (Section 2.7.2). The PCR products were electrophoresed on a

2% (w/v) agarose gel and visualised by UV transillumination (Section 2.6.1.4). The

positive control PCR (A) and the PCR for early and late generation cultures (B) are

shown. The product bands were analysed using Image J software, and the ratio of

spliced XBP-1 mRNA to total XBP-1 mRNA (C) is also shown. Error bars represent

SEM for three biological replicates. ♦ indicates p<0.10, using independent samples t-

test to compare late generation cultures to early generation cultures (on the same day

of batch culture).


Early generation

Late generation

126

Figure 3.17 Analysis of XBP-1 mRNA during culture

A.

B.

C.

3 9 3 9

Early Late

GAPDH XBP-1

XBP-1(u)

XBP-1(s)

0

10

20

30

40

50

60

3 9 Positive

Am

ou

nt

of

spli

ced

XB

P-1

mR

NA

to to

tal

XB

P-1

mR

NA

(%

)

Day

Day

Generation

♦

127

3.5 METABOLIC ANALYSIS OF CELL LINE 3.90

3.90 was shown to be unstable with a 40% decrease in antibody titre between early and

late generation cultures (Section 3.2). The instability of 3.90 was thought not to be due

to a loss in recombinant antibody gene copy number, antibody mRNA expression or

protein synthesis (Section 3.3), but was attributed to less protein secretion (Figure

3.11B). As the protein expression of the UPR markers (ATF4, GADD153, BiP and PDI)

all increased during batch culture, I suggested that some recombinant antibody was

failing to fold correctly, initiating an UPR (Section 3.4). It has also been previously

reported that increased expression of ATF4 and GADD153 mRNA can result from

nutrient deprivation (Bruhat et al, 1997), and that unfolded proteins can accumulate

with glucose starvation (Pouysségur et al, 1977). Therefore, changes in the availability

of nutrients (including glucose) may influence the UPR status of the cell, and may

potentially affect the mechanisms involved in protein folding. Metabolite profiles of

early generation and late generation cultures were investigated to determine possible

nutrient limitations between early and late generation cultures.

Most amino acids analysed only decreased during the first five days of batch culture,

with no further decline in their relative concentrations after day 5 of batch culture

(Appendix 2). However, asparagine, serine, leucine and lysine were found to be utilised

during the entire batch culture (Figure 3.18). Asparagine decreased rapidly during batch

culture and was depleted from both early generation and late generation cultures by day

9 of batch culture (Figure 3.18A). Serine consumption by 3.90 cultures was also rapid.

There was a five-fold decrease in serine by day 5, and complete serine depletion by day

13 of batch culture, for both early and late generation cultures (Figure 3.18B). Leucine

(Figure 3.18C), and lysine (Figure 3.18D) concentrations were also decreased during

batch culture, but neither of these amino acids were completely depleted.

As well as investigating metabolites that were utilised during culture, metabolites

released from the cell were also considered. By day 13 of batch culture both early and

late generation cultures showed a relative thirty-fold increase in glycerol (Figure

3.19A), and a relative one hundred and twenty-fold increase in glycine (Figure 3.19B).

The accumulation of glycine and glycerol during batch culture was not altered in

response to LTC. The profile of alanine accumulation, however, was dependent on the

128

generation time of culture. Alanine concentration was greatest for early generation

cultures on day 9 of batch culture, after which alanine decreased slightly between days 9

and 13 of batch culture (Figure 3.19C). For late generation cultures alanine

concentration was maximal by day 7 of batch culture, followed by a steady decrease in

relative alanine concentration. The observed decrease in relative alanine concentration

during the end phase of batch culture suggests that following production of alanine, as a

by-product, cells were possible re-utilising alanine at a crucial point during culture

when nutrients were limiting. On days 5 and 7 of batch culture relative alanine

concentration was greater for late generation cultures than early generation cultures.

Late generation cultures were potentially producing more alanine, resulting in the

increased accumulation of extracellular alanine. Increased alanine production has been

previously stated to be an indication of poor energy usage by CHO cells (Bonarius et al,

2001; Goudar et al, 2009; Lu et al, 2005), and may suggest altered metabolic flux for

3.90 cultures in response to LTC, resulting in poor energy usage for late generation

cultures. Poor energy usage for late generation cultures may account for lower antibody

titres for these cultures.

129

Figure 3.18 Analysis of amino acid utilisation during culture



Section 2.3.2). Supernatant samples, taken on days 5, 7, 9 and 13 of batch culture

(Section 2.3.2), and a medium control sample, were spiked with the internal standard

myristic acid d27 and lyophilised. Chemical derivatization was performed in two stages,

with methyloxyamine hydrochloride in pyridine, before the addition of MSTFA and

TMCS (Section 2.10.3.1). All samples were analysed using GC-MS analysis, within 24

hrs of derivatization. Raw data processing was performed using ChemStation and

AMDIS (Section 2.10.3.2). The relative percentage of asparagine (A), serine (B),

leucine (C) and lysine (D) are shown. All values were normalised to the internal

standard, myristic acid d27. Error bars represent SD for two biological replicates.


Early generation

Late generation

130

Figure 3.18 Analysis of amino acid utilisation during culture

A.

B.

D.

C.

0

1

2

3

4

5

6

Medium 5 7 9 13

Rela

tiv

e a

spa

ra

gin

e

(%)

Day

0

5

10

15

20

25

30

35

Medium 5 7 9 13

Rela

tiv

e s

erin

e

(%)

Day

0

5

10

15

20

Medium 5 7 9 13

Rela

tiv

e ly

sin

e

(%)

Day

0

20

40

60

80

Medium 5 7 9 13

Rela

tiv

e leu

cin

e

(%)

Day

131

Figure 3.19 Effect of culture generation time on metabolite accumulation




(Section 2.3.2), and a medium control sample, were lyophilised, and derivatized as

stated in Figure legend 3.18 and Section 2.10.3. All samples were analysed using GC-

MS analysis, within 24 hrs of derivatization. Raw data processing was performed using

ChemStation and AMDIS (Section 2.10.3.2). The relative percentage of glycerol (A),

glycine (B), and alanine (C) are shown. All values were normalised to the internal

standard, myristic acid d27. Error bars represent SD for two biological replicates.


Early generation

Late generation

132

Figure 3.19 Effect of culture generation time on metabolite accumulation

A.

B.

C.

0

20

40

60

80

100

Medium 5 7 9 13

Rela

tiv

e g

lycero

l

(%)

Day

0

50

100

150

Medium 5 7 9 13

Rela

tiv

e g

lycin

e

(%)

Day

0

5

10

15

20

Medium 5 7 9 13

Rela

tiv

e a

lan

ine

(%)

Day

133

The TCA cycle has been described as the central regulator in cell energy homeostasis

and cell metabolism (Krebs 1970). The TCA cycle uses acetyl CoA, created from

pyruvate, as a result of glycolysis. Pyruvate can also act as a precursor for the

production of alanine and lactate. Concentrations of glucose were completely depleted

by day 13 of batch culture, with no change in glucose consumption between early and

late generation cultures (Figure 3.20A). As the cell utilised glucose, lactate was created

and released from the cell. For early generation cultures lactate reached a maximum

concentration on day 9 of batch culture, after which lactate was re-utilised by the cell, as

glucose concentrations were depleted (Figure 3.20B). For late generation cultures

lactate was greatest on day 7 of batch culture, after which lactate was re-utilised. On

days 9 and 13 of batch culture lactate concentrations were lower for late generation

cultures than early generation cultures, possibly due to increased rates of lactate re-

utilisation.

To confirm and fully quantitate rates of glucose and lactate utilisation, glucose and

lactate concentrations were determined enzymatically. Glucose consumption determined

enzymatically mirrored the glucose consumption seen during batch culture using GC-

MS analysis (Figure 3.21A). Although glucose concentration was similar for both early

and late generation batch cultures late generation cultures had a higher rate of glucose

utilisation than early generation cultures (Figure 3.21B) due to lower CCT values as a

result of LTC. The rate of glucose metabolism possibly influenced lactate production

(Figure 3.21C). Late generation cultures re-utilised lactate at a faster rate than early

generation cultures during the end (decline) phase of batch culture (Figure 3.21D).

Potentially due to inefficient glucose metabolism during LTC, late generation cultures

utilised glucose at a faster rate then switched to using lactate as an energy source. The

metabolic changes between early and late generation cultures may have influenced

antibody titre loss as a result of LTC.

134

Figure 3.20 Analysis of glucose and lactate concentrations during culture




(Section 2.3.2), and a medium control sample, were lyophilised, and derivatized as

stated in Figure legend 3.18 and Section 2.10.3. All samples were analysed using GC-

MS analysis, within 24 hrs of derivatization. Raw data processing was performed using

ChemStation and AMDIS (Section 2.10.3.2). A, shows the relative percentage of

glucose, and B, shows the relative percentage of lactate. All values were normalised to

the internal standard, myristic acid d27. Error bars represent SD for two biological

replicates.


A.

B.

0

20

40

60

80

100

Medium 5 7 9 13

Rela

tiv

e g

luco

se

(%)

Day

0

50

100

150

200

250

300

350

Medium 5 7 9 13

Rela

tiv

e la

cta

te

(%)

Day

Early generation

Late generation

135

Figure 3.21 Investigating rates of glucose and lactate utilisation during culture



Section 2.3.2). Supernatant samples taken during batch culture (Section 2.3.2), and a

medium control sample, were analysed for glucose and lactate using enzymatic assays.

Glucose was converted to glucoeimine in a two-step reaction, measured

spectrophotometrically at 505nm (as described in Section 2.10.1). Glucose standards

were also analysed. The concentration of lactate in samples was determined based on

the catalysis of pyruvate by LDH with the reduction of NAD+. The reduction of NAD

+ to

NADH was measured spectrophometrically at 340nm (Section 2.10.2). Lactate

standards were also analysed. The concentrations of glucose (A) and lactate (C) for

early and late generation batch cultures are shown. The rate of utilisation was

calculated using the CCT values in Figure 3.2B (Section 2.11.2). The rates of glucose

(B) and lactate (D) utilisation for early and late generation batch cultures are also

shown. Error bars represent SEM for three biological replicates. ♦ indicates p<0.10,

using independent samples t-test to compare late generation cultures to early

generation cultures.


Early generation

Late generation

136

Figure 3.21 Investigating rates of glucose and lactate utilisation during culture

A.

B.

D.

C.

0

10

20

30

40

50

Medium 5 7 9 13

Glu

co

se c

on

cen

tra

tio

n

(mM

)

Day

0

10

20

30

40

50

60

Medium 5 7 9 13

La

cta

te c

on

cen

tra

tio

n

(mM

)

Day

0

0.5

1

1.5

2

Ra

te o

f la

cta

te u

tili

sati

on

(dd

9-d

13

)

(pM

/cell

/da

y)

0

0.2

0.4

0.6

0.8

1

Ra

te o

f g

luco

se u

tili

sati

on

(d0

-d1

3)

(pM

/cell

/da

y)

♦

137

3.6 DISCUSSION

Results show that recombinant CHO cell line 3.90 was unstable in response to LTC.

Instability was characterised with a 40% decrease in final antibody titre and a 30%

decrease in Qp (d0-d15) between early generation (generations 20 and 40) and late

generation cultures (generations 60, 80 and 100, Section 3.2). Any insight into the

reason for instability provides valuable information, potentially presenting

characteristics of an optimal cell line (which could be chosen at the screening stage),

and/or highlighting markers of instability that could be used to exclude cell lines before

proceeding with extensive characterisation studies.

During the initial stages of batch culture there was no evidence of instability in response

to LTC. Antibody titre values, for all generations, were similar until day 7 of batch

culture (Figure 3.1A), with no change in viable cell densities (Figure 3.1B), CCT

(Figure 3.2B), cell cycle distribution (Figure 3.3), Qp (Figure 3.4) or recombinant

mRNA expression (Figure 3.8) between early and late generation cultures during the

exponential phase of batch culture. Differences in antibody titre between early and late

generation cultures only became significant from day 11 of batch culture (Figure 3.1A).

The main questions from the characterisation investigations were why did late

generation cultures express less antibody than early generation cultures, and why did the

decline in antibody titre only occur during the end phase of batch culture.

The loss in antibody titre in response to LTC was accompanied with lower viable cell

densities. Late generation cultures had lower viable cell densities (Figure 3.1A) and

CCT values (Figure 3.2B) than early generation cultures during the end (decline) phase

of batch culture. As CCT is determined by the rates of cell growth and cell death, with

the balance between these two fates dependent on the rate of cell division (Lloyd & Al-

Rubeai, 1999), it was not surprising that cell cycle distribution was also altered in

response to LTC (Figure 3.3). By day 11 of batch culture early generation cultures had

significantly higher antibody titres and a greater proportion of cells in G0/G1. A trend

which has been previously reported for other CHO and hybridoma cells (Al-Rubeai &

Emery, 1990; Dutton et al, 2006; Kromenaker & Srienc, 1991). However, this trend was

only observed during the end (decline) phase of batch culture. Figure 3.22 shows that

there was no correlation between antibody titre and proportion of cells in G0/G1 on

138

days 3 and 7 of batch culture. The figure also highlights the increase in the percentage

of cells in G0/G1 as batch culture progresses. The accumulation of late generation cells

in G0/G1 halts on day 7 of batch culture (Figure 3.22B), whilst the accumulation of

early generation cells in G0/G1 continues, until at least day 11 of batch culture (Figure

3.22C). It is highly possible that the changes in cell cycle distributions were due to the

factors altering the cell biomass between early and late generation cultures (Lodish et al,

2004; Morgan, 2007).

The decrease in antibody titres values in response to LTC was not solely due to

alterations in cell biomass as Qp values were also affected (Figure 3.4). However, the

changes in cell biomass and antibody titre in response to LTC were potentially linked to

similar mechanisms. A decrease in viable cells suggests late generation cultures were

experiencing enhanced cell death compared to early generation cultures. Increased cell

death could be due to stimuli involved in receptor-mediated, mitochondrial, or ER-stress

mediated apoptosis (reviewed in Arden & Betenbaugh, 2006), or due to a loss of

protective factors, such as nutrients. Apoptosis as a result of ER stress occurs due to the

accumulation of mis/unfolded proteins in the ER. Late generation cultures may have

experienced changes to its cell biomass due to the accumulation of more mis/unfolded

proteins in the ER, resulting in an enhanced UPR. The UPR pathway is initially

activated to lower ER stress by preventing global protein translation (Harding et al,

2002; Harding et al, 1999), with the exception of ATF4 (Harding et al, 2000a; Lu et al,

2004). Although the expression of ATF4 (mRNA and protein expression) was not

dependent on generation time (Figure 3.13A and Figure 3.14A), the expression of

ATF4‟s downstream targets, GADD34 (Ma & Hendershot, 2003) and GADD153

(Harding et al, 2000a) were altered in response to LTC, resulting in greater GADD153

mRNA and less GADD34 mRNA for late generation cultures (Figure 3.13B). As

GADD34 is required for feedback inhibition of the UPR, mediating dephosphorylation

of eIF2α (Novoa et al, 2001), decreased GADD34 mRNA for late generation cultures

(Figure 3.13B) potentially suggests less feedback inhibition on the UPR pathway, which

could result in enhanced ER stress for late generation cultures. Protein expression of

GADD34 needs to be examined but initial protein investigations found eIF2α

phosphorylation was not altered in response to LTC (data not shown). Studies

examining eIF2α phosphorylation found that addition of tunicamycin, a known ER

stress inducer, did not further enhance eIF2α phosphorylation within this cell line (data

139

not shown). These findings suggest that phosphorylation of eIF2α was possibly elevated

by the process of recombinant protein production, and it may have taken sufficient

GADD34 protein to alter the feedback pathway. Analysis of GADD34 protein would be

needed to determine if expression was altered in response to LTC.

Less feedback inhibition of the UPR would allow the up-regulation of proteins to either

assist in protein folding or, during excessive stress, commit the cell to cell death

(McCullough et al, 2001; Yoshida et al, 2003). The mRNA expression of such proteins,

XBP-1(s) (Figure 3.17C) and GADD153 (Figure 3.13C), were increased for late

generation cultures, during the late exponential and stationary phase of batch culture. As

no change in ATF4 protein was observed between early and late generation cultures

(Figure 3.14) regulation of GADD153 mRNA was possibly under the control of other

ER stress proteins, such as ATF6 and XBP-1 (Oyadomari & Mori, 2003). Protein

analysis of ATF6 and XBP-1 may have shown altered regulation between early and late

generation culture, which could have further supported the suggestion that late

generation cultures were potentially experiencing prolonged, and enhanced ER stress.

Unfortunately there is a lack of commercially available good quality antibodies to detect

ATF6 and spliced XBP-1 proteins.

As previously mentioned in Section 1.5 protein production, folding and secretion are

energy dependent processes (highlighted in Figure 1.7). Any impact on the cells ability

to provide ATP will adversely impact these processes (Scriven et al, 2007; Simone &

Roberto, 2007). Previously findings have linked glucose concentrations to ATP

production in CHO cultures (Lu et al, 2005). Glucose was used rapidly by 3.90, for both

early and late generation cultures (Figure 3.20A and 3.21A). I suggest lactate, produced

as a by-product of glycolysis, was re-utilised by 3.90 cultures upon glucose depletion

(Figure 3.20B and Figure 3.21C). Lactate can be exploited by cells as it can be

converted back to pyruvate and used within the TCA cycle. The re-utilisation of lactate

has also been highlighted for other CHO cultures (Altamirano et al, 2009; Altamirano et

al, 2006; Ma et al, 2009; Tsao et al, 2005). Another glycolysis by-product which also

appeared to be re-utilised during the decline phase of batch culture was alanine. Alanine

is produced from the reversible transamination of glutamate and pyruvate. Low

extracellular glutamate concentrations were determined using GC-MS but

140

concentrations were similar for both early and late generation cultures (data not shown).

Although re-utilisation of alanine was apparent for both early and late generation

cultures during the decline phase of culture, differences in extracellular alanine

concentration was observed on day 5 and 7 of culture between early and late generation

cultures (Figure 3.19C). The increased alanine production could be an indication of

poor energy usage of late 3.90 cultures (particularly on day 5 and 7 of batch culture,

Bonarius et al, 2001; Goudar et al, 2009; Lu et al, 2005). Enhanced alanine production

may be a potential marker of lower protein production within this cell line.

Intracellular metabolite analysis would have been useful to identify intracellular

changes in metabolism. However, investigating intracellular metabolites has limitations,

including medium contamination, and the requirement for intact viable cells, ideally

from the exponential or stationary phase of culture. Unfortunately during these phases

no significant difference in antibody titre was observed between early and late

generation cultures (Figure 3.1A).

I propose that in response to LTC the metabolic profiles of 3.90 were altered, resulting

in lower antibody titres for late generation cultures. I suggest the equilibrium of

pyruvate conversion favours by-product formation, with less pyruvate available for

acetyl CoA conversion. The proposed alterations in metabolic pathways in response to

LTC are shown in Figure 3.23. These changes would limit flux through the glycolytic

pathway, and TCA cycle, ultimately producing less intermediates for ATP production.

Intracellular concentrations of ATP and metabolic co-enzymes, NAD+ and NADH, are

discussed in the next paragraph.

Initial investigations highlighted alterations for intracellular ATP, NAD+ and NADH

between early and late generation cultures (Figure 3.24). During exponential phase of

batch culture ATP, NAD+ and NADH were greater for late generation cultures than

early generation cultures, however, all concentrations were similar to CHO ATP, NAD+

and NADH concentrations reported by Sellick, et al. (Sellick et al, 2008). Enhanced

NADH for late generation cultures during the exponential phase of batch culture may

have favoured increased lactate and NAD+ production by LDH. By the stationary phase

of batch culture late generation cultures had less NAD+ and NADH, potentially

indicating the requirement for enhanced recycling of NAD/NADH (using re-utilised

141

lactate as a substrate) to maintain ATP production. Less NAD+

may also be related to

the stress status of the cell and nutrient availability (Bordone et al, 2006; Imai, 2009;

Yang & Sauve, 2006). The ratio of NAD+ and NADH provides information on the state

of a cell in the form of the catabolic reduction charge (crc), which is defined as

[NADH]/([NAD+]+[NADH]). In growing cells the crc is always low, as NAD

+, in the

oxidized form provides an oxidizing power for catabolism. On day 5 of batch culture

the crc charge for early and late generation cultures was between 0.03-0.04, consistent

with growing cells (Sellick et al, 2008). However by day 9 of batch culture early

generation cultures had a crc of 0.017 whilst late generation cultures had a crc of 0.001.

The difference of crc provides another indication of metabolic changes to 3.90 cultures

in response to LTC.

Maintaining redox homeostasis is necessary for a high producing cell line. The

production of secretory proteins, rich in disulphide bonds, can result in oxidative stress

(Cenci & Sitia, 2007; Harding et al, 2003). Disulphide bonds are required for correct

protein folding, and require the oxidation and reduction of ERO1α and PDI. If redox

intermediates become limiting (potentially due to altered glycolytic and TCA flux)

reduced proteins would accumulate in the ER (Masciarelli & Sitia, 2008). These

reduced, unfolded proteins would result in an UPR and eventual degradation.

Fluorescent protein reporters have been used to measure ER redox status and UPR

activity in single yeast cells (Kang & Hegde, 2008; Merksamer et al, 2008). The

methods could potentially be used within mammalian cells to determine the real-time

ER redox status during culture.

Increased protein degradation may also reflect the metabolic state of the cell (Vabulas &

Hartl, 2005). Increased ERAD for late generation cultures could be a possible

explanation for the loss in antibody titre observed in response to LTC. However, ERAD

is also an ATP-dependent mechanism (Goldberg, 2003; Meusser et al, 2005). Enhanced

degradation may maintain the amino acid pool (which undergoes a net-loss during

protein secretion), but it would deplete the cellular stocks of ATP, and consequently

limit protein translation. The association of monosomes and dissociation of polysomes

were observed during batch culture (Figure 3.9), consistent with attenuation of an early

step in translation (Prostko et al, 1993). Late generation cultures also exhibited greater

polysome dissociation than early generation cultures (Figure 3.10), suggesting late

142

generation cultures had lower rates of translation than early generation cultures

(Harding et al, 2000b), and although the relative concentrations of intracellular

recombinant protein were similar between early and late generation cultures (Figure

3.11A and 3.12) these assays were unable to detect the recombinant protein in a fully-

folded conformation.

From the investigations it was clear that ER stress markers and nutrient utilisation were

altered in response to LTC, ultimately resulting in lower antibody titres and CCT values

for late generation cultures. The alterations in response to LTC are summarised in

Figure 3.25. It may be possible that two different situations are influencing antibody

titre stability in response to LTC (Figure 3.1A). One explanation could be the possibility

that older cells (late generation cells) have a lower resistance to ER stress (Li &

Holbrook, 2004), resulting in an enhanced UPR, observed with increased expression of

GADD153 mRNA (Figure 3.13C) and XBP-1(s) mRNA (Figure 3.17C), and decreased

antibody titre values. However, if the age of cells were the key determinant of instability

why was antibody titre loss between early and late generation cultures only limited to

the end (decline) phase of batch culture. During the decline phase of batch culture

asparagine (Figure 3.18A), serine (Figure 3.18B), and glucose (Figure 3.20A and Figure

3.21A) were depleted from cultures. Nutrient availability and usage during batch culture

may be the key determining factor in antibody stability. I have previously suggested that

in response to LTC the metabolic profile of 3.90 becomes altered, observed with

increased rates of glucose and lactate utilisation for late generation cultures (Figure

3.21). Greater rates of glucose and lactate utilisation may be needed to continue flux

through the glycolytic pathway and TCA cycle in order to maintain production of ATP.

Any limitations in the production of ATP, and redox intermediates, could potentially

inhibit protein production and protein folding, resulting in lower antibody titres and

increased ER stress, both observed for late generation cultures. Any protein unable to

fold correctly would eventually experience ERAD, and cultures unable to rectify the ER

stress would instead commit to apoptosis (Merksamer & Papa, 2010), resulting in lower

antibody titres and viable cell densities, also observed for late generation cultures.

To determine if metabolic profiles of cultures were altered in response to LTC the flux

of CHO culture could be examined using 13

C labelled glucose addition and MS/NMR

spectroscopy. Labelled glucose has been used to assess fluxes, but there are

143

disadvantages in determining flux through the TCA cycle especially as most 13

C atoms

tend to be released in the form of lactate and alanine (Goudar et al, 2009; Omasa et al,

2010; Quek et al, 2010). Although this approach could be undertaken to determine the

metabolic flux of 3.90 cultures the limitations have to be addressed.

3.7 SUMMARY

Characterisation studies showed 3.90 to be unstable in response to LTC, observed with

a 40% decrease in final antibody titre and a 30% decrease in Qp values. I propose the

instability observed for 3.90 was due to metabolic changes as a result of LTC.

Metabolic flux alterations, seen with enhanced alanine accumulation, and increased

rates of lactate utilisation for late generation cultures, potentially resulted in less energy

and intermediates needed to maintain high levels of recombinant protein production,

folding and secretion for late generation cultures. Less metabolic intermediates as a

consequence of changes to metabolic profiles possibly limited protein folding, resulting

in stressed cultures. An increase in the mRNA expression of UPR markers, GADD153

and XBP-1(s), was also observed as a consequence of LTC. These characteristics of ER

stress were not absent from early generation cultures, but they were often delayed, or

expressed at lower levels, compared to that seen for late generation cultures. These

stress markers may also limit antibody production and secretion for early generation

cultures.

144

Figure 3.22 Correlation between antibody titre and proportion of cells in G0/G1

3.90 was cultured as previously described (Figure legend 3.1). On days 3, 7 and 11 of

batch culture supernatant samples were analysed by ELISA (Section 2.5.1) for

determination of antibody titre. Fixed cells on the same days of batch culture were

analysed by flow cytometry using PI excitation for determination of G0/G1 cell cycle

phase transition (as described in Section 2.4.1). Antibody titre values are shown

together with the percentage cells in G0/G1 for days 3 (A), 7 (B) and 11 (C) of batch

culture. Error bars represent SEM for three biological replicates.


20

40

60

80

100

145

Figure 3.22 Correlation between antibody titre and proportion of cells in G0/G1

A.

B.

C.

0

200

400

600

800

1000

40 50 60 70 80 90

An

tib

od

y t

itre

(mg

/L)

G0/G1 cell cycle phase

(%)

0

200

400

600

800

1000

40 50 60 70 80 90

An

tib

od

y t

itre

(mg

/L)


(%)

0

200

400

600

800

1000

40 50 60 70 80 90

An

tib

od

y t

itre

(mg

/L)


(%)

146

Figure 3.23 Potential metabolic changes in response to LTC

A basic metabolic profile showing flux through the glycolytic pathway and the TCA

cycle. Red arrows indicate the potential metabolic changes in response to LTC. I

suggest that as a result of LTC the metabolic equilibrium is altered in favour of alanine

and lactate production, limiting metabolic flux through the TCA cycle. This process

would ultimately result in less intracellular ATP intermediates needed for protein

production and protein folding.


Glyceraldehyde-3-phosphate

Acetyl CoA

Pyruvate

Oxaloacetate

TCA Cycle

Lactate Lactate

Alanine

Serine

Glycine

Asparagine

Glycolysis

Glycine

Succinyl

CoA

Alanine

Asparagine

Oxidative

Phosphorylation

NADH

NAD+

O2

ADP

ATP

NADH

NAD+

ADP

ATP

ATP

ADP

Mitochondria

Cytosol

147

Figure 3.24 Investigating ATP, NAD+ and NADH concentrations for 3.90 cultures



Section 2.3.2). 1x107 cells, taken from days 5 and 9 of batch culture (Section 2.3.2),

were quenched in 60% methanol with 0.85% (w/v) AMBIC (at -40 °C). The metabolites

were extracted by resuspension of the cell pellet in 100% methanol followed by flash

freezing in liquid nitrogen (Section 2.10.4). Once the samples were thawed and re-

extracted the methanol extracts were pooled and lyophilised. Dried metabolite extracts

were resuspended in ddH20 for use in each of the metabolite assays. ATP assays were

performed using an ATP Bioluminescence Assay Kit CLS II (Section 2.10.5) and

NAD+/NADH assays were performed using an NAD

+/NADH Quantification Kit (Section

2.10.6). Both kits were utilised according to manufacturer’s instructions.


Early generation

Late generation

148

Figure 3.24 Investigating ATP, NAD+ and NADH concentrations for 3.90 cultures

A.

B.

C.

0

2

4

6

8

10

5 9

AT

P c

on

cen

tra

tio

n (

mM

)

Day

0.00

0.05

0.10

0.15

0.20

0.25

5 9

NA

D+

co

ncen

tra

tio

n (

mM

)

Day

0.000

0.002

0.004

0.006

0.008

0.010

5 9

NA

DH

co

ncen

tra

tio

n (

mM

)

Day

149

Figure 3.25 Alterations to nutrient utilisation, UPR stress markers, cell biomass

and antibody titre in response to LTC

Characteristics of antibody titre loss were examined in response to LTC. The changes in

these markers were possibly related to the decrease in CCT and antibody titre values

seen for late generation cultures during the end (decline) phase of batch culture.

indicates a significant change for late generation cultures compared to generation 20

cultures (p<0.10 using independent samples t-test).


Day 0 3 5 7 9 11 13 15

Antibody titre

CCT

Percentage of cells in

G0/G1

GADD153 mRNA

GADD34 mRNA

XBP-1 (s) mRNA

Rates of glucose and lactate utilisation

Polysome peak area

*

*

*

**

*

Alanine accumulation

Late generation cultures

Generation 60

Generation 80

Generation 100

Increased values

Decreased values

150

6

CHAPTER 4

7


LINE 3.90 IN RESPONSE TO

FEED ADDITION

151

4. CHARACTERISATION OF CELL LINE 3.90 IN REPSONSE TO FEED

ADDITION


Cell line 3.90 was initially characterised in Chapter 3. Instability was observed between

early generation and late generation cultures, characterised with a 40% loss in final

antibody titre and a 30% decrease in Qp (d0-d15, Section 3.2.1). Characterisation

studies suggested that decreased antibody titre was due to changes in the metabolic flux

profiles of cultures in response to LTC, which potentially impacted protein production

and secretion from the cell.

In this Chapter a CD feed was added to cultures with the aim to further enhance

antibody titre and to identify factors involved in improving antibody titre, whilst

maintaining stable protein production during LTC. Throughout the investigations the

term early generation cultures refers to batch cultures created at ≤ 40 generations and

the term late generation cultures refers to batch cultures created at ≥ 60 generations. In

this Chapter the batch cultures defined as the control cultures with no addition, are the

cultures previously characterised in Chapter 3, and are used as a control within this

Chapter to determine the effects of feed addition on cell line 3.90.


CELL LINE 3.90 IN RESPONSE TO FEED ADDITION

Batch cultures were created at generations 20, 40, 60, 80 and 100. A CD feed (2% [v/v])

was added to cultures during the exponential phase of growth (days 3-7 of batch

culture). Supernatant samples were taken routinely throughout batch culture and

analysed for antibody titre. During the exponential stage of batch culture, antibody titres

for cultures with feed addition (Figure 4.1A) were similar to antibody titres for cultures

without feed addition (Figure 3.1A). The increase in antibody titres in response to feed

addition was only observed from the stationary phase of culture. Feeding enhanced final

antibody titres for all generation cultures by approximately 300 mg/ml. Although final

antibody titres were increased in response to feed addition, cultures supplemented with

152

feed still experienced instability in response to LTC, observed with a significant 30%

decrease in final antibody titre between early and late generation cultures (Figure 4.1A).

Growth analysis was performed in parallel to ensure that changes in cell densities were

not responsible for the changes in antibody titre. The patterns of growth for cultures in

the presence of feed were similar at all stages of LTC (Figure 4.1B). At all generations,

cultures were in exponential phase until day 7 of batch culture. After day 7 cultures

entered, and remained in stationary phase until day 11, following which cultures entered

a decline phase. Viable cell densities were maximal on day 9 for all generation cultures.

Culture viability declined from day 11, and was ≤ 30% or below by day 15 of batch

culture. Viability measurements for batch cultures with feed addition (Figure 4.1C)

were similar to viability measurements for the control batch cultures without feed

addition (Figure 3.1C). Final CCT values for all batch cultures with feed addition were

between 50-60x106 cells x day/ml (Figure 4.2). These CCT findings were similar to the

CCT values determined for control cultures in the absence of feed addition (Figure

3.2B).

Although feeding increased final antibody titre without significantly altering viable cell

densities cell cycle phase distribution was analysed as previous findings have shown

CHO cells to accumulate in G0/G1 after nutrient addition (Sitton & Srienc, 2008).

Throughout fed batch culture the percentage of cells in G0/G1 phase increased (Figure

4.3A), the percentage of cells in S phase decreased (Figure 4.3B), and the percentage of

cells in G2/M phase remained relatively unchanged (Figure 4.3C). The percentage of

cells in G0/G1 was similar for both early generation cultures with or without feed

addition (Figure 4.4A). However, late generation day 11 cultures without feed addition

had a significantly lower percentage of cells in G0/G1 than day 11 cultures with feed

addition. An example is shown for generation 60 cultures showing the influence of feed

addition on the proportion of late generation cells in G0/G1 phase cells (Figure 4.4B).

As feed addition increased antibody titres (Figure 4.1A), without enhancing viable cell

densities (Figure 4.1B), Qp was calculated to determine the effect of feed addition on

antibody production rate, per cell, per day. Qp was calculated using viable cell densities

and antibody titre measurements at different stages of batch culture, as explained in

Section 3.2. In response to feed addition Qp (d0-d15) values for early and late

153

generation cultures were increased by approximately 5 pg/cell/day (Figure 4.5A).

Maximal Qp values were observed during the early (exponential) phase of batch culture.

Qp (d0-d7) values were similar for cultures regardless of feed addition (Figure 4.5B).

Although feed was added to cultures during the exponential phase of batch culture

feeding had the greatest impact on Qp during the end (decline) phase of batch culture.

At generations 20, 40 and 60 feed addition increased Qp (d9-d15) values by

approximately 20-40%, with a significant 65% increase in Qp (d9-d15) for cultures

created at generations 80 and 100 (Figure 4.5C). Qp (d9-d15) values for cultures

supplemented with feed addition decreased by approximately 30% in response to LTC

(Figure 4.5C). However despite the decline in Qp between early and late generation

cultures, Qp was still enhanced in response to feed addition.

It has been reported that some cell phenotypes, such as cell size and viable cell

densities, are altered by changes to medium osmolality (Kutz & Burg, 1998). Initial

analysis showed osmolality to increase from approximately 0.310 Osm/kg to 0.450

Osm/kg after feed addition (Appendix 3). Although viable cell densities of 3.90 cultures

were not affected by the addition of feed (Figure 4.1B), the average cell diameter for

both early and late generation cells was increased by 2 µm in response to feed addition

(Figure 4.5A). However, this increase was not statistically different.

154

Figure 4.1 Effect of feed addition on recombinant antibody titre, viable cell

densities and cell viability during batch culture

3.90 was subject to long-term culture in suspension using MSX supplemented CD-CHO

media. Batch growth analysis was performed in shake flasks at generation numbers 20,

40, 60, 80 and 100, ± 4 generations. Batch cultures were created at 0.2x106 cells/ml,

and maintained at 37oC, 140 rpm and with a manual supply of 5% CO2 in air. A CD

feed was added during the exponential stage of batch culture (as described in Section

2.3.2). Cells were cultured under these conditions until viability was ≤ 30%. Antibody

titres (A), viable cell densities (B), and cell viabilities (C) are shown. Antibody titre was

measured by ELISA (Section 2.5.1) and viable cell densities and cell viabilities were

determined by light microscopy and trypan blue exclusion (Section 2.3.3) from samples

taken routinely during batch culture. Error bars represent SEM for three biological


repeats. * indicates p<0.05, using independent samples t-test to compare cultures

created at generations 40, 60, 80 and 100 to generation 20 cultures (on the same day of

batch culture).


20

40

60

80

100

155

Figure 4.1 Effect of feed addition on recombinant antibody titre, viable cell

densities and cell viability during batch culture

B.

C.

A.

0

2

4

6

8

10

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

200

400

600

800

1000

1200

1400

1600

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

0

20

40

60

80

100

0 5 10 15

Percen

tag

e v

iab

ilit

y

(%)

Day

*** ***

156

Figure 4.2 Effect of feed addition on CCT

Viable cell densitites was determined using the trypan blue exclusion (as described in

Section 2.3.3). The CCT was determined from the growth of the batch cultures with feed

addition (Figure 4.1B). This figure shows the CCT for batch cultures created at

generations 20, 40, 60, 80 and 100. For calculation of CCT see Section 2.11.2. Error

bars represent SEM for three biological replicates. Each biological replicate value is

an average from duplicate technical repeats. ♦ indicates p<0.10, using independent

samples t-test to compare cultures created at generations 40, 60, 80 and 100 to

generation 20 cultures (on the same day of batch culture).


0

10

20

30

40

50

60

70

80

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

l)

Day

20

40

60

80

100

♦♦ ♦♦

157

Figure 4.3 Effect of feed addition on cell cycle phase distribution during batch

culture

3.90 was cultured as previously described (Figure legend 4.1). A CD feed was added

during the exponential stage of batch culture (as described in Section 2.3.2). 1x106

cells, taken on days 3, 5, 7, 9 and 11 of batch culture, were analysed by flow cytometry

using PI excitation by a 488 nm laser, and emission measured by a 613/20 nm bandpass

filter (Section 2.4.1). The data was analysed by Summit 4.3 and ModFit LT software.

The percentage of cells in A, G0/G1 phase, B, S phase, and C, G2/M phase are shown.

Error bars represent SEM for three biological replicates.


20

40

60

80

100

158

Figure 4.3 Effect of feed addition on cell cycle phase distribution during batch

culture

0

20

40

60

80

100

3 5 7 9 11

S c

ell

cy

cle

ph

ase

(%)

Day

0

20

40

60

80

100

3 5 7 9 11

G2

/M c

ell

cy

cle

ph

ase

(%)

Day

B.

C.

A.

0

20

40

60

80

100

3 5 7 9 11

G0

/G1

cell

cy

cle

ph

ase

(%)

Day

159

Figure 4.4 The percentage of cells in G0/G1 cell cycle phase for cultures with and

without feed addition

3.90 was cultured as previously described (Figure legend 3.1 and Figure legend 4.1). A

CD feed was added to the relevant cultures during the exponential stage of batch

culture (as described in Section 2.3.2). 1x106 cells, taken from days 3, 5, 7, 9 and 11 of

batch culture, were analysed by flow cytometry using PI excitation by a 488 nm laser,

and emission measured by a 613/20 nm bandpass filter (Section 2.4.1). The data was

analysed by Summit 4.3 and ModFit LT software. A, shows the percentage of generation

20 cells in G0/G1 phase, and B, shows the percentage of generation 60 cells in G0/G1

phase. Error bars represent SEM for three biological replicates. * indicates p<0.05,

and ♦ indicates p<0.10, using independent samples t-test to compare cultures with feed

addition to the corresponding control culture with no addition (on the same day of

batch culture).


B.

A.

0

20

40

60

80

100

3 5 7 9 11

Gen

era

tio

n 2

0

G0

/G1

cell

cy

cle

ph

ase

(%)

Day

0

20

40

60

80

100

3 5 7 9 11

Gen

era

tio

n 6

0

G0

/G1

cell

cy

cle

ph

ase

(%)

Day

Control (no addition)

+ feed addition

* ♦

160

Figure 4.5 Effect of feed addition on specific productivity

Specific productivity (Qp) was determined from the antibody titre and the growth of

control batch cultures (with no addition) shown in Figure 3.1A and Figure 3.1B,

respectively, and from the antibody titre and the growth of batch cultures with feed

addition, shown in Figure. 4.1A and Figure 4.1B, respectively. Qp was calculated for

the entire batch culture, using antibody titre and cell density measurements from days 0

to 15 of culture, Qp (d0-d15). Qp was also calculated for the early (exponential) phase

of batch culture, using antibody titre and cell density measurements from days 0 to 7 of

culture, Qp (d0-d7), and for the end (decline) phase of batch culture, using antibody

titre and cell density measurements from days 9 to 15 of culture, Qp (d9-d15). For

determination of Qp see Section 2.11.2. Qp (d0-d15) (A), Qp (d0-d7) (B), and Qp (d9-

d15) (C) are shown. Error bars represent SEM for three biological replicates. Each

biological replicate value is an average from duplicate technical repeats. * indicates

p<0.05, and ♦ indicates p<0.10, using independent samples t-test to compare cultures

with feed addition to the corresponding control culture with no addition.



+ feed addition

161

Figure 4.5 Effect of feed addition on specific productivity

0

10

20

30

40

50

20 40 60 80 100

Qp

(d0

-d7

)

(pg

/cell

/da

y)

Generation

0

5

10

15

20

25

20 40 60 80 100

Qp

(d9

-d1

5)

(pg

/cell

/da

y)

Generation

B.

C.

A.

0

5

10

15

20

25

20 40 60 80 100

Qp

(d0

-d1

5)

(pg

/cell

/da

y)

Generation

* *

* *

*

♦

♦

162

Figure 4.6 Effect of feed addition on cell diameter

3.90 was cultured as previously described (Figure legend 3.1 and Figure legend 4.1).

On day 9 of batch culture, early generation (≤ 40 generations) and late generation (≥

60 generations) cells, with and without feed addition, were prepared for cell counting

using trypan blue dye (as described in Section 2.3.3). 100 cells were counted and

measured for cell diameter using a Widefield Axiovision microscope, and using the

Axiovision software. The average cell diameter, for early and late generation cultures

in the presence and absence of feed, is shown. Error bars represent the SD for 100 cells

counted.

0

5

10

15

20

no addition + feed addition no addition + feed addition

Early Late

Cell

dia

mete

r

(µm

)

Culture condition/Generation

163


IN RESPONSE TO FEED ADDITION

Feed addition during batch culture increased antibody titres by approximately 25%,

producing on average 5 pg/cell/day more antibody than batch cultures in the absence of

feed (Section 3.2.1 and Section 4.2.1). However, cultures with feed addition still

experienced instability in response to LTC, presenting a 30% loss in final antibody titre

and a 25% decrease in Qp (d0-d15) for late generation cultures (Section 4.2.1). In the

work described in Section 4.3, antibody mRNA expression was investigated to

determine how feed affects productivity and instability relating to mRNA expression.

The work also examined polysome profiles and the prevalence of intracellular

recombinant protein in response to feed addition.

4.3.1 Analysis of recombinant gene mRNA expression from cultures with feed

addition

For cultures in the presence of feed, GS mRNA was increased by approximately 50%

from day 3 to day 9 of batch culture for all generations, with no difference in GS

mRNA expression between early and late generation cultures (Figure 4.7A). Heavy

chain mRNA increased two-fold from day 3 to day 9 of batch culture, for all generations

(Figure 4.7B). Light chain mRNA also increased during batch culture, but the fold

change was dependent on the culture generation (Figure 4.7C). Light chain mRNA

expression for generations 20, 40, and 60 fed cultures increased approximately four-

fold, whilst expression for generations 80 and 100 fed cultures increased two-fold from

day 3 to day 9 of culture, however, the changes in light chain mRNA expression

between early and late generation cultures were not significant. As antibody titre and Qp

values were enhanced in response to feed it was also important to compare the relative

recombinant mRNA expression for cultures with and without feed addition (shown in

Figure 4.8).

Although the relative GS mRNA expression was greater for cultures with feed addition

than for the control cultures, the increase in GS mRNA was not statistically significant

(Figure 4.8A). Expression of heavy chain mRNA (Figure 4.8B) and the light chain

mRNA (Figure 4.8C) was similar for all cultures, regardless of feed addition. As

164

cultures in the presence of feed had greater antibody titres and similar expression of

recombinant mRNA than cultures in the absence of feed addition, the cultures

supplemented with feed may have utilised the available recombinant transcripts in a

more productive manner.

4.3.2 Investigating characteristics of polysome profiles in response to feed addition

To investigate the possibility that the mRNA could be utilised more efficiently

polysome profiles were investigated. For both early (Figure 4.9A) and late (Figure

4.9B) generation cultures, feed addition enhanced the polysome peak area, compared to

polysome peak area from profiles for cultures without feed addition (previously shown

in Figure 3.9). To quantify monosome and polysome peak areas, polysome profiles in

response to feed addition were analysed using Image J software and the resultant data

are shown in Figure 4.10.

The quantified 40S peak area (Figure 4.10A), 60S peak area (Figure 4.10B), 80S peak

area (Figure 4.10C) all increased slightly in response to feed addition for both early and

late generation cultures. The polysome peak area also increased in response to feed

addition. (Figure 4.10D). In the presence of feed the polysome peak area increased

approximately two-fold for early generation cultures and a significant three-fold for late

generation cultures compared to the polysome peak area for the respective generation

cultures in the absence of feed. Harding et al, have shown polysome dissociation to

occur in murine embryonic stem cells as a result of ER stress, these cultures also had

decreased translational ability (Harding et al, 2000b). The increase in polysome peak

area for cultures with feed addition indicated potentially greater rates of translation for

these cultures, a feature that may be consistent with cells experiencing less cell stress.

4.3.3 Analysis of intracellular recombinant protein in response to feed addition

Analysis of the intracellular protein within cells may give an indication of increased

protein translation. L-[4,5-3H] leucine incorporation, previously used to measure global

protein synthesis in Section 3.2.2, was not a chosen method for analysis due to unknown

concentrations of leucine within the CD feed. To investigate intracellular protein levels

in response to feeding, antibody-conjugated dyes, which specifically detect both the

165

recombinant heavy and light chain proteins were used in the immunofluorescent assays.

Both early and late generation cultures had less intracellular heavy chain and light chain

protein after feeding (Figure 4.11). Although intracellular recombinant protein levels

were lower in response to feed addition this was not necessarily indicative of protein

synthesis rates. Lower levels of intracellular recombinant protein could be the result of

enhanced rates of protein secretion in response to feed addition. Protein secretion would

be enhanced if feeding allowed a greater concentration of correctly-folded proteins that

match the fidelity required for protein secretion. More fully-folded proteins and

potentially less mis/unfolded proteins would also result in less ER stress. To determine

if feed addition was affecting the cellular stress status UPR markers were examined.

These data are described in the next Section.

166

Figure 4.7 Effect of feed addition on recombinant mRNA expression


during the exponential stage of batch culture (as described in Section 2.3.2). mRNA

levels were compared using q-RTPCR from samples taken on days 3, 5, 7 and 9 of batch

culture (as detailed in Section 2.7.1), using the mRNA specific primer sets for A, GS, B,

heavy chain, and C, light chain. Samples were normalised using mRNA β-Actin primers.



20

40

60

80

100

167

Figure 4.7 Effect of feed addition on recombinant mRNA expression

A.

B.

C.

0

50

100

150

200

3 5 7 9

Hea

vy

ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A ex

press

ion

)

Day

0

50

100

150

200

250

3 5 7 9

Lig

ht ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A ex

press

ion

)

Day

0

50

100

150

200

3 5 7 9

GS

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A ex

press

ion

)

Day

168

Figure 4.8 Analysis of recombinant mRNA expression between cultures with and


3.90 was cultured as previously described (Figure legend 3.1 and Figure legend 4.1). A

CD feed was added to the relevant cultures during the exponential stage of batch

culture (as described in Section 2.3.2). mRNA levels were compared using q-RTPCR

from samples taken on day 9 of batch culture (as detailed in Section 2.7.1), using the

mRNA specific primer sets for A, GS, B, heavy chain, and C, light chain. Samples were

normalised using mRNA β-Actin primers. Error bars represent SEM for three biological

replicates.


20

40

60

80

100

169

Figure 4.8 Analysis of recombinant mRNA expression between cultures with and


A.

B.

C.

0

50

100

150

200

250

300

no addition + feed addition

Lig

ht ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Culture condition

0

50

100

150

200

250

300

no addition + feed additionHea

vy

ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Culture condition

0

50

100

150

200


GS

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Culture condition

170

Figure 4.9 Investigating characteristics of polysome profiles in response to feed

addition

3.90 was cultured as previously described (Figure legends 3.1 and Figure legend 4.1)

Polysomes were extracted on day 7 of batch culture, for both early generation (≤ 40

generations) and late generation (≥ 60 generations) cultures, with and without feed

addition (Section 2.8.2). The extracts were analysed on a UV/Vis machine at 254nm

after sucrose gradient centrifugation, as described in Section 2.8.3. The peaks that

contain the small ribosomal subunit (40S), the large ribosomal subunit (60S), and both

subunits (80S) are indicated by arrows. The polysome peaks generated by 2, 3, 4 etc.

80S ribosomes on a single mRNA are also indicated by an arrow. Figure 4.9A,

represents a typical 3.90 early generation polysome profile, (i) shows an example of a

polysome profile from a control culture (no addition) and (ii) shows an example of a

polysome profile from a culture with feed addition. Figure 4.9B, represents a typical

3.90 late generation polysome profile, (i) shows an example of a polysome profile from

a control culture (no addition) and (ii) shows an example of a polysome profile from a

culture with feed addition.

171

Figure 4.9 Investigating characteristics of polysome profiles in response to feed

addition

Polysomes

60S

40S

80S

40S

80S

60S

Polysomes

40S

80S

60S

Polysomes

60S

40S

80S

Polysomes

A.

B.

(i)(ii)

(i)(ii)

Early Generation Culture

Late Generation Culture


Polysomes

172

Figure 4.10 Quantification of monosome and polysome peak areas

Polysomes were extracted and analysed as described in Figure 4.9 and Section 2.8, for

both early generation (≤ 40 generations), and late generation (≥ 60 generations)

cultures, with and without feed addition. The peaks that contain the small ribosomal

subunit (40S), the large ribosomal subunit (60S), and both subunits (80S), and the

polysome peaks were analysed using Image J software to provide a relative

representation of peak variation. The change in the 40S (A), 60S (B), 80S (C) and

polysome (D) peak areas between early and late generation day 7 polysome profiles are

shown. Error bars represent SEM for three biological replicates. * indicates p<0.05,

using independent samples t-test to compare cultures with feed addition to the

corresponding control culture with no addition.



+ feed addition

173

Figure 4.10 Quantification of monosome and polysome peak areas

A.

B.

C.

D.

0

20

40

60

80

Early Late

Rela

tiv

e 6

0S

pea

k a

rea

Generation

0

50

100

150

200

250

Early Late

Rela

tiv

e 8

0S

pea

k a

rea

Generation

0

20

40

60

80

100

120

Early Late

Rela

tiv

e p

oly

som

e

pea

k a

rea

Generation

0

10

20

30

40

Early Late

Rela

tiv

e 4

0S

pea

k a

rea

Generation

*

174

Figure 4.11 Analysis of intracellular heavy chain and light chain protein after feed

addition


Batch cultures created at early generations (≤ 40 generations) and late generations (≥

60 generations). A CD feed was added to the relevant cultures during the exponential

stage of batch culture (as described in Section 2.3.2). 2x106 fixed cells on day 9 of batch

culture were washed, blocked and incubated with 10µg goat anti-human IgG, Fcγ-APC

and 6µg goat anti-human lambda light chain-FITC (Section 2.4.2). The samples were

then analysed by a CyAn ADP flow cytometer, using the 488nm and infra-red excitation

lasers to excite the FITC and APC conjugates. Unstained 3.90 cells and stained

parental cells were also required for setting initial parameters with the CyAn-ADP flow

cytometer. The data was gated to select single cells, and was analysed by Summit 4.3

software. Error bars represent SEM for three biological replicates.


0

20

40

60

80


Early Late

Rela

tiv

e m

ea

n f

luo

resc

en

ce i

nte

nsi

ty

(co

mp

ared

to

th

e p

aren

tal

cell

lin

e)




175

4.4 DETERMINING THE REGULATION OF UPR MARKERS IN RESPONSE

TO FEED ADDITION

Feed addition to batch cultures increased antibody titres by approximately 25%, but did

not prevent antibody titre loss in response to LTC (Section 3.2 and Section 4.2).

Molecular characterisation studies showed that increased antibody titre and Qp in

response to feed addition were not due to changes in expression of recombinant

antibody transcript (Section 4.3.1). Increased polysome association (Section 4.3.2) gave

an indication of greater translational capacity for cultures in the presence of feed.

Analysis of intracellular recombinant protein showed that cultures had less intracellular

protein in response to feed addition, however, this is not an indicator of decreased

protein translation (Section 4.3.3). I suggested that cultures supplemented with feed had

less intracellular protein due to enhanced rates of protein secretion. Protein secretion

would be increased for cultures that had a greater concentration of proteins in their

correctly-folded conformation. More correctly-folded proteins, and potentially less

mis/unfolded proteins, would also result in less ER stress. The expression of UPR

markers were investigated to determine potential changes in response to feed addition.

As previously mentioned in Section 3.4 once mis/unfolded proteins are detected in the

ER, expression of ATF4, GADD34 and GADD153 are upregulated via the activation of

the PERK pathway. The mRNA expression of UPR markers were compared between

cultures with and without feed addition on day 9 of batch culture. The mRNA

expression of ATF4 (Figure 4.12A), GADD34 (Figure 4.12B) and GADD153 (Figure

4.12C) was approximately two to three-fold lower for cultures with feed addition

compared to expression measured for the control cultures. As the expression of

GADD34 and GADD153 mRNA were altered in response to LTC (Figure 3.13), the

expression of UPR markers were investigated between early and late generation cultures

in the presence of feed. These studies are discussed below.

Investigations found that the expression of ATF4 (Figure 4.13A) and GADD34 (Figure

4.13B) mRNA for fed cultures was not altered in response to LTC, but the expression of

GADD153 mRNA was dependent on the generation time of culture. GADD153 mRNA

expression was two-fold greater for late generation cultures than early generation

cultures (Figure 4.13C). This increase, however, was not statistically different.

176

In order to extend the observations made at the mRNA level, cellular ATF4 and

GADD153 protein were examined by western blot analysis. The relative ATF4 protein

expression on day 9 of batch culture was similar for cultures with or without feed

addition, with no difference in expression of ATF4 protein (Figure 4.14A) or

GADD153 protein (Figure 4.14B) between early and late generation cultures. However,

a significant six-fold decrease in GADD153 protein was observed in response to feed

addition, regardless of generation time of culture (Figure 4.14B). As GADD153 protein

was lowered in response to feed addition other UPR markers were also investigated.

Although the expression of PDI protein and BiP protein (ER chaperones) was not

affected by feeding (data not shown), spliced XBP-1 mRNA was significantly lowered

as a result of feed addition (Figure 4.15).

Feed addition, by an unknown mechanism, increased antibody titre whilst lowering cell

stress, shown with a lower expression of ATF4, GADD34, GADD153 and XBP-1(s)

mRNA and less GADD153 protein. The decreased expression of such UPR markers

was possibly the result of the passage of less mis/unfolded proteins in the ER. I propose

that after feed addition cultures were not subjected to nutrient depletion, so nutrients

were available for the maintenance of relevant polypeptides needed for correct antibody

formation, allowing for greater rates of protein secretion and higher antibody titres.

Metabolic investigations were undertaken to provide an insight into the differences in

productivity in response to feed addition (Section 4.5).

177

Figure 4.12 Effect of feed addition on the mRNA expression of ATF4, GADD34,

and GADD153

3.90 was cultured as previously described (Figure legends 3.1 and Figure legend 4.1).

A CD feed was added to the relevant cultures during the exponential stage of batch



mRNA specific primer sets for A, ATF4, B, GADD34, and C, GADD153. Samples were


replicates. * indicates p<0.05, and ♦ indicates p<0.10, using independent samples t-test

to compare cultures with feed addition to the corresponding generation with no

addition.


20

40

60

80

100

178

Figure 4.12 Effect of feed addition on the mRNA expression of ATF4, GADD34,

and GADD153

A.

B.

C.

0

50

100

150

200

250

300


GA

DD

15

3 m

RN

A e

xp

ress

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Culture condition

0

50

100

150

200

250

300

350

400


GA

DD

34

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Culture condition

0

50

100

150

200

250

300

350


AT

F4

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Culture condition

*

* * * * *

♦ ♦

♦ ♦

* ♦ ♦ ♦ ♦ ♦

179

Figure 4.13 Effects of LTC on the mRNA expression of UPR markers from

cultures supplemented with feed addition


during the exponential stage of batch culture (as described in Section 2.3.2). mRNA

levels were compared using q-RTPCR from samples taken on days 3, 5, 7, and 9 of

batch culture (as detailed in Section 2.7.1), using the mRNA specific primer sets for A,

ATF4, B, GADD34, and C, GADD153. Samples were normalised using mRNA β-Actin

primers. Error bars represent SEM for three biological replicates.


20

40

60

80

100

180

Figure 4.13 Effects of LTC on the mRNA expression of UPR markers from

cultures supplemented with feed addition

A.

B.

C.

0

50

100

3 5 7 9

GA

DD

15

3 m

RN

A e

xp

ress

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A ex

press

ion

)

Day

0

50

100

150

200

3 5 7 9

GA

DD

34

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A ex

press

ion

)

Day

0

50

100

150

200

3 5 7 9

AT

F4

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A ex

press

ion

)

Day

181

Figure 4.14 Analysis of ATF4 and GADD153 protein in response to feed addition




stage of batch culture (as described in Section 2.3.2). Protein was extracted on day 9 of

batch culture (detailed in Section 2.5.3.1). 60µg of protein was separated by SDS-

PAGE (Section 2.5.3.2), transferred (Section 2.5.3.3) and then detected using anti-

rabbit polyclonal ATF4 and GADD153 antibodies. Membranes were stripped and re-

probed using an anti-mouse pan ERK antibody (Section 2.5.3.4). Bands were analysed

using Image J software and calculated relative to ERK (Section 2.5.3.5). The relative

intensities of ATF4 (A) and GADD153 (B) are shown. Error bars represent SEM for

three biological replicates. * indicates p<0.05, using independent samples t-test to

compare cultures with feed addition to the corresponding control culture with no

addition.


A.

B.

0

50

100

150

Early Late

AT

F4

pro

tein

in

ten

sity

(rela

tiv

e t

o E

RK

sta

nd

ard

)

Generation

0

50

100

150

200

250

Early Late

GA

DD

15

3 p

ro

tein

in

ten

sity

(rela

tiv

e to

ER

K s

tan

da

rd

)

Generation


+ feed addition

* *

182

Figure 4.15 Analysis of XBP-1 mRNA splicing in response to feed addition




stage of batch culture (as described in Section 2.3.2). cDNA was synthesised from RNA

extracted on day 9 of batch culture (Section 2.7.1.3) and the PCR was performed using

the newly synthesised cDNA and the XBP-1(s) primers (Section 2.7.2). The PCR

products were electrophoresed on a 2% (w/v) agarose gel and visualised by UV

transillumination (Section 2.6.1.4). The product bands were analysed using Image J

software and the ratio of spliced XBP-1 mRNA to total XBP-1 mRNA is shown. Error

bars represent SEM for three biological replicates. * indicates p<0.05, using

independent samples t-test to compare cultures with feed addition to the corresponding

control culture with no addition.


0

10

20

30

40

50

Early Late

Ra

tio

of

spli

ced

XB

P-1

mR

NA

to t

ota

l X

BP

-1 m

RN

A

(%)

Generation


+ feed addition

*

*

183

4.5 METABOLIC ANALYSIS OF CELL LINE 3.90 IN RESPONSE TO FEED

ADDITION

Cultures with feed addition had enhanced final antibody titre and Qp values than

cultures without feed addition (Section 3.2 and Section 4.2). In order to obtain greater

antibody titres protein secretion must also have been increased. I suggest that antibody

titre and secretion were the result of greater protein folding as a result of feed addition.

Feeding possibly provided the cultures with the concentrations of nutrients needed for

the production of relevant antibody protein/chains required for complete folding and

effective post-translational modifications.

Fed cultures also experienced instability during LTC (Section 4.2.1), characterised by a

30% loss in final antibody titre and a 25% decrease in Qp (d0-d15). Metabolic

alterations were also considered as contributing factors to the apparent instability,

especially as alanine accumulation, and lactate utilisation rates were altered in response

to LTC (observed for control cultures in Section 3.5). Metabolite profiles of early

generation and late generation batch cultures have been generated to characterise

potential differences in nutrient usage in response to feed addition. These profiles are

compared to metabolic profiles from cultures without feed addition (previously

discussed in Section 3.5).

Asparagine, serine, and lysine were all previously shown to decrease during batch

culture in the absence of feed addition, with the complete depletion of asparagine by

day 9 of batch culture, and depletion of serine by day 13 of batch culture (Figure 3.18).

Extracellular asparagine concentrations were increased for cultures with feed addition

(due to the presence of asparagine within the feed). However, despite feed addition,

asparagine concentration declined during batch culture, and became limiting by day 13

of batch culture (Figure 4.16A). Serine (Figure 4.16B) and lysine (Figure 4.16C)

concentrations were also enhanced after feed addition, and were maintained at

concentrations similar to that of the medium sample during the entire batch culture.

Leucine concentrations were greatly increased in the presence of feed, with

concentrations two- to three-fold greater than the medium sample (Figure 4.17A). It was

also apparent that even in the presence of feed late generation cultures had lower leucine

concentrations by day 13 of batch culture, compared to the corresponding early

184

generation culture. This may suggest altered metabolic flux in response to LTC, with

late generation cultures experiencing greater rates of leucine utilisation despite feed

additions. Isoleucine and valine concentrations were also enhanced by feed addition,

and by day 13 of batch culture the concentrations of isoleucine (Figure 4.17B) and

valine (Figure 4.17C) were three- to four-fold greater than concentrations observed for

the medium sample, regardless of generation time of culture. The increased

concentrations of leucine, isoleucine and valine were the result of feed accumulation

during the exponential phase of culture and a lack of utilisation of these amino acids,

possibly as feeding supplies the cell with other more favourable amino acids to

metabolise. Glycerol, glycine and alanine also accumulated during batch culture.

However, the increase in glycerol, glycine and alanine concentrations were not a result

of feed addition as these metabolites were not present in the CD feed. Glycerol

accumulation for batch cultures with feed addition was similar to the glycerol

accumulation observed for batch cultures without feed addition (Figure 4.17D).

However, glycine and alanine concentrations were altered in cultures which had

undergone a feeding regime. Although glycine still increased fifty-fold during fed batch

culture the relative concentration of glycine on day 13 of culture was approximately half

the concentration analysed from the control cultures without feed addition (Figure

4.17E), and whilst relative alanine concentrations by day 13 of batch culture were

similar for all cultures (regardless of feed addition) addition of feed to cultures resulted

in a steady increase of alanine during batch culture, with no apparent re-utilisation of

alanine (which was observed for the control cultures in the absence of feed addition,

Figure 4.17F).

Glucose and lactate utilisation rates were also investigated in response to feed addition.

In the presence of feed glucose concentration was greatest on day 7 of batch culture (the

last day of feed addition) and was slightly utilised over the remainder of the batch

culture (Figure 4.18A). For cultures supplemented with feed glucose concentrations

(Figure 4.18A) and rates of glucose utilisation (Figure 4.18B) were similar between

early and late generation batch cultures. There was, however, a decrease in the rate of

glucose utilisation for cultures in the presence of feed compared to the relative control

culture. As glucose was not depleted from batch cultures in the presence of feed

addition, lactate re-utilisation was not required. Lactate accumulated during the entire

batch culture, and by day 13 of culture lactate concentrations were three-fold greater for

185

cultures with feed addition than for cultures without feed addition (Figure 4.18C). As

lactate was not re-utilised after feeding the rate of lactate utilisation/production was

significantly altered, observed with a „shift‟ towards lactate production in response to

feed addition (Figure 4.18D).

For cultures in the presence of feed addition the majority of nutrients were not limited,

glucose utilisation rates were decreased, lactate was not re-utilised, and less glycine and

alanine were released from the cell. These adaptations may have generated a more

energy-efficient cell, allowing for greater antibody titres in a fed environment.

186

Figure 4.16 Effects of feed addition on amino acid concentrations


Batch cultures were created at early generations (≤ 40 generations) and late

generations (≥ 60 generations). A CD feed was added to the relevant cultures during

the exponential stage of batch culture (as described in Section 2.3.2). Supernatant

samples, taken on days 5, 7, 9 and 13 of batch culture (Section 2.3.2), and a medium

control sample, were spiked with the internal standard myristic acid d27 and lyophilised.

Chemical derivatization was performed in two stages, with methyloxyamine

hydrochloride in pyridine, before the addition of MSTFA and TMCS (Section 2.10.3.1).

All samples were analysed using GC-MS analysis, within 24 hrs of derivatization. Raw

data processing was performed using ChemStation and AMDIS (Section 2.10.3.2). The

relative percentage of asparagine (A), serine (B), and lysine (C) are shown. All values

were normalised to the internal standard myristic acid d27. Error bars represent SD for

two biological replicates.


Early generation control (no addition)

Early generation + feed addition

Late generation control (no addition)

Late generation + feed addition

187

Figure 4.16 Effects of feed addition on amino acid concentrations

A.

B.

C.

0

1

2

3

4

5

6

Medium 5 7 9 13

Rela

tiv

e a

spa

ra

gin

e

(%)

Day

0

10

20

30

40

Medium 5 7 9 13

Rela

tiv

e s

erin

e

(%)

Day

0

5

10

15

20

Medium 5 7 9 13

Rela

tiv

e ly

sin

e

(%)

Day

188

Figure 4.17 Increased metabolites in response to feed addition





samples, taken on days 5, 7, 9 and 13 of batch culture (Section 2.3.2), and a medium

control sample, were lyophilised and derivatized as stated in Figure legend 4.16 and

Section 2.10.3.1. All samples were analysed using GC-MS analysis, within 24 hrs of

derivatization. Raw data processing was performed using Chemstation and AMDIS

(Section 2.10.3.2). The relative percentage of leucine (A), isoleucine (B), valine (C),

glycerol (D), glycine (E) and alanine (E) are shown. All values were normalised to the

internal standard myristic acid d27. Error bars represent SD for two biological

replicates.






189

Figure 4.17 Increased metabolites in response to feed addition

A.

B.

C.

0

50

100

150

200

250

300

Medium 5 7 9 13

Rela

tiv

e iso

leu

cin

e

(%)

Day

0

50

100

150

200

250

300

Medium 5 7 9 13

Rela

tiv

e leu

cin

e

(%)

Day

0

50

100

150

200

Medium 5 7 9 13

Rela

tiv

e v

ali

ne

(%)

Day

190

D.

E.

F.

0

20

40

60

80

Medium 5 7 9 13

Rela

tiv

e g

lycero

l

(%)

Day

0

50

100

150

Medium 5 7 9 13

Rela

tiv

e g

lycin

e

(%)

Day

0

5

10

15

Medium 5 7 9 13

Rela

tiv

e a

lan

ine

(%)

Day

191

Figure 4.18 Effect of feed addition on glucose and lactate concentrations





samples taken on days 5, 7, 9 and 13 (Section 2.3.2), and a medium control sample,

were analysed for glucose and lactate using enzymatic assays. Glucose was measured

spectrophotometrically at 505nm (as described in Section 2.10.1). Glucose standards

were also analysed. The concentration of lactate in samples was determined based on

the catalysis of pyruvate by LDH with the reduction of NAD+. The reduction of NAD

+ to

NADH was measured spectrophometrically at 340nm (as described in Section 2.10.2).

Lactate standards were also analysed. The concentrations of glucose (A) and lactate

(C) for early and late generation batch cultures are shown. The rates of production and

utilisation were calculated from days 5-13 of batch culture using the CCT values in

Figure 4.2 (Section 2.11.2). The rates of glucose utilisation (B) and lactate production

(D) for early and late generation batch cultures are also shown. Error bars represent

SEM for three biological replicates. * indicates p<0.05, and ♦ indicates p<0.10, using


generation control culture.






192

Figure 4.18 Effect of feed addition on glucose and lactate concentrations

A.

B.

C.

D.

0

10

20

30

40

50

Medium 5 7 9 13

Glu

co

se c

on

cen

tra

tio

n

(mM

)

Day

0

10

20

30

40

50

60

70

80

Medium 5 7 9 13

La

cta

te c

on

cen

tra

tio

n

(mM

)

Day

-1

-0.5

0

0.5

1

1.5

Ra

te o

f la

cta

te p

ro

du

cti

on

( p

M/c

ell

/da

y)

0

0.2

0.4

0.6

0.8

1

Ra

te o

f g

luco

se u

tili

sati

on

(pM

/cell

/da

y)

* *

* *

* *

* *

* *

♦

193

4.6 DISCUSSION

Analysis showed final antibody titres were increased in response to a CD feed addition

(Figures 3.1 and 4.1), consistent with previous findings for recombinant protein

production in mammalian fed cultures (Bibila et al, 1994; deZengotita et al, 2000;

Kuwae et al, 2005; Ma et al, 2009). Antibody titre was greater for cultures in the

presence of feed, without alteration to cell biomass (Figure 4.1B and Figure 4.2) or

recombinant mRNA expression (Figure 4.8). Figure 4.19 summarises the changes to

cultures in response to feed addition. Increased antibody titre and Qp values (Figure

4.5A) for cultures in the presence of feed occurred alongside changes to polysome

profiles. Greater polysome association as a result of feed addition (Figure 4.10D) is

possibly be due to less cellular stress (Harding et al, 2000b; Talvas et al, 2006), also

observed with decreased mRNA expression of UPR markers, ATF4, GADD34,

GADD153 (Figure 4.13), and XBP-1(s) (Figure 4.15). Although expression of ATF4

mRNA was lower for cultures supplemented with feed (Figure 4.13A), interestingly

ATF4 protein was not altered in response to feed addition (Figure 4.14A). As ATF4

protein intensity was similar for all cultures, regardless of feed addition, I propose that

all cultures encountered some degree of ER stress during recombinant protein

production. ATF4 protein may be essential for efficient recombinant protein production

and secretion. This suggestion will also be addressed again in Chapter 7.

Although the relative intensity of ATF4 protein was not altered in response to feed

addition, cultures supplemented with feed expressed significantly less GADD153

protein than cultures without feed addition (Figure 5.14B). As discussed in Section

1.8.3.3.2 GADD153 is regulated by both ER stress and amino acid deprivation. ATF4

translation, subsequent to ER stress, is not sufficient to fully activate AARE-dependent

transcription of GADD153 (Averous et al, 2004; Oyadomari & Mori, 2003) and that

ATF2 phosphorylation is necessary for AARE-dependent GADD153 transcription

(Averous et al, 2004). ATF2 phosphorylation is increased during cellular stress by

mitogen-activated protein kinase (MAPK) cascades (Ouwens et al, 2002). Initial

investigations showed no definite association between ATF2 phosphorylation and the

fed status of the cell (data not shown). The phosphorylation status of ATF2 alone may

not be enough to regulate GADD153 transcription and may require binding partners

such as Jun dimerisation protein 2 (JDP2), a bZIP protein known to interact with ATF2

194

(Jin et al, 2001). It has been suggested that the JDP2 may recruit HDAC to silencing

GADD153 transcription during periods when amino acids are not limited (Cherasse et

al, 2008; Chaveroux et al, 2010). However, further analysis would be needed to confirm

the regulation of ATF2, and JDP2, in response to feed addition. Other activating

transcription factors are also regulated by amino acid availability within the cell,

including ATF3. ATF3 expression increases during ER stress and amino acid

deprivation (Chen et al, 1996; Chen et al, 2004; Hashimoto et al, 2002), and has been

shown to modulate the transcription of asparagine synthetase (ASNS, Chen et al, 2004;

Pan et al, 2003). ASNS is discussed in more detail below.

Feed addition had the greatest impact on Qp during the end (decline) phase of batch

culture. It was during this phase of culture when nutrients, such as asparagine, serine,

and glucose became depleted in cultures without feed addition (Section 3.5). Previous

data has shown that glucose limitation alters recombinant glycosylation patterns (Hayter

et al, 1992). Potentially excess glucose, in response to feed addition, enhanced

glycosylation, allowing for greater antibody secretion (glycosylation profiles of the

secreted protein in response to the different culture conditions will be discussed in

Chapter 5). Feed addition maintained the majority of amino acids, and glucose, to a

similar concentration as observed for the medium control sample (Figure 4.16). One

exception was for the concentration of asparagine. Despite the addition of feed to

cultures the concentration of asparagine was not maintained at high concentrations

during batch culture due to the rapid utilisation of asparagine by 3.90 cultures (Figure

4.16A). Previous findings have also highlighted rapid utilisation of asparagine by CHO

cells (Hayter et al, 1991). In the presence of feed asparagine concentrations became

limiting by day 13 batch culture, four days after depletion in cultures without feed

addition. It was during this end (decline) phase of culture when Qp and antibody titres

were significantly increased in response to feed addition. It may be possible that the

increased productivity in response to feed addition was primarily due to enhanced

availability of asparagine.

Asparagine is needed for polypeptide synthesis and N-linked glycan formation. It can be

generated by cells from aspartate using ASNS, an enzyme up-regulated during amino

acid starvation (Gong et al, 1991; Guerrini et al, 1993; Hutson et al, 1997). Andrulis et

al, have shown that asparagine-starved CHO cells had decreased asparaginyl-tRNAAsn

195

and increased ASNS activity (Andrulis et al, 1979). It is possible that intracellular

asparagine concentrations were increased in response to feed addition, potentially

enhancing the rates of protein production for fed cultures. Intracellular metabolite

analysis would be needed to confirm intracellular concentrations of asparagine. As

asparagine was available for cultures during a crucial time of productivity it would be

tempting to state the importance of this single amino acid in relation to increased

antibody titre and Qp. However, the effects of a commercially available feed, CHO CD

Efficient Feed™ A (Invitrogen), were investigated to determine if another feed, which

contained asparagine, could also enhance productivity. The commercial feed did not

enhance antibody titre or Qp values above those attained for cultures in the absence of

feed (data not shown). Enhancing the productivity of 3.90 cultures was dependent on

creating an optimum specific feed based on the nutritional requirement of the specific

cultures.

Although cultures in the presence of feed utilised a small number of amino acids after

day 5 of batch culture the release of metabolites during the entire batch culture was

affected by feed addition. Glycine (Figure 4.17E), alanine (Figure 4.17F) and lactate

(Figure 4.18C) accumulation all altered in response to feed addition. Glycine

concentration was lower for cultures in the presence of feed, and alanine and lactate

were not re-utilised as a result of feed addition. In response to feed addition 3.90

cultures could be experiencing improved metabolism. Improved metabolic flux has been

shown for other CHO cultures in response to feed addition (Ma et al, 2009; Omasa et al,

2009; Chong et al, 2010). For example, the addition of pyruvate to CHO cultures was

shown to increase productivity whilst also enhancing ATP and NADH production

(Omasa et al, 2009). Initial investigations showed ATP, NAD+ and NADH

concentrations for 3.90 cultures were increased in response to feed addition (Figure

4.20). As discussed in Section 3.6 greater concentrations of ATP, NAD+ and NADH

may allow for greater protein production and protein folding. The determination of

correct/greater protein folding prior to protein secretion will be discussed further in

Chapter 7. Although concentrations of ATP, NAD+ and NADH were increased with

feed addition, lower ATP and NADH concentrations were seen for the late generation

fed cultures compared to the early generation culture. Low levels of energy

intermediates may be related to the instability observed for this cell line.

196

Instability was still apparent for cultures in the presence of feed. Changes in antibody

titre between early and late generation cultures were observed from day 13 of batch

culture (Figure 4.1A). Feed addition did not prevent antibody titre loss between early

and late generation cultures but it did appear to delay the effects of instability, and the

associated characteristics (shown in Figure 4.21). For example, significant antibody titre

loss between early and late generation cultures in the presence of feed occurred on day

13 of batch culture (Figure 4.1A), whilst significant antibody titre loss between early

and late generation cultures in the absence of feed was observed on day 11 of batch

culture (Figure 3.1A). Late generation cultures with feed addition also had increased

GADD153 mRNA on day 9 of batch culture compared to early generation cultures

(Figure 4.12C), whilst alterations in GADD153 mRNA between early and late

generation batch cultures, in the absence of feed addition, was observed on day 7 of

batch culture (Section 3.4). Unlike batch cultures without feed addition, the cell cycle

phase distribution of cultures supplemented with feed addition was not altered between

early and late generation cultures (Figure 4.3). As feed addition delayed characteristics

of instability, the changes in cell cycle distribution, particularly a decrease in the

percentage of cells in G0/G1, may have occurred after day 11 of batch culture (as

indicated in Figure 4.21). The delay in antibody titre loss between early and late

generation cultures in the presence of feed was during a period of culture when

extracellular asparagine (Figure 4.16A) and glucose (4.18A) were still available for

cultures in the presence of feed. As mentioned further investigations showed that Qp

was not entirely dependent on asparagine availability.

4.7 SUMMARY

Antibody titre and Qp was enhanced in response to feed addition, potentially due to

altered cellular metabolism, which resulted in less lactate and alanine re-utilisation and

less glycine accumulation. These potential metabolic flux alterations in response to feed

addition allowed for greater concentrations of ATP, NAD+ and NADH, potentially

increasing protein production and protein folding. Either due to enhanced protein

folding, or as a direct result of nutrient availability, cultures with feed addition had

lower mRNA expression of UPR markers, ATF4, GADD34, GADD153 and XBP-1(s),

and less GADD153 protein than cultures without feed addition. However, despite

197

increased antibody titre and Qp values as a result of feed addition these cultures still

experienced instability in response to LTC. Late generation fed cultures had

significantly less antibody titre and Qp values that early generation fed cultures,

possibly as a result of metabolic flux changes, observed with lower ATP and NADH

concentrations. Intracellular metabolite analysis may provide conclusive data regarding

the changes to metabolites and the metabolic flux of 3.90 cultures in response to feed

addition and LTC.

198

Figure 4.19 Alterations to 3.90 cultures in response to feed addition

The changes to productivity, expression of UPR stress markers and nutrient utilisation

in the presence of feed. In response to feed addition the expression of UPR markers and

lactate re-utilisation were decreased, viable cell densities, CCT and recombinant mRNA

expression remained unaltered, and antibody titre and Qp values were increased.

Polysome peak area

Antibody titre

Qp

Viable cell densities

CCT

Recombinant mRNA

ATF4, GADD34 mRNA

GADD153 mRNA and protein

XBP-1(s) mRNA

Glycine accumulation

Alanine re-utilisation

Lactate re-utilisation

Enhanced in response to feed addition

Lowered in response to feed addition

199

Figure 4.20 Concentrations of ATP, NAD and NADH in response to feed addition




the exponential stage of batch culture (as described in Section 2.3.2). 1x107 cells, taken

on day 9 of batch culture (Section 2.3.2), were quenched in 60% methanol with 0.85%

(w/v) AMBIC (at -40 °C). The metabolites were extracted by resuspension of the cell

pellet in 100% methanol followed by flash freezing in liquid nitrogen (Section 2.10.4).

Once the samples were thawed and re-extracted the methanol extracts were pooled and

lyophilised. Dried metabolite extracts were resuspended in ddH20 for use in each of the

metabolite assays. ATP assays were performed using an ATP Bioluminescence Assay

Kit CLS II (Section 2.10.5) and NAD+/NADH assays were performed using an

NAD+/NADH Quantification Kit (Section 2.10.6). Both kits were utilised according to

manufacturer’s instructions.



+ feed adidition

200

Figure 4.20 Concentrations of ATP, NAD+ and NADH in response to feed addition

A.

B.

C.

0

2

4

6

8

10

Early Late

AT

P c

on

cen

tra

tio

n (

mM

)

0.00

0.05

0.10

0.15

0.20

Early Late

NA

D+

co

ncen

tra

tio

n (

mM

)

0.000

0.005

0.010

0.015

0.020

Early Late

NA

DH

co

ncen

tra

tio

n (

mM

)

201

Figure 4.21 Time-line of changes to late generation cultures with feed addition

Although feed addition increased productivity for all generation cultures, a significant

decrease in antibody titre between early and late generation cultures in the presence of

feed was apparent. During batch culture alterations in the mRNA expression of ER

stress markers, and a decrease in CCT, were observed between early and late

generation cultures. These markers of instability were delayed in response to feed

addition but were still evident during the end (decline) phase of batch culture. ?

suggests the possibility that changes to cell cycle distribution may have occurred

between early and late generation culture during the decline phase of batch culture.

indicates a significant change for late generation cultures compared to generation 20

cultures (p<0.10, using independent samples t-test).


Day 0 3 5 7 9 11 13 15

Antibody titre

CCT

Percentage of cells in

G0/G1

GADD153 mRNA

XBP-1 (s) mRNA

*

?


Generation 60

Generation 80

Generation 100

Increased values

Decreased values

202

8

CHAPTER 4

9


LINE 3.90 IN RESPONSE TO

DIMETHYL SULFOXIDE

ADDITION

203

5. CHARACTERISATION OF CELL LINE 3.90 IN RESPONSE TO DIMETHYL

SULFOXIDE (DMSO) ADDITION


Cell line 3.90 was previously characterised in Chapter 3 and found to be unstable in

terms of a loss in final antibody titre and Qp between early and late generation cultures.

The protein expression of UPR markers ATF4 and GADD153, and ER chaperones BiP

and PDI were all upregulated during batch culture, with enhanced expression of

GADD153 and XBP-1(s) mRNA for late generation cultures (Section 3.4). Metabolite

analysis also showed differences in alanine accumulation, and altered rates of lactate

utilisation between early and late generation cultures, potentially indicating altered

metabolic flux in response to LTC (Section 3.5). Investigations were continued to

determine if antibody titres and Qp values could be improved further by addition of a

CD feed (Chapter 4). Feeding did enhance final antibody titres and Qp values (Section

4.2.), whilst lowering the mRNA expression of UPR markers ATF4, GADD34 and

GADD153, and XBP-1(s) (Section 4.4). As the effects of feeding maintained a high

concentration of most nutrients and ensured, for example, that during batch culture

glucose was never depleted and lactate was not re-utilised (Section 4.5), I suggested that

feeding altered the metabolic flux of the cell allowing for increased ATP and greater

protein secretion. Although feed addition increased final antibody titres, feed addition to

cultures did not prevent instability as a result of LTC (Figure 4.1A). My next aim was to

increase antibody titre and Qp, whilst maintaining stable antibody production during

LTC, using addition of simple supplements.

Previous literature has shown that addition of simple low molecular weight chemicals,

including sodium butyrate, hexanohydroxamic acid, glycerol, DMSO and sorbitol,

increased productivity for CHO and hybridoma cells (Allen et al, 2008; Kim & Lee,

2000; Li et al, 2006b; Li et al, 2006c; Petch & Butler, 1996; Rodriguez et al, 2005;

Zhou & Sharfstein, 2008). I initially investigated the consequences of glycerol, sorbitol,

and DMSO addition at various concentrations to cultures and identified that 2% (v/v)

DMSO addition had the greatest impact on antibody titre (Appendix 4), and have

therefore focused upon the consequence and mechanisms in response to the addition of

2% (v/v) DMSO on day 5 of batch culture.

204

In this Chapter, consistent with the format in Chapters 3 and 4, the term early generation

cultures will refer to batch cultures created at ≤ 40 generations and the term late

generation cultures will refer to batch cultures created at ≥ 60 generations. Where

appropriate, 2% (v/v) DMSO was added on day 5 of batch culture. As described in

Chapter 4 appropriate cultures were also supplemented with a 2% (v/v) CD feed

addition on days 3 to 7 of batch culture. The data obtained for cultures in the absence

and presence of feed addition within this Chapter represent control cultures performed

in parallel to the cultures with DMSO addition. The majority of the control cultures

discussed in this Chapter have not been described in the previous Chapters. However, I

will state when I am comparing the cultures supplemented with DMSO to cultures that

have been previously mentioned in Chapters 3 and 4. The main aim of Chapter 5 is to

determine the influence of DMSO on antibody titre, Qp values, the expression of UPR

markers and metabolic utilisation/production rates.


CELL LINE 3.90 IN RESPONSE TO DMSO

The growth characteristics and antibody titre of cultures with and without DMSO

addition were assessed for early generation (Figure 5.1) and late generation (Figure 5.2)

cultures. Early generation control cultures had similar antibody titres to values observed

in Chapters 3 and 4. DMSO addition significantly enhanced antibody titres from day 13

of batch culture, increasing final antibody titre by approximately 200-300 mg/L

(regardless of feed addition, Figure 5.1A). The addition of DMSO to early generation

cultures increased antibody titres whilst inhibiting cell growth. Early generation cultures

without DMSO addition had maximal cell densities of approximately 6x106 cells/ml,

whilst cultures supplemented with DMSO only achieved maximal cell densities of

approximately 4x106 cell/ml (Figure 5.1B). As a result of DMSO addition viable cell

densities were statistically lower on day 9 of batch culture. The alteration in viable cell

densities was also reflected in the CCT values for cultures. From day 9 of batch culture

the CCT for cultures with DMSO was less than the CCT for cultures without DMSO,

and significantly lower for fed cultures in the presence of DMSO from day 11 of batch

culture (Figure 5.3C).

205

Late generation cultures were also analysed to determine the influence of DMSO on

productivity and stability on cultures after LTC. Late generation cultures also had

increased final antibody titres (by approximately 200-300 mg/L) in response to DMSO

addition (Figure 5.2A), with lower viable cell densities (Figure 5.2B), and CCT values

(Figure 5.2C). No changes in cell biomass were observed for late generation cultures as

a result of DMSO addition.

The addition of DMSO to cultures increased antibody titre values, but only during the

end (decline) phase of batch culture (also seen for antibody titres in response to feed

addition, Section 4.2). Early generation cultures with DMSO had significantly increased

antibody titres from day 11 of batch culture, compared to the relative control culture

(Figure 5.1A), whereas enhanced antibody titres for late generation cultures in response

to DMSO addition were dependent on the fed status of the culture. Without feed

addition antibody titres for late generation cultures supplemented with DMSO were

significantly increased from day 13 of batch culture in response to DMSO, whilst in the

presence of feed the antibody titre for late generation cultures was significantly

enhanced from day 9 of batch culture (Figure 5.2A). Although final antibody titres were

greater for cultures in response to DMSO addition, DMSO addition did not prevent

antibody titre loss in response to LTC. Final antibody titres between early and late

generation cultures with DMSO decreased by 372 mg/L, and final antibody titres

between early and late generation cultures with feed and DMSO addition decreased by

246 mg/L, however, this change was not statistically significant. Therefore, in order to

increase productivity and maintain stable antibody titres during LTC it may be

advantageous to utilise cultures in a fed environment in the presence of DMSO.

206


for early generation cultures


culture analysis was performed in shake flasks for early generation (≤ 40 generations)

cultures. Batch cultures were created at 0.2x106 cells/ml, and maintained at 37

oC, 140

rpm and with a manual supply of 5% CO2 in air. A CD feed was added to cultures

during the exponential stage of batch culture, whilst 2% (v/v) DMSO was added on day

5 of batch culture (as described in Section 2.3.2). Antibody titres were measured by

ELISA (Section 2.5.1) and viable cell densities were determined by light microscopy

and trypan blue exclusion (Section 2.3.3) from samples taken routinely during batch

culture. Antibody titres (A), viable cell densities (B), and CCT (C) are shown. For

determination of CCT see Section 2.11.2. Error bars represent SEM for three biological


repeats. * indicates p<0.05, using independent samples t-test to compare the cultures

with DMSO to the corresponding control culture without DMSO addition (on the same

day of batch culture).

Annotation of early generation batch cultures in Figure 5.1


+ DMSO addition

Control (+ feed addition)

+ feed + DMSO addition

207


for early generation cultures

B.

C.

A.

0

2

4

6

8

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

10

20

30

40

50

60

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

L)

Day

0

400

800

1200

1600

2000

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

*

**

* *

*

*

*

*

208


for late generation cultures

3.90 was cultured as described previously (Figure legend 5.1), for batch cultures

created at late generations (≥ 60 generations). A CD feed and 2% (v/v) DMSO was

added to the relevant cultures (as described in Section 2.3.2). Antibody titres were

measured by ELISA (Section 2.5.1) and viable cell densities were determined by light

microscopy and trypan blue exclusion (Section 2.3.3) from samples taken routinely

during batch culture. Antibody titres (A), viable cell densities (B), and CCT (C) are

shown. For determination of CCT see Section 2.11.2. Error bars represent SEM for

three biological replicates. Each biological replicate value is an average from duplicate

technical repeats. * indicates p<0.05, using independent samples t-test to compare the

cultures with DMSO to the corresponding control culture without DMSO addition (on

the same day of batch culture).

Annotation of late generation batch cultures in Figure 5.2


+ DMSO addition



209


for late generation cultures

B.

C.

A.

0

400

800

1200

1600

0 3 5 7 9 11 13 15

An

tib

od

yti

tre

(mg

/L)

Day

0

2

4

6

8

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

10

20

30

40

50

60

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

L)

Day

*

*

*

*

*

*

210

From analysis it was apparent that antibody titres were increased and viable cell

densities were lowered as a result of DMSO addition, as a consequence to these changes

Qp (d0-d15) was significantly enhanced (Figure 5.3). In response to DMSO addition the

fold change in Qp (d0-d15) was increased by 30-40% for both early and late generation

cultures. Although cell growth was altered by DMSO addition cell cycle distribution

was not. The percentage of cells G0/G1 cell cycle phase was not altered by the addition

of DMSO to either early generation (Figure 5.4A) or late generation (Figure 5.4B)

cultures. The proportion of cells in S cell cycle phase and G2/M cell cycle phase were

also unaffected by DMSO addition (data not shown). My findings were contradictory to

the findings reported by Li et al, and Liu et al, who found CHO cell cycle distribution to

change in presence of DMSO (Li et al, 2006c; Liu & Chen, 2007b). Although Li et al,

and Liu et al, added lower DMSO concentrations per culture (1-1.5% [v/v] DMSO

addition), the viable cell densities upon DMSO addition were less than the viable cell

densities for my 3.90 cultures at the time of DMSO addition. The changes to DMSO

concentration on a per cell basis may have resulted in greater alterations to cell cycle

distribution.

During investigations I also found that DMSO addition to cultures (in the absence of

feed) increased medium osmolality by approximately 0.25 Osm/kg (Appendix 4). As

previously mentioned in Section 4.2.1 changes to medium osmolality can affect cell

growth and cell size. Larger cells may be more productive merely due to increased

cellular volume (Lloyd et al, 2000). I found cell diameters for early and late generation

cultures with DMSO were approximately 13-14µm (Figure 5.5), similar to diameter

measurements of cells from cultures previously described in Section 3.2.1. Therefore

the enhancement in productivity in response to DMSO addition was not due to changes

within cellular volume. The alterations in productivity were possibly due to other

mechanisms potentially at the site of mRNA transcription, protein translation, post-

translation modification and/or protein secretion.

211

Figure 5.3 Effect of DMSO on specific productivity (Qp)

Specific productivity (Qp) was determined from the antibody tire and the CCT values

for early generation (≤ 40 generations) and late generation (≥ 60 generations) batch

cultures seen in Figure 5.1 and Figure 5.2, respectively. Qp was calculated for the

entire batch culture, using antibody titre and cell density measurements on days 0 to 15

of culture, Qp (d0-d15). For determination of Qp see Section 2.11.2. The fold change in

Qp (d0-d15) relative to early generation control cultures (without feed or DMSO

additions) is shown. Error bars represent SEM for three biological replicates. Each


p<0.05, using independent samples t-test to cultures with DMSO to the corresponding

control culture without DMSO addition.


0

0.5

1

1.5

2

2.5

- DMSO + DMSO - DMSO + DMSO

- feed addition + feed addition

Fo

ld c

ha

ng

e i

n Q

p(d

0-d

15

)

(rela

tiv

e to

ea

rly

gen

era

tio

n c

ult

ures

wit

ho

ut

feed

or D

MS

O a

dd

itio

ns)

Culture condition

Early generation

Late generation

*

*

*

*

212

Figure 5.4 Effect of DMSO addition on G0/G1 cell cycle phase transition

3.90 was cultured as described previously (Figure legend 5.1). A CD feed and 2% (v/v)

DMSO was added to the relevant cultures (as described in Section 2.3.2). 1x106 cells,

taken on days 7, 9, and 11 of batch culture, were analysed by flow cytometry using PI

excitation by a 488nm laser, and emission measured by a 613/20nm bandpass filter

(Section 2.4.1). The data was analysed by Summit 4.3 and ModFit LT software. The

percentage of early generation (≤ 40 generations) cells in G0/G1 cell cycle phase (A)

and the percentage of late generation (≥ 60 generations) cells in G0/G1 cell cycle phase

(B) are shown. Error bars represent SEM for three biological replicates.


B.

A.

0

20

40

60

80

100

7 9 11

G0

/G1

cell

cy

cle

ph

ase

(%)

Day

0

20

40

60

80

100

7 9 11

G0

/G1

cell

cy

cle

ph

ase

(%)

Day


+ DMSO addition



213

Figure 5.5 Cell size comparisons for cultures with and without DMSO addition

3.90 was cultured as previously described (Figure legend 5.1). On day 9 of batch

culture, early generation (≤ 40 generations) and late generation (≥ 60 generations)

cells, with and without DMSO addition (2% [v/v] added on day 5 of culture), were

prepared for cell counting using trypan blue dye (as described in Section 2.3.3). 100

cells were counted and measured for cell diameter using a Widefield Axiovision

microscope, and analysed using the Axiovision software. The average cell diameter for

early and late generation cells is shown. Error bars represent the SD for 100 cells

counted.

0

5

10

15

20


Early Late

Cell

Dia

mete

r

(µm

)

Culture condition/

Generation

214


IN RESPONSE TO DMSO ADDITION

DMSO addition on day 5 of batch culture increased final antibody titre regardless of fed

status or generation time of the culture (Section 5.2.1). The increased antibody titres in

response to DMSO addition were not due to changes in cell size or cell cycle

distribution. As previous studies highlighted changes to mRNA transcription as a result

of DMSO addition (Liu et al, 2001), investigations were continued to determine

molecular changes of 3.90 cultures as a result of DMSO addition. These investigations

included analysis of antibody mRNA expression (Section 5.3.1) and polysome profiles

(Section 5.3.2).

5.3.1 Effect of DMSO addition on recombinant gene mRNA expression

As no change in GS mRNA expression was observed in response to LTC (Section

3.3.2) or feed addition (Section 4.3.1) I did not examine GS mRNA in response to

DMSO addition. The expression of both heavy chain mRNA (Figure 5.6A) or light

chain mRNA (Figure 5.6B) was not altered in response to DMSO addition, regardless of

generation time of culture. Analysis of mRNA supported findings shown in Chapters 3

and 4 that recombinant mRNA expression was not affected by LTC or feed addition.

5.3.2 Effect of DMSO addition on polysome profiles

Polysome profiles were also investigated for cultures supplemented with DMSO and

compared to the polysome profiles previously described in Chapters 3 and 4. Analysis

showed that DMSO addition did not influence the relative 40S and 60S monosome peak

areas (data not shown) but did result in a lower 80S peak area and a lower polysome

peak area (for early and late cultures in the presence of feed, Figure 5.7). Increased 80S

peaks have been previously associated with culture conditions which promote cell stress

and halt protein synthesis (Ashe et al, 2000; Demeshkina et al, 2007; Shenton et al,

2006; Talvas et al, 2006; Volarevic et al, 2000). DMSO addition to 3.90 cultures may

create a culture environment which encourages protein synthesis (and potentially lowers

cellular stress).

215

5.3.3 Effect of DMSO addition on intracellular recombinant protein

To determine if protein synthesis was enhanced in response to DMSO addition

intracellular heavy chain and light chain proteins were analysed using specific antibody-

conjugated dyes. The mean fluorescence intensity of both the heavy chain protein and

the light chain protein were not altered in response to DMSO addition (when compared

to the respective control cultures previously described in Chapters 3 and 4, Figure 5.8).

DMSO addition could have stimulated both the rate of protein synthesis and protein

secretion to a similar degree so that no overall change in levels of intracellular protein

was observed. DMSO addition may have potentially encouraged correct protein folding

within the ER, lowering the concentration of mis/unfolded proteins and ER stress,

allowing for increased rates of protein secretion. The UPR markers were measured to

determine any changes in response to DMSO addition. These data are shown in the next

Section.

216

Figure 5.6 Effect of DMSO addition on recombinant mRNA expression


created at early generations (≤ 40 generations) and late generations (≥ 60 generations).

A CD feed and 2% (v/v) DMSO was added to the relevant cultures (as described in

Section 2.3.2). mRNA levels were compared using q-RTPCR from samples taken on

days 7 and 9 of batch culture (as detailed in Section 2.7.1), using the mRNA specific

primer sets for A, heavy chain, and B, light chain. Samples were normalised using

mRNA β-Actin primers. Error bars represent SEM for three biological replicates.


A.

B.

0

50

100

150

200

250

300

7 9 7 9

Early Late

Hea

vy

ch

ain

mR

NA

ex

pre

ssio

n

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

ctin

mR

NA

ex

pre

ssio

n)

Day/Generation

0

50

100

150

200

250

300

350

400

7 9 7 9

Early Late

Lig

ht c

ha

in m

RN

A e

xp

ress

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

ctin

mR

NA

ex

pre

ssio

n)

Day/Generation


+ DMSO addition



217

Figure 5.7 Quantification of monosome and polysome peaks in response to DMSO

addition




Section 2.3.2). Polysomes were extracted on day 7 of batch culture (Section 2.8.2). The

extracts were analysed on a UV/Vis machine at 254nm after sucrose gradient

centrifugation, as described in Section 2.8.3. The peaks were analysed using Image J

software to provide a relative representation of peak variation. The fold change in the

80S peak area and the polysome peak area between early and late generation day 7

polysome profiles is shown. Error bars represent SEM for three biological replicates. *

indicates p<0.05, using independent samples t-test to compare cultures with DMSO to

the corresponding control culture without DMSO addition.


0.00

0.50

1.00

1.50

2.00

2.50

80S Polysome 80S Polysome

Early Late

Fo

ld c

ha

ng

e i

n p

ea

k a

rea

(rela

tiv

e to

ea

rly

gen

era

tio

n c

on

tro

l

cu

ltu

res

wit

ho

ut

DM

SO

ad

dit

ion

)

Peak /Generation


+ DMSO addition



*

* *

*

218

Figure 5.8 Analysis of intracellular recombinant protein in response to DMSO

addition

3.90 was cultured as previously described (Figure legend 5.1). 2x106 of fixed cells from

early generation (≤ 40 generations) and late generation (≥ 60 generations) day 9

cultures (with and without feed and DMSO additions, Section 2.3.2) were washed,

blocked and incubated with 10 µg goat anti-human IgG, Fcγ-APC and 6 µg goat anti-

human lambda light chain-FITC. The samples were then analysed by a CyAn ADP flow

cytometer, using the 488nm and infra-red excitation lasers to excite the FITC and APC

conjugates (Section 2.4.2). Unstained 3.90 cells and stained parental cells were also

required for setting initial parameters with the CyAn-ADP flow cytometer. The data was

gated to select single cells, and was analysed by Summit 4.3 software. Error bars



0.0

0.5

1.0

1.5

2.0

- DMSO + DMSO - DMSO + DMSO - DMSO + DMSO - DMSO + DMSO

- feed addition + feed addition - feed addition + feed addition

Early Late

Fo

ld c

ha

ng

e i

n m

ea

n f

luo

resc

en

ce i

nte

nsi

ty

(rela

tiv

e to

ea

rly

gen

era

tio

n c

on

tro

l cu

ltu

res)




219

5.4 THE UPR STATUS OF CULTURES AFTER DMSO ADDITION

As antibody titres were greater in response to DMSO (Section 5.2) the rates of protein

secretion must also have also increased accordingly. Lower levels of UPR stress, either

due to less mis/unfolded proteins, or by mechanisms which resolve UPR stress, would

encourage both protein synthesis and secretion.

Initial analysis of UPR markers indicated that DMSO addition had no impact on the

expression of ATF4 or GADD34 mRNA (data not shown). However, it was evident that

GADD153 mRNA was altered in response to DMSO addition. Early generation

cultures, on day 9 of batch culture, had approximately 40% less GADD153 mRNA in

response to DMSO addition, regardless of the fed state of the culture (Figure 5.9). Late

generation cultures, also had significantly lower GADD153 mRNA in response to

DMSO addition, however, the percentage decrease in GADD153 was dependent on the

fed culture environment. For late generation cultures in the presence of DMSO

GADD153 mRNA was approximately 60% and 40% lower for cultures with and

without feed addition, respectively (Figure 5.9).

The protein expressions of UPR markers were also analysed. Protein expression of PDI

(Figure 5.10A), ATF4 (Figure 5.10B) and GADD153 (Figure 5.10C) were not altered in

response to DMSO addition. A significant six-fold decrease in GADD153 protein

expression was observed for the fed cultures but this was irrespective of DMSO

addition. This data confirmed the change in GADD153 protein expression previously

observed in response to feed addition (Section 4.4), and again identified no differences

in PDI, ATF4 or GADD153 protein in response to LTC (Section 3.4).

Although the expression of ATF4 and GADD153 at protein level was not altered by

DMSO addition it could be possible that DMSO addition was affecting expression of

other UPR components, including ATF6 (Section 1.8.3.2), and IRE-1 (Section 1.8.3.1),

and their downstream target, XBP-1. Investigations found the ratio of spliced XBP-1

mRNA to total XBP-1 mRNA was lower for cultures supplemented with DMSO, with

the percentage decrease dependent on the fed status of the culture. In response to

DMSO addition the fold change in XBP-1(s) mRNA to total XBP-1 mRNA was 35%

lower in cultures without feed addition, and 50% lower in cultures with feed addition

220

(Figure 5.11). The degree of spliced XBP-1 mRNA also highlighted differences

between early and late generation cultures. For each culture condition the ratio of XBP-

1(s) mRNA was greater for the late generation cultures than early generation cultures.

The changes in antibody titre, Qp, GADD153 mRNA and XBP-1(s) mRNA in response

to DMSO addition were similar to the changes observed in response to feed addition (as

described in Chapter 4). DMSO addition to fed cultures further increased productivity,

whilst lowering GADD153 mRNA expression and XBP-1 splicing to levels below those

observed for cultures with separate feed or DMSO additions. As components of the

UPR are affected by both feed and DMSO addition I suggest that these culture

conditions result in less ER stress due to a greater degree of correctly-folded proteins

within the ER, suitable for secretion. However, it may also be possible that by adding

feed and DMSO to cultures any mis/unfolded proteins within the ER by-pass the

regulation of the UPR, and become unintentionally secreted. Protein conformations,

including correct post-translational modifications are extremely important for protein

functionality. Mis/unfolded proteins which are secreted may not contain these essential

modifications and therefore may impact protein quality. The functionality of the

secreted protein is discussed in Section 5.5.

221

Figure 5.9 Effect of DMSO addition on GADD153 mRNA expression




Section 2.3.2). mRNA levels were compared using q-RTPCR from samples taken on day

7 and 9 of batch culture (as detailed in Section 2.7.1), using the GADD153 mRNA

specific primer set. Samples were normalised using mRNA β-Actin primers. Error bars

represent SEM for three biological replicates. ♦ indicates p<0.10, using independent

samples t-test to compare cultures with DMSO to the corresponding control culture

without DMSO addition.


0

100

200

300

400

7 9 7 9

Early Late

GA

DD

15

3 m

RN

A e

xp

ress

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Day/Generation


+ DMSO addition



♦

♦

♦

♦

♦

222

Figure 5.10 Effect of DMSO addition on expression of PDI, ATF4 and GADD153

protein




Section 2.3.2). Protein was extracted on day 9 of batch culture (as detailed in Section

2.5.3.1). 60µg of protein was separated by SDS-PAGE (Section 2.5.3.2), transferred

(Section 2.5.3.3) and then detected using anti-rabbit polyclonal PDI, ATF4, GADD153

antibodies. All membranes were stripped and re-probed using an anti-mouse pan ERK


amount of PDI, ATF and GADD153 protein expression was calculated relative to ERK

expression (Section 2.5.3.5). The relative protein intensities of PDI (A), ATF4 (B), and

GADD153 (C) are shown. Error bars represent SEM for three biological replicates.



+ DMSO addition



223

Figure 5.10 Effect of DMSO addition on expression of PDI, ATF4 and GADD153

protein

A.

B.

C.

0

50

100

150

200

250

Early Late

PD

I p

ro

tein

in

ten

sity

(rela

tiv

e t

o E

RK

sta

nd

ard

)

Generation

0

50

100

150

200

250

Early Late

GA

DD

15

3 p

ro

tein

in

ten

sity

(rela

tiv

e t

o E

RK

sta

nd

ard

)

Generation

0

50

100

150

Early Late

AT

F4

pro

tein

in

ten

sity

(rela

tiv

e t

o E

RK

sta

nd

ard

)

Generation

224

Figure 5.11 Analysis of XBP-1(s) mRNA in response to DMSO addition




Section 2.3.2). cDNA was synthesised from RNA extracted on day 9 of batch culture

(Section 2.7.1.3) and the PCR was performed using the newly synthesised cDNA and the

XBP-1(s) primers (Section 2.7.2). The PCR products were electrophoresed on a 2%

(w/v) agarose gel and visualised by UV transillumination (Section 2.6.1.4). The product

bands were analysed using Image J software, and the quantified ratio of spliced XBP-1

mRNA to total XBP-1mRNA is shown (A). The visualised ratio of spliced XBP-1 mRNA

to total XBP-1 mRNA for an early generation culture (B) is also shown. Error bars

represent SEM for three biological replicates. * indicates p<0.05, using independent

samples t-test to compare cultures with DMSO to the corresponding control culture

without DMSO addition.


Early generation

Late generation

225

Figure 5.11 Analysis of XBP-1(s) mRNA in response to DMSO addition

A.

B.

- + - +

Feed addition

DMSO

- - + +

0

0.5

1

1.5


- feed addition + feed addition

Fo

ld c

ha

ng

e o

f sp

liced

XB

P-1

mR

NA

to t

ota

l X

BP

-1 m

RN

A

(Rela

tiv

e to

ea

rly

gen

era

tio

n co

ntr

ol

cu

ltu

res

wit

ho

ut

ad

dit

ion

s)

Culture condition

* *

* *

226

5.5 FUNCTIONALITY OF THE SECRETED ANTIBODY

The glycan characteristics for the secreted antibody protein were investigated to

determine if LTC, feed or DMSO addition altered the oligosaccharide profiles of the

secreted protein. The main glycan detected, with approximately 80% of the relative

oligosaccharide area, was the fucosylated G0F. The other glycans identified

(representing approximately 20% of the relative oligosaccharide area) were G1 and

G1F. There was no statistical difference in the relative percentage areas of the three

main oligosaccharides in response to feed or DMSO addition, or generation time of

culture (Figure 5.12). Product characterisation data, which included whole molecule

analysis, oligosaccharide analysis, peptide map analysis and potency analysis, was also

provided by colleagues at MedImmune. The product characterisation data showed the

secreted antibody was not functionally compromised as a result of DMSO addition (or

feed addition, or generation time, data not shown). The addition of DMSO to cultures

improves antibody titres for a valuable protein with therapeutic potential.

227

Figure 5.12 Effects of culture conditions on secreted glycan profiles




Section 2.3.2). The antibody was purified (Section 2.5.4.1) and deglycosylated using

PNGase F (Section 2.5.4.2). Proteins were removed using ethanol precipitation and the

remaining glycans were lyophilised (Section 2.5.4.3). The resuspended glycans were

desalted (Section 2.2.4.4) and analysed by MALDI-ToF-MS for relative glycan form

quantification and glycan structural determination (Section 2.2.4.5). Error bars



0

20

40

60

80

100

120

- DMSO + DMSO - DMSO + DMSO - DMSO + DMSO - DMSO + DMSO

- feed additon + feed addition - feed addition + feed addition

Early Late

Rela

tiv

e a

rea

of

oli

go

sacch

arid

es

(%)


G0F

G1

G1F

228

5.6 METABOLISM OF 3.90 CULTURES IN RESPONSE TO DMSO ADDITION

DMSO addition to culture increased final antibody titre and Qp values (Section 5.2). As

I have previously suggested in Chapter 4, with regards to feed addition, protein

synthesis and secretion would be enhanced if the cells contained a greater concentration

of correctly-folded proteins and less mis/unfolded proteins. The ability of cultures to

produce antibody protein/chains, and to ensure the correct post-translation

modifications required for complete folding, may be greatly linked to the metabolic

profile of the cell. Previous literature has shown that DMSO addition to CHO cultures

decreased glucose and amino acid utilisation, and lactate production (Li et al, 2006b; Li

et al, 2006c). It may be possible that metabolic flux pathways were altered in response

to DMSO addition, allowing for the overall consequence of increased antibody titres.

The metabolite profiles of early and late generation cultures in the presence of DMSO

were compared to the metabolic profiles of control cultures in the absence of DMSO.

The metabolic profiles for the control cultures were previously described in Sections 3.5

and 4.5.

5.6.1 Effects of DMSO addition on the production of metabolites

For both early and late generation cultures the relative concentrations of isoleucine,

leucine, valine, methionine, threonine, lysine, serine, and asparagine were not altered in

response to DMSO (data not shown). However, DMSO addition did affect the

accumulation of glycerol, glycine, lactate and alanine for early generation (Figure 5.13)

and late generation (Figure 5.14) cultures. On days 7 and 9 of batch culture the

accumulation of glycerol and glycine for early generation cultures in the presence of

DMSO was similar to glycine and glycerol accumulation from cultures in the absence of

DMSO. However, by day 13 of batch culture extracellular glycerol (Figure 5.13A) and

glycine (Figure 5.13B) concentrations were 50% lower for cultures supplemented with

DMSO compared to the corresponding control culture. Alterations to alanine and lactate

in response to DMSO were also apparent. In fed conditions alanine accumulation was

increased by day 13 of batch culture in response to DMSO addition (Figure 5.13C),

whilst lactate accumulation was less as a result of DMSO addition (regardless of fed

status, Figure 5.13D).

229

The changes in glycerol and glycine accumulation for late generation cultures were

similar to the differences observed for the early generation cultures, with glycerol

(Figure 5.14A) and glycine (Figure 5.14B) concentrations approximately 50% lower for

cultures supplemented with DMSO by day 13 of batch culture. Although changes were

apparent for extracellular accumulation of glycerol and glycine, the accumulations of

alanine (Figure 5.14C) and lactate (Figure 5.14D) in late generation cultures were not

affected by the addition of DMSO.

For early and/or late generation cultures glycerol, glycine, lactate and alanine

accumulation were altered in response to DMSO, independent of feed addition or

generation time of culture. Metabolic variations in response to DMSO potentially

altered the conversion balance of pyruvate, with less pyruvate used in the by-product

production of glycerol, glycine and lactate, allowing for more pyruvate to be converted

to acetyl CoA for use within the TCA cycle. If DMSO addition was adjusting the

glycolytic and TCA flux pathways changes in glucose uptake may be apparent.

5.6.2 Effects of DMSO addition on rates of glucose and lactate utilisation

The rate of glucose utilisation from days 0 to 13 of batch culture were not affected by

DMSO addition (Figure 5.15A). As the feeding regime created difficulties in

determining glucose usage for fed cultures during the exponential phase of batch

culture, rates of glucose utilisation were also calculated for the end (decline) phase of

batch culture. Both early and late generation cultures had greater rates of glucose

utilisation from days 9 to 13 of batch culture in response to DMSO addition (Figure

5.15B). This may be an indication that cultures with DMSO had a greater requirement

for glucose metabolism during the decline phase of batch culture than cultures without

DMSO. The rate of lactate production was also altered in response to DMSO addition

(Figure 5.16). The changes in the rate of lactate utilisation was an indication that either

lactate production was low, or that any lactate produced and released from the cell was

quickly re-utilised. Lactate concentration was 50% lower for cultures supplemented

with both feed and DMSO addition compared to the concentration from the control

cultures. As fed cultures did not re-utilise lactate changes in the rates of lactate

production/utilisation in response to DMSO was probably due to less lactate production.

230

In response to DMSO addition differences in glycine and glycerol accumulation, and

changes in the rates of glucose utilisation and lactate production/utilisation, were

observed. Increased rates of glucose utilisation during the decline phase of batch

cultures in response to DMSO may suggest that cultures in the presence of DMSO were

still metabolically active during the end phase of batch culture, whilst lower glycine,

glycerol and lactate accumulation in response to DMSO could be an indication of

enhanced metabolic flux through the TCA cycle. Greater flux through the TCA cycle

could increase ATP availability for the DMSO cultures. Altered metabolism in response

to DMSO may allow for increased protein synthesis and secretion. Both events would

ultimately result in higher antibody titres and Qp values, as observed for cultures in the

presence of DMSO.

231

Figure 5.13 Analysis of glycerol, glycine, alanine and lactate accumulation from

early generation cultures in the presence of DMSO


created at early generations (≤ 40 generations). A CD feed and 2% (v/v) DMSO was

added to the relevant cultures (as described in Section 2.3.2). Supernatant samples,

taken on days 5, 7, 9 and 13 of batch culture (Section 2.3.2), and a medium control

sample, were spiked with the internal standard myristic acid d27 and lyophilised.

Chemical derivatization was performed in two stages, with methyloxyamine

hydrochloride in pyridine, before the addition of MSTFA and TMCS (Section 2.10.3.1).

All samples were analysed using GC-MS analysis, within 24 hrs of derivatization. Raw

data processing was performed using ChemStation and AMDIS (Section 2.10.3.2). The

fold change was calculated relative to the medium control. The fold change in the

production of glycerol (A), glycine (B), alanine (C), and lactate (D) are shown. All

values were normalised to the internal standard myristic acid d27. Error bars represent

SD for two biological replicates.



+ DMSO addition



232


early generation cultures in the presence of DMSO

A.

B.

C.

0

5

10

15

20

25

30

7 9 13

Fo

ld c

ha

ng

e i

n a

lan

ine

(rela

tiv

e to

med

ium

)

Day

0

50

100

150

7 9 13

Fo

ld c

ha

ng

e i

n g

lycin

e

(rela

tiv

e to

med

ium

)

Day

0

50

100

150

200

250

300

7 9 13

Fo

ld c

ha

ng

e i

n l

acta

te

(rela

tiv

e to

med

ium

)

Day

D.

0

10

20

30

40

7 9 13

Fo

ld c

ha

ng

e i

n g

lycero

l

(rela

tiv

e to

med

ium

)

Day

233


late generation cultures in the presence of DMSO



added to the relevant cultures (as described in Section 2.3.2). Supernatant samples,

taken on days 5, 7, 9 and 13 of batch culture (Section 2.3.2), and a medium control

sample, were spiked with the internal standard myristic acid d27 and lyophilised as

stated in Figure legend 5.13. All samples were analysed using GC-MS analysis, within

24 hrs of derivatization. Raw data processing was performed using ChemStation and

AMDIS (Section 2.10.3.2). The fold change was calculated relative to the medium

control. The fold change in the production of glycerol (A), glycine (B), alanine (C), and

lactate (D) are shown. All values were normalised to the internal standard myristic acid

d27. Error bars represent SD for two biological replicates.



+ DMSO addition



234


late generation cultures in the presence of DMSO

A.

B.

C.

D.

0

10

20

30

40

7 9 13

Fo

ld c

ha

ng

e i

n g

lycero

l

(rela

tiv

e to

med

ium

)

Day

0

5

10

15

7 9 13

Fo

ld c

ha

ng

e i

n a

lan

ine

(rela

tiv

e to

med

ium

)

Day

0

50

100

150

7 9 13

Fo

ld c

ha

ng

e i

n g

lycin

e

(rela

tiv

e to

med

ium

)

Day

0

50

100

150

200

250

300

7 9 13

Fo

ld c

ha

ng

e i

n l

acta

te

(rela

tiv

e to

med

ium

)

Day

235

Figure 5.15 Investigating glucose utilisation rates in response to DMSO addition




Section 2.3.2). Supernatant samples taken on days 5, 7, 9 and 13, were analysed for

glucose using an enzymatic assay (described in Section 2.10.1). Rates of utilisation

were determined using the relevant CCT values (for calculations see Section 2.11.2). A,

shows the rate of glucose utilisation during batch culture (d5-d13), and B, shows the

rate of glucose utilisation during the decline phase of batch culture (d9-d13). Error



A.

B.

-0.5

0.0

0.5

1.0

1.5

Early LateRa

te o

f g

luco

se u

tili

sati

on

(d5

-d1

3)

(pM

/cell

/da

y)

Generation

-0.5

0.0

0.5

1.0

1.5

Early LateRa

te o

f g

luco

se u

tili

sati

on

(d

9-d

13

)

(pM

/cell

/da

y)

Generation


+ DMSO addition



236

Figure 5.16 Investigating lactate production rates in response to DMSO addition




Section 2.3.2). Supernatant samples taken on days 5, 7, 9 and 13, were analysed for

lactate using an enzymatic assay (described in Section 2.10.2). Rates of production

were determined using the relevant CCT values (for calculations see Section 2.112).

The rate of lactate utilisation is shown. Error bars represent SEM for three biological

replicates. * indicates p<0.05, and ♦ indicates p<0.10, using independent samples t-test

to compare cultures with DMSO to the corresponding control culture without DMSO

addition.


-1.0

-0.5

0.0

0.5

1.0

1.5

Early Late

Ra

te o

f la

cta

te p

ro

du

cti

on

(pM

/cell

/da

y)

Generation


+ DMSO addition



*

♦

♦ *

237

5.7 DISCUSSION

Final antibody titres and Qp (d0-d15) values were increased in response to DMSO, by

approximately 30-50%, regardless of the generation time or fed status of the culture

(Figure 5.1A, Figure 5.2A and Figure 5.3). A similar enhancement of productivity in

response to DMSO addition has been reported for other CHO cultures, with changes in

cell growth as a result of DMSO addition (Li et al, 2006b; Li et al, 2006c; Liu & Chen,

2007b; Ma et al, 2008). Growth arrest was observed for 3.90 cultures supplemented

with DMSO (Figure 5.1B and Figure 5.2B). DMSO-induced growth arrest of CHO and

hybridoma cells has been previously associated with increased expression of CKI p27

and inactivation of Rb kinases, cyclin D2/CDK4 and cyclin E/CDK2, resulting in

induction of p21cip1

(Fiore & Degrassi, 1999; Ponzio et al, 1998). Although G1/G0 cell

cycle inhibition was not apparent for 3.90 cultures in response to DMSO addition

(Figure 5.4), cell cycle arrest has been shown for other CHO cultures as a result of

DMSO addition (Fiore & Degrassi, 1999; Fiore et al, 2002; Li et al, 2006c; Liu & Chen,

2007b; Ponzio et al, 1998). G1 cycle arrest may have been observed for other CHO cell

cultures and not for 3.90 cultures due to differences in culture conditions, including time

of DMSO addition. DMSO (1% [v/v]) addition on day 0 of batch culture was also

investigated (a similar supplement procedure used in previous literature) and although it

suppressed cell growth, it did not altered cell cycle distribution (data not shown).

Changes in G1 phase may not be apparent for 3.90 cultures as the PI staining, used in

determination of cell cycle distribution, does not distinguish G0 and G1 as individually

phases. Although the proportion of cells in G0/G1 were not altered in response to

DMSO addition it may be possible that the ratio of cells in G0 and G1 were affected,

but these changes were not be detected (Sitton & Srienc, 2008). Protocols which

separate cells in G0 and G1, for example, using stains such as acridine orange,

flurorescent antibodies, or staining for specific cyclins, may provided a more useful

method for the elucidation of changes to cell cycle progression in response to DMSO

addition (Darzynkiewicz et al, 1996; Darzynkiewicz et al, 1980a; Darzynkiewicz et al,

1980b; Gerdes et al, 1983).

Previous investigations have suggested that increased Qp as a result of DMSO addition

was due to increased mRNA transcription (Liu et al, 2001). Stratling, found that DMSO

addition increased RNA synthesis and put forward that the effects of DMSO on RNA

238

synthesis were mediated by weakening the interactions between histones and chromatin

subunits (Stratling, 1976). My findings indicate that recombinant mRNA, for both early

and late generation cultures, was not affected by DMSO addition (Figure 5.6), but

changes to polysome profiles suggest protein synthesis may have been increased for

cultures supplemented with DMSO. The increased dissociation, or less association, of

the 80S ribosomal subunit for cultures supplemented with DMSO (Figure 5.7) may have

allowed for increased rates of protein translation (Ashe et al, 2000; Demeshkina et al,

2007; Shenton et al, 2006; Talvas et al, 2006; Volarevic et al, 2000). Although there are

limitations with the quantative measurement of the polysome peaks, such as the

potential variation in the manual baseline of the peaks, initial tritiated leucine

incorporation assays confirmed that global protein synthesis was increased in response

to DMSO addition (Figure 5.17). However, further investigations would be needed to

confirm specific recombinant protein synthesis rates for 3.90 cultures in the presence

and absence of DMSO.

As cultures supplemented with DMSO had greater antibody titres, rates of antibody

secretion must also have been increased. I previously suggested that 3.90 cultures were

experiencing an UPR during batch culture observed with the increased expression of ER

stress markers ATF4, GADD34, GADD153 and XBP-1(s) mRNA and BiP, PDI, ATF4

and GADD153 protein (Section 3.5). In response to DMSO addition GADD153 (Figure

5.9) and XBP-1(s) mRNA (Figure 5.11) was less, possibly as cultures supplemented

with DMSO had a higher degree of protein folding, avoiding an elevated UPR

compared to cultures without DMSO. DMSO has been previously shown to stabilise

proteins in their native conformation and influence protein folding (Yoshida et al,

2002). Investigating protein expression of XBP-1, and the proteins which trigger XBP-1

splicing, such as ATF6, may provide greater insight into regulation of ER stress in

response to DMSO addition. However, as mentioned in Section 4.6 the commercially

available antibodies against these proteins are poor quality.

Although DMSO addition lowered expression of GADD153 mRNA (Figure 5.9) it did

not affect GADD153 protein or the protein expression of PDI or ATF4 (Figure 5.10).

The translation control of GADD153 was only dependent on the „fed status‟ of the

culture. Although the expression of GADD153 mRNA was lower in the presence of

DMSO sufficient transcript was available to allow for GADD153 translation. As

239

discussed in Section 1.8.3.3.2 the regulation of GADD153 is not only dependent on ER

stress but also amino acid limitations within cultures (Bruhat et al, 1997).

Previous literature has shown that DMSO addition to CHO cultures significantly

decreased rates of glucose and amino acid utilisation, and lactate production (Li et al,

2006b; Li et al, 2006c). Although amino acid utilisation for 3.90 cultures was not

affected by DMSO addition the accumulation of glycerol and glycine were lowered in

response to DMSO addition, for both early generation (Figure 5.13) and late generation

(Figure 5.14) cultures. As viable cell densities were also altered as a result of DMSO

addition, relative rates of glycerol and glycine productions (per cell, per day) were

calculated. The average relative rates of both glycerol and glycine productions were

lower for cultures in the presence of DMSO, but the changes were not significant (data

not shown). Decreased extracellular glycerol and glycine accumulation suggests

metabolic changes in response to DMSO addition, with less by-product formation and

altered rates of lactate production/utilisation (Figure 5.16). Lactate concentration in

cultures has been shown to alter with LDH activity (Kim & Lee, 2007). Investigating

LDH may provide a clearer understanding of lactate usage in response to DMSO

addition.

The rates of glucose utilisation were also increased for cultures supplemented with

DMSO, but only during the end (decline) phase of batch culture (Figure 5.15B).

Correlation between glucose utilisation and antibody titres were investigated and it was

found that cultures with the greatest rates of glucose utilisation during the decline phase

of batch culture also had the highest final antibody titres (Figure 5.18). The rates of

glucose utilisation were also dependent on the generation time of culture. Late

generation cultures (Figure 5.18B) had lower rates of glucose utilisation, and lower final

antibody titres, than early generation cultures (Figure 5.18A), with the exception of

cultures supplemented with both feed and DMSO addition. These cultures had similar

rates of glucose utilisation regardless of culture generation time and also had similar

final antibody titres. Enhanced rates of glucose utilisation during the end phase of batch

culture may have allowed for increased glycolytic flux to maintain production of ATP

and relevant intermediates needed for protein synthesis and secretion.

DMSO addition to hybridoma and CHO cultures has also been shown to affect

glycosylation patterns (Hayter et al, 1992; Rodriguez et al, 2005; Tachibana et al, 1994).

240

The glycan profiles for the secreted antibody from 3.90 cultures was not affected by

DMSO, feed addition or LTC, and was similar to other monoclonal antibodies

expressed from CHO cells (Hansen et al, 2010; Wai Lam et al, 2003).

The effects of DMSO addition on productivity, expression of UPR markers and

metabolism are highlighted in Figure 5.19. These alterations in response to DMSO

addition may have been dependent on the culture conditions used for this suspension

cell line, or the time of DMSO addition, or cell line specificity. As there is little

literature discussing the response of DMSO to suspension CHO cells, and to ensure that

the effects seen for 3.90 were not cell line specific, studies were continued with another

suspension antibody-secreting CHO cell line. These findings are discussion in the next

Chapter.

5.8 SUMMARY

DMSO addition increased final antibody titres and Qp values for both early and late

generation cultures. Initial investigations indicated that protein synthesis was increased

in response to DMSO addition, also reflected with changes to the

association/dissociation of the 80S ribosomal subunit. Enhanced protein synthesis may

have been possible due to changes in the metabolic flux of the cultures, observed with

increased rates of glucose utilisation, during the decline phase of batch cultures, and less

by-product accumulation. Active glucose metabolism during the end phase of batch

culture may have allowed cultures supplemented with DMSO to generate more ATP,

potentially enhancing the rates of protein synthesis, protein folding and protein

secretion within this cell line

241

Figure 5.17 Effect of DMSO addition on global protein synthesis

3.90 was cultured as previously described (Figure legend 5.1) Cell suspensions (500µl)

from early generation (≤ 40 generations) and late generation (≥ 60 generations) day 7

cultures (with and without 2% (v/v) DMSO addition) were transferred to 24 well plates

and incubated with L-[4,5-3H] leucine. Protein synthesis and secretion was measured

as the rate of incorporation of L-[4,5-3H] leucine into TCA precipitable-material over a

48 hr time period (Section 2.5.2) The fold increase for intracellular protein was

determined relative to early generation cultures at 0 hrs. Error bars represent SD for

two biological replicates.

Annotation of time-points in Figure 5.17

0

10

20

30

40

50

60

70

no addition + DMSO no addition + DMSO

Early Late

Fo

ld in

crea

se i

n i

ntr

acell

ula

r p

ro

tein

( rela

tiv

e to

ea

rly

gen

era

tio

n v

alu

es

at

0h

rs)


0 hrs

24 hrs

48 hrs

242

Figure 5.18 Correlation between antibody titre and rates of glucose utilisation




Section 2.3.2). Batch culture supernatant samples were analysed by ELISA (Section

2.5.1) for determination of antibody titre and for glucose concentrations using the

enzymatic assay described in Section 2.10.1. The rate of glucose utilisation was

determined from the relevant CCT values (for calculations see Section 2.11.2). Final

antibody titre values are shown together with rates of glucose utilisation (d9-d13) for

early generation cultures (A), and late generation cultures (B). Error bars represent

SEM for three biological replicates.



+ DMSO addition



243

Figure 5.18 Correlation between antibody titre and rates of glucose utilisation

B.

A.

0

200

400

600

800

1000

1200

1400

1600

1800

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Fin

al a

nti

bo

dy

tit

re

(mg

/L)

Utilisation rates (d9-d13)

(pM/cell/day)

0

200

400

600

800

1000

1200

1400

1600

1800

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4

Fin

al a

nti

bo

dy

tit

re

(mg

/L)

Utilisation rates (d9-d13)

(pM/cell/day)

244

Figure 5.19 Alterations to 3.90 cultures in response to DMSO addition

The changes to productivity, expression of UPR stress markers and nutrient utilisation

in the presence of DMSO. In response to DMSO viable cell densities, CCT, the 80S

peak area, the expression of GADD153 and XBP-1(s) mRNA, and glycerol and glycine

accumulation were lowered, recombinant mRNA expression remained unaltered, and

final antibody titres, Qp values and rates of glucose utilisation during the decline (end)

phase of batch cultures were increased. ? indicates that potentially lactate

accumulation was either lowered or lactate re-utilisation was increased in response to

DMSO addition.

Antibody titre

Qp

Glucose utilisation (decline phase)

Lactate re-utilisation ?

Recombinant mRNA


CCT

80S peak area

GADD153 mRNA

XBP-1(s) mRNA

Glycerol accumulation

Glycine accumulation

Lactate accumulation ?

Enhanced in response to DMSO addition

Lowered in response to DMSO addition

245

10

CHAPTER 6

11

CELL LINE 51.69 HAS

CHARACTERISTICS SIMILAR

TO THOSE OF CELL LINE 3.90

246

6. CELL LINE 51.69 HAS CHARACTERISTICS SIMILAR TO THOSE OF

CELL LINE 3.90


The recombinant CHO cell line, 3.90, has been previously characterised in response to

LTC (Chapter 3), feed addition (Chapter 4) and DMSO addition (Chapter 5). 3.90 was

found to be unstable, with a loss in final antibody titre and Qp values between early

generation (generations 20 and 40) and late generation (generations 60, 80 and 100)

cultures (Section 3.2). CD feed (Section 4.2) and DMSO (Section 5.2) additions

increased final antibody titre but did not prevent antibody titre loss in response to LTC.

Data shown in Section 3.4 highlighted an UPR during 3.90 batch culture, indicating

problems in protein folding, or problems in the mechanisms resolving mis/unfolded

proteins. From investigations detailed in Chapters 3, 4 and 5 I found that expression of

GADD153 and XBP-1(s) mRNA were enhanced in response to LTC, and lowered in

response to feed and DMSO addition. The metabolic profiles of cultures were also

altered in response to different culture conditions. Late generation cultures had greater

rates of glucose and lactate utilisation than early generation cultures (Section 3.5).

These data gave indications of altered cellular metabolism between early and late

generation cultures, potentially resulting in less glycolytic and TCA flux, and lower

ATP production. Changes in rates of glucose and lactate production/utilisation were

also observed in response to feed (Section 4.5) and DMSO (Section 5.5) addition.

It is possible that the expression of UPR markers and changes in lactate and glucose

utilisation for 3.90 cultures, in response to LTC, feed and/or DMSO addition were

specific to 3.90. My findings would have greater generic relevance if such events were

characterised for another recombinant CHO cell line. Cell line 51.69 (51.69) was chosen

for the investigations. Although 51.69 was derived from transfections of the same

parental cell host as 3.90, with the same plasmid DNA, 51.69 was generated from a

separate transfection. In this Chapter I have characterised 51.69 in response to LTC,

feed addition and DMSO addition.

247

Characterisation of 51.69 involved investigating growth and antibody titre properties for

both early generation and late generation cultures. As described in the earlier Chapters

the term early generation cultures will refer to batch cultures created at ≤ 40

generations, and the term late generation cultures will refer to batch cultures created at ≥

60 generations.

6.2 ANALYISIS OF CELL LINE 51.69 IN RESPONSE TO LTC

6.2.1 Final antibody titres and viable cell densities were lower as a result of LTC

Cell line 51.69 was shown to be unstable in terms of antibody titre between early and

late generation cultures. Early generation cultures had final antibody titres of

approximately 1000 mg/L (similar to early generation 3.90 cultures, Section 3.2). A

significant 25% decrease in final antibody titre values were observed for late generation

cultures Figure 6.1A). Changes in antibody titres between early and late generation

cultures were apparent from day 11 of batch culture. Growth analysis for cultures

showed that the maximal viable cell densities for early and late generation 51.69

cultures were approximately 4x106 cells/ml (Figure 6.1B). Although the growth patterns

during the exponential and stationary phase of cultures were similar for both early and

late generation cultures it was noticed that during the end (decline) phase of batch

culture late generation cultures had lower viable cell densities than early generation

cultures (observed on day 13 of batch culture). Despite the change in viable cell

densities there was no significant alteration in CCT between early and late generation

cultures (Figure 6.1C). Alterations to viable cell densities and antibody titres were also

observed with decreased Qp (d0-d15) values in response to LTC (Figure 6.1D).

Changes to cell growth in response to LTC were also seen with variations to cell cycle

distribution. Late generation 51.69 cultures, on days 9 and 11 of batch culture, had

fewer cells in G0/G1 cell cycle phase than early generation cultures (Figure 6.2A).

Alterations in the percentage of cells in G0/G1 reflected changes in the proportion of

cells in S and G2/M phase. Late generation cultures had a greater percentage of cells in

S phase on day 11 of batch culture (Figure 6.2B), and a greater percentage of cells in

G2/M phase on day 9 of batch culture (Figure 6.2C). Alterations to cell cycle phase

248

distribution in response to LTC were similar for both 51.69 cultures and 3.90 cultures

(Figure 3.3).

6.2.2 Antibody titre loss was not at the level of recombinant mRNA expression for

51.69

The instability observed for 51.69 cultures in response to LTC was not due to changes

in the expression of heavy chain or light chain mRNA (Figure 6.3). Expression of heavy

chain (Figure 6.3A) and light chain (Figure 6.3B) mRNA increased approximately two-

fold from days 3 to 9 of batch culture, but was similar for both early and late generation

cultures. As the decrease in antibody titre observed between early and late generation

cultures was not due to changes at mRNA level again it was proposed the resultant

decrease in antibody titre in response to LTC was due to changes in cellular events

acting between mRNA expression and protein secretion. These include actions

regulating protein translation and protein folding.

Intracellular protein was measured on day 9 of batch culture using specific antibody-

conjugated dyes which detect the recombinant heavy chain and light chain protein. The

relative intensities of both intracellular heavy chain and light chain protein were not

altered in response to LTC (data not shown). The expression of UPR markers were also

investigated to determine if antibody titre loss between early and late generation

cultures was seen with enhanced cellular stress. From days 3 to 9 of batch culture,

ATF4 (Figure 6.4A) and GADD153 (Figure 6.4B) mRNA increased four-fold and

seven-fold, respectively, but the expression of ATF4 and GADD153 mRNA and protein

(data not shown) was similar for both early and late generation cultures. The up-

regulation of UPR markers suggested that both early and late generation 51.69 cultures

experienced ER stress during batch culture.

249

6.2.3 Late generation 51.69 cultures had greater rates of lactate utilisation

ER stress has been previously linked changes in the metabolic state of the cell during

nutrient deprivation (Lee, 2001; Okada et al, 2002: Harding et al, 2003). Nutrient

starvation occurs during rapid utilisation of amino acids and glucose. Although the rates

of glucose utilisation were similar for early and late generation batch cultures (data not

shown), the rates of lactate utilisation were greater for late generation cultures than for

early generation cultures (Figure 6.5). A greater rate of lactate utilisation for late

generation cultures may be an indication of a metabolic „shift‟ as a result of LTC. A

metabolic „shift‟ could be linked to altered energy production between early and late

generation culture, and the resultant loss in productivity.

250

Figure 6.1 Analysis of recombinant antibody titre, viable cell densities, CCT and

Qp for cell line 51.69

51.69 was subject to long-term culture in suspension using MSX supplemented CD-

CHO media. Batch growth analysis was performed in shake flasks at early generations

(≤ 40 generations) and late generations (≥ 60 generations). Batch cultures were created

at 0.2x106 cells/ml, and maintained at 37

oC, 140 rpm and with a manual supply of 5%

CO2 in air. Antibody titres were measured by ELISA (Section 2.5.1), and viable cell

densities were determined by light microscopy and trypan blue exclusion (Section 2.3.3)

from samples taken routinely during batch culture. Antibody titres (A), viable cell

densities (B), CCT (C) and Qp (d0-d15) (D) are shown. For determination of CCT and

Qp see Section 2.11.2. Error bars represent SEM for three biological replicates. Each


p<0.05, and ♦ indicates p<0.10, using independent samples t-test to compare late

generation cultures to early generation cultures (on the same day of batch culture).


Early generation

Late generation

251

Figure 6.1 Analysis of recombinant antibody titre, viable cell densities, CCT and

Qp for cell line 51.69

B.

C.

A.

0

1

2

3

4

5

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

5

10

15

20

25

30

35

40

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

l)

Day

0

400

800

1200

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

D.

0

5

10

15

20

25

30

Qp (d0-d15)

Sp

ecif

ic p

ro

du

cti

vit

y (

Qp

)

(pg

/cell

/da

y)

* * *

*

♦

252

Figure 6.2 The percentage of cells in G0/G1 was lower for late generation 51.69

cultures

51.69 was cultured as previously stated (Figure legend 6.1). Batch cultures were created

at early generations (≤ 40 generations) and late generations (≥ 60 generations). 1x106

cells, taken on days 3, 5, 7, 9, and 11 of batch culture, were analysed by flow cytometry

using PI excitation by a 488nm laser, and emission measured by a 613/20nm bandpass

filter (Section 2.4.1). The data was analysed by Summit 4.3 and ModFit LT software. The

percentage of cells in A, G0/G1 phase, B, S phase, and C, G2/M phase are shown. Error

bars represent SEM for three biological replicates. * indicates p<0.05, using independent

samples t-test to compare late generation cultures to early generation cultures (on the

same day of batch culture).


Early generation

Late generation

253

Figure 6.2 The percentage of cells in G0/G1 was lower for late generation 51.69

cultures

B.

C.

A.

0

25

50

75

100

3 5 7 9 11

G0

/G1

cell

cy

cle

ph

ase

(%)

Day

0

25

50

75

100

3 5 7 9 11

S cell

cy

cle

ph

ase

(%)

Day

0

25

50

75

100

3 5 7 9 11

G2

cell

cy

cle

ph

ase

(%)

Day

* *

*

*

254

Figure 6.3 Expression of recombinant mRNA was not altered in response to LTC



mRNA levels were compared using q-RTPCR from samples taken on days 3, 5, 7 and 9

of batch culture (as detailed in Section 2.7.1), using the mRNA specific primer sets for

A, heavy chain, and B, light chain. Samples were normalised using mRNA β-Actin

primers. Error bars represent SEM for three biological replicates.


B.

A.

0

50

100

150

200

3 5 7 9

Hea

vy

ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Day

0

50

100

150

200

3 5 7 9

Lig

ht ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

Day

Early generation

Late generation

255

Figure 6.4 ATF4 and GADD153 mRNA increased during batch culture



mRNA levels were compared using q-RTPCR from samples taken on days 3, 5, 7 and 9

of batch culture (Section 2.7.1), using the mRNA specific primer sets for A, ATF4, and

B, GADD153. Samples were normalised using mRNA β-Actin primers. Error bars



B.

A.

0

50

100

150

200

250

3 5 7 9

AT

F4

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Day

0

50

100

150

200

3 5 7 9

GA

DD

15

3 m

RN

A e

xp

ress

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Day

Early generation

Late generation

256

Figure 6.5 Late generation cultures had greater rates of lactate utilisation



Supernatant samples taken on days 5, 7, 9 and 13 of batch culture (Section 2.3.2) were

analysed for lactate using an enzymatic assay (described in Section 2.10.2). Rates of

lactate utilisation were determined using the relevant CCT values (for calculations see

Section 2.11.2). The rate of lactate utilisation is shown for days 5 to 13 of batch culture.



-0.1

0

0.1

0.2

0.3

0.4

0.5

Ra

te o

f la

cta

teu

tili

sati

on

(pM

/cell

/da

y)

Generation

Early generation

Late generation

257

6.3 ANALYSIS OF CELL LINE 51.69 IN RESPONSE TO FEED ADDITION

Initial productivity analysis showed that 51.69 was unstable in response to LTC. The

change in productivity between early and late generation 51.69 cultures was similar to

the decrease in productivity observed between early and late generation 3.90 cultures.

The upregulation of UPR markers during batch culture and the differences in viable cell

densities, cell cycle distribution and lactate utilisation rates between early and late

generation cultures were also similar for exemplar cell lines 51.69 and 3.90. Analysis

was continued to determine if the characteristics described in Section 6.2 were altered in

response to different culture conditions. In this Section growth, productivity, markers of

ER stress and rates of glucose and lactate utilisation were investigated in response to a

CD feed addition (the same feed and feeding regime previously described in Section

2.3.2 and Chapter 4).

6.3.1 Feed addition increased recombinant protein production

Feed addition significantly increased antibody titres from day 11 and day 9 of batch

culture for early generation cultures and late generation cultures, respectively (Figure

6.6A). In response to feed addition final antibody titres were increased by

approximately 30% for both early and late generation cultures. However, cultures

supplemented with feed still experienced an approximate 25% decrease in antibody titre

in response to LTC. For both early and late generations cultures feed addition increased

antibody titres without affecting viable cell densities (Figure 6.6B). Qp was also

significantly increased, as a consequent of feed addition enhancing antibody titres

without altering viable cell growth. Qp (d0-d15) was approximately two-fold greater in

response to feed addition, for both early and late generation cultures (Figure 6.7).

Although the CD feed was added during the exponential phase of culture Qp (d0-d7)

was not affected by feeding, instead the feed addition had the greatest influence on Qp

during the decline phase of batch culture. In the presence of feed Qp (d9-d15) was

increased three-fold and two-fold for early and late generation cultures, respectively

(Figure 6.7).

258

6.3.2 Feed addition significantly lowered GADD153 mRNA and protein expression

Investigations were continued to determine the regulation of events at molecular level in

response to feed addition. Recombinant mRNA was similar for all cultures, regardless

of feed addition (data not shown). However, ATF4, GADD34 and GADD153 mRNA

expression was found to be significantly three-fold to four-fold lower in response to

feed addition (Figure 6.8). Although the expression of PDI and ATF4 protein was not

affected by feeding, GADD153 protein was significantly less in response to feed

addition (Figure 6.9). GADD153 protein was approximately four-fold lower for cultures

in the presence of feed compared to cultures without feed addition. Lower XBP-1(s)

mRNA (Figure 6.10) and GADD153 protein expression indicates 51.69 cultures in the

presence of feed had less ER stress than cultures without feed addition.

6.3.3 Metabolic profiles were altered for 51.69 in response to feed addition

As productivity and ER stress was altered in response to feed addition, metabolic

changes were also investigated at the level of glucose and lactate utilisation. As the

feeding regime involved addition of feed to cultures during the exponential stage of

batch culture I only investigated the rates glucose utilisation from the stationary phase

of batch culture. The rates of glucose utilisation were slightly greater during this period

of batch culture in response to feed addition (Figure 6.11A). Lactate re-utilisation was

also not apparent for cultures supplemented with feed, but rates of lactate production

were ultimately dependent on generation time of culture. In the presence of feed late

generation cultures either had lower rates of lactate production than early generation

cultures, or late generation cultures produced lactate then utilised lactate at a greater rate

than early generation cultures. However, due to changes in the net rate of lactate

production, alterations to lactate utilisation were not seen (Figure 6.11B). Either

suggestion indicates the possibility of a metabolic „shift‟ as a result of LTC.

259

Figure 6.6 Feed addition increased final antibody titres for cell line 51.69



A CD Feed was added to the relevant cultures during the exponential phase of batch

culture (as described in Section 2.3.2). Antibody titres were measured by ELISA

(Section 2.5.1), and viable cell densities were determined by light microscopy and

trypan blue exclusion (Section 2.3.3) from samples taken routinely during batch culture.

Antibody titres (A) and viable cell densities (B) are shown. Error bars represent SEM

for three biological replicates. Each biological replicate value is an average from

duplicate technical repeats. * indicates p<0.05, and ♦ indicates p<0.10, using


control culture with no addition (on the same day of batch culture).


B.

A.

0

400

800

1200

1600

2000

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

0

1

2

3

4

5

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day





* *

*

♦

* *

*

260

Figure 6.7 Feed addition enhanced specific productivity (Qp) for cell line 51.69

This figure compares Qp values for early generation (≤ 40 generations) and late

generation (≥ 60 generations) cultures, with and without feed addition. Qp was

calculated for the entire batch culture, using antibody titre and cell density

measurements from days 0 to 15 of culture, Qp (d0-d15). Qp was also calculated for the

early (exponential) phase of batch culture, using antibody titre and cell density

measurements from days 0 to 7 of culture, Qp (d0-d7), and for the end (decline) phase

of batch culture, using antibody titre and cell density measurements from days 9 to 15

of culture, Qp (d9-d15). For determination of Qp see Section 2.11.2. Error bars

represent SEM for three biological replicates. Each biological replicate value is an

average from duplicate technical repeats. * indicates p<0.05, using independent

samples t-test to compare cultures with feed addition to the corresponding control

culture with no addition (during the same period of batch culture).

Annotation for Figure 6.7

0

10

20

30

40

50

60

70


Early Late

Sp

ecif

ic p

ro

du

cti

vit

y (

Qp

)

(pg

/cell

/da

y)


Qp (d0-d15)

Qp (d0-d7)

Qp (d9-d15)

*

*

*

*

261

Figure 6.8 The mRNA expression of UPR markers were lower for cultures with

feed addition



A CD feed was added to the relevant cultures during the exponential phase of batch



mRNA specific primer sets for ATF4, GADD34 and GADD153. Samples were


replicates. * indicates p<0.05, using independent samples t-test, to compare cultures

with feed addition to the corresponding control culture with no addition.


0

50

100

150

200

250

300

ATF4 GADD34 GADD153

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

UPR maker





* * *

* *

*

262

Figure 6.9 GADD153 protein was significantly lowered in response to feed addition



A CD feed was added to the relevant cultures during the exponential stage of batch

culture (Section 2.3.2). Protein was extracted on day 9 of batch culture (as detailed in

Section 2.5.3.1). 60µg protein was separated by SDS-PAGE (Section 2.5.3.2),

transferred (Section 2.5.3.3) and then detected using anti-rabbit polyclonal PDI, ATF4

and GADD153 antibodies. All membranes were stripped and re-probed using an anti-

mouse pan ERK antibody (Section 2.5.3.4). Bands were examined using Image J

software, and the amount of PDI, ATF and GADD153 protein expression was

calculated relative to ERK expression (Section 2.5.3.5). Error bars represent SEM for

three biological replicates. * indicates p<0.05, using independent samples t-test, to

compare cultures with feed addition to the corresponding control culture with no

addition.


0

50

100

150

200

250

300

PDI ATF4 GADD153

Pro

tein

in

ten

sity

(rela

tiv

be to

ER

K s

tan

da

rd

)

UPR maker





* *

263

Figure 6.10 XBP-1(s) mRNA was less after feed addition




culture (as described in Section 2.3.2). cDNA was synthesised from RNA extracted on

day 9 of batch culture (Section 2.7.1.3) and the PCR was performed using the newly

synthesised cDNA and the XBP-1(s) primers (Section 2.7.2). The PCR products were

electrophoresed on a 2% (w/v) agarose gel and visualised by UV transillumination

(Section 2.6.1.4). The product bands were analysed using Image J software, and the

quantified ratio of spliced XBP-1mRNA to total XBP-1mRNA is shown Error bars

represent SEM for three biological replicates. ♦ indicates p<0.10, using independent

samples t-test to compare cultures with feed addition to the corresponding control

culture with no addition.


0

10

20

30

40

50

Early Late

Am

ou

nt

of

spli

ced

XB

P-1

mR

NA

to t

ota

l X

BP

-1 m

RN

A

(%)

Generation


+ feed adidition

♦

264

Figure 6.11 Analysis of glucose and lactate utilisation rates in response to feed

addition




culture (as described in Section 2.3.2). Supernatant samples taken on days 5, 7, 9 and

13 of batch culture were analysed enzymatically to determine the concentration of

glucose (Section 2.10.1) and lactate (Section 2.10.2). The rates of utilisation and

production were determined using the relevant CCT values (using the calculation

described in Section 2.11.2). Error bars represent SEM for three biological replicates.


B.

A.

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Early Late

Ra

te o

f la

cta

te p

ro

du

cti

on

(d5

-d1

3)

(pM

/cell

/da

y)

Generation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Early Late

Ra

te o

f lg

luco

seu

tili

sati

on

(d9

-d1

3)

(pM

/cell

/da

y)

Generation


+ feed adidition

265

6.4 ANALYSIS OF CELL LINE 51.69 IN RESPONSE TO DMSO ADDITION

Characterisation analysis showed that 51.69 was unstable in response to LTC. Late

generation cultures had 25% lower final antibody titres than early generation cultures

(Section 6.2.1). Late generation cultures also had less cells in G0/G1 cell cycle phase

(Figure 6.2), and greater rates of lactate utilisation (Figure 6.5) than early generation

cultures. Although the expression of UPR markers (including expression of ATF4,

GADD34, and GADD153 mRNA and PDI, ATF4 and GADD153 protein) were

increased during batch culture, the expression remained similar for both early and late

generation cultures (Section 6.2.2). In response to feed addition the expression levels of

ATF4, GADD34, GADD153, and XBP-1(s) mRNA and GADD153 protein were lower

(Section 6.3.2), and changes in lactate production and re-utilisation were observed

(Section 6.3.3). In this Section growth, productivity, markers of ER stress and rates of

glucose and lactate metabolism were investigated in response to a DMSO addition, to

determine if antibody titres for 51.69 cultures could be further enhanced, and

maintained at a stable concentration during LTC

6.4.1 Cell line 51.69 encountered growth arrest in response to DMSO addition

The final antibody titres for early generation cultures were not affected by DMSO

addition (Figure 6.12A), but changes in cell growth were observed in response to

DMSO (Figure 6.12B). Cultures supplemented with DMSO had altered patterns of

growth, observed with an extended stationary phase of batch culture and lower maximal

viable cell densities. The effect of DMSO on cell growth consequently altered cell

biomass, resulting in lower CCT values (Figure 6.12C).

Antibody titres for late generation cultures were not enhanced with DMSO addition

alone and were only increased for cultures supplemented with both feed and DMSO

additions (Figure 6.13A, observed from day 11 of batch culture). Late generation

cultures also had decreased viable cell densities (observed on day 9 of batch culture,

Figure 6.13B), and CCT values (Figure 6.13C), in response to DMSO addition. The

effect of DMSO on cell growth and antibody titres subsequently altered antibody

production rates per cell. Qp (d0-d15) was increased by approximately 30% for both

early and late generation cultures in response to DMSO addition (Figure 6.14).

266

6.4.2 GADD153 mRNA and protein expression was significantly lowered in

response to DMSO addition

Cultures with DMSO had relatively similar expression of heavy chain mRNA (Figure

6.15A) and light chain mRNA (Figure 6.15B) compared to the mRNA expression from

the respective control cultures. Therefore changes in productivity were not due to

differences in the expression of the antibody transcript. As mentioned in previous

Chapters an increase in antibody titre, in response to feed and DMSO addition, was

dependent on cultures undergoing increased rates of antibody secretion. Secretion may

be enhanced for cultures experiencing low levels of ER stress. The expressions of ER

stress markers were investigated and although the expression of ATF4 (Figure 6.15C)

and GADD34 (Figure 6.15D) mRNA was not altered in response to DMSO, the

expression of GADD153 mRNA and protein was less (Figure 6.16). In response to

DMSO addition GADD153 mRNA was 39% and 34% lower for early generation and

late generation cultures (with no additions), respectively (Figure 6.16A). GADD153

mRNA, however, was not altered when DMSO was added to cultures already

supplemented with feed, possibly as feeding alone was sufficient in maintaining low

expression of GADD153 mRNA.

The effect of DMSO addition on GADD153 mRNA was also reflected at protein level.

GADD153 protein for both early and late generation cultures was approximately 50%

less in response to DMSO addition. Cultures supplemented with feed had similar levels

of GADD153 protein, independent of DMSO addition (Figure 6.16B). An example of

the regulation of GADD153 protein in response to the different culture conditions is

shown in Figure 6.16C. The alteration in GADD153 suggested that cultures with

DMSO had less cellular stress than cultures without DMSO.

Other cell stress pathways were also altered in response to DMSO. Ratios of XBP-1(s)

mRNA to total XBP-1 mRNA were also lower for cultures supplemented with DMSO

(Figure 6.17A). The regulation of XBP-1(s) mRNA in response to DMSO addition is

shown in Figure 6.17B. Investigations showed that DMSO addition to cultures resulted

in lower expression of ER stress markers, potentially due to greater protein folding in

267

response to DMSO. Increased protein folding would result in less ER stress and

enhanced protein secretion.

6.4.3 DMSO addition increased the rates of glucose utilisation for 51.69 cultures

As cellular stress was lowered in response to DMSO addition metabolic changes were

also investigated to determine if possible metabolic changes could be responsible for a

higher degree of protein folding. The rate of glucose utilisation (d9-d13) was increased

in response to DMSO (Figure 6.18A), possibly as glucose metabolism was still active

during the end (decline) phase of batch culture for cultures with DMSO. In response to

DMSO addition glucose utilisation rates were increased two-fold for early generations

cultures (without feed supplements), and three-fold for late generation cultures

(regardless of feed addition).

Early generation cultures in the presence of DMSO had slightly increased lactate

utilisation rates, whilst late generation cultures had similar rates of lactate

production/utilisation, independent of DMSO addition (Figure 6.18B). The increased

rate of glucose utilisation and the changes to lactate production in response to DMSO

are either directly or indirectly related to a more productive cell.

268

Figure 6.12 DMSO addition to for early generation 51.69 cultures did not enhance

antibody titres


created at early generations (≤ 40 generations). A CD feed and 2% (v/v) DMSO was

added to the relevant cultures, as described in Section 2.3.2. Antibody titres were

measured by ELISA (Section 2.5.1), and viable cell densities were determined by light





technical repeats. * indicates p<0.05, using independent samples t-test to compare





+ DMSO addition



Figure 6.12 DMSO addition to for early generation 51.69 cultures did not enhance

antibody titres

B.

C.

A.

0

1

2

3

4

5

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

0

5

10

15

20

25

30

35

40

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

l)

Day

0

400

800

1200

1600

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

* *

270

Figure 6.13 Antibody titres were enhanced for late generation 51.69 cultures in the

presence of feed and DMSO



added to the relevant cultures, as described in Section 2.3.2. Antibody titres were






technical repeats. * indicates p<0.05, using independent samples t-test to compare





+ DMSO addition



271

Figure 6.13 Antibody titres were enhanced for late generation 51.69 cultures in the

presence of feed and DMSO

B.

C.

A.

0

400

800

1200

1600

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

0

5

10

15

20

25

30

35

40

0 3 5 7 9 11 13 15

Cu

mu

lati

ve c

ell

tim

e

(x1

06

cell

s x

da

y/m

l)

Day

0

1

2

3

4

5

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

* *

* *

*

272

Figure 6.14 Qp values were increased in response to DMSO addition

Specific productivity (Qp) was determined from the antibody tire and the growth of

early generation (≤ 40 generations) and late generation (≥ 60 generations) batch

cultures seen in Figure 6.13 and Figure 6.14, respectively. Qp was calculated for the

entire batch culture, using antibody titre and cell density measurements from days 0 to

15 of culture, Qp (d0-d15). For determination of Qp see Section 2.11.2. Error bars

represent SEM for three biological replicates. Each biological replicate value is an

average from duplicate technical repeats. * indicates p<0.05 and ♦ indicates p<0.10,

using independent samples t-test to compare cultures with DMSO to the corresponding



0

10

20

30

40

50

60

70

Early Late

Sp

ecif

ic p

ro

du

cti

vit

y (

Qp

)

(pg

/cell

/da

y)

Generation


+ DMSO addition



*

♦ ♦

273

Figure 6.15 Recombinant mRNA expression was not altered in response to DMSO



A CD feed and 2% (v/v) DMSO was added to the relevant cultures, as described in

Section 2.3.2. mRNA levels were compared using q-RTPCR from samples taken on day

9 of batch culture (as detailed in Section 2.7.1), using the mRNA specific primer sets for

A, heavy chain, B, light chain, C, ATF4 and D, GADD34. Samples were normalised

using mRNA β-Actin primers. Error bars represent SEM for three biological replicates.



+ DMSO addition



274

Figure 6.15 Recombinant mRNA expression was not altered in response to DMSO

A.

B.

C.

0

50

100

150

200

250

300

350

Early Late

GA

DD

34

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Generation

D.

0

50

100

150

200

250

Early LateLig

ht ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Generation

0

50

100

150

200

250

300

Early Late

AT

F4

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Generation

0

50

100

150

200

250

Early Late

Hea

vy

ch

ain

mR

NA

ex

press

ion

(% r

ela

tiv

e to

sta

nd

ard

an

d

β-A

cti

n m

RN

A e

xp

ress

ion

)

Generation

275

Figure 6.16 DMSO addition to 51.69 cultures lowered GADD153 expression

51.69 was cultured as previously described (Figure legend 6.1). A CD feed and 2%

(v/v) DMSO was added to the relevant culture, as described in Section 2.3.2. mRNA and

protein was isolated on day 9 of batch culture. mRNA levels were compared using q-

RTPCR (as detailed in Section 2.7.1), using the mRNA specific primer sets for

GADD153. Samples were normalised using mRNA β-Actin primers. Protein was

extracted on days 9 of batch culture (as detailed in Section 2.5.3.1). 60µg protein was

separated by SDS-PAGE (Section 2.5.3.2), transferred (Section 2.5.3.3) and then

detected using an anti-rabbit polyclonal GADD153 antibody. Membranes were stripped

and re-probed using an anti-mouse pan ERK antibody (Section 2.5.3.4). Bands were

analysed using Image J software, and the amount of GADD153 protein expression was

calculated relative to ERK expression (Section 2.5.3.5). A, shows the relative intensity

of GADD153 mRNA expression, and B, shows the relative intensity of GADD153

protein expression. The regulation of GADD153 protein for the different culture

conditions is also shown (C) Error bars represent SEM for three biological replicates. *

indicates p<0.05 and ♦ indicates p<0.10, using independent samples t-test to compare

cultures with DMSO to the corresponding control culture without DMSO addition.



+ DMSO addition



276

Figure 6.16 DMSO addition to 51.69 cultures lowered GADD153 expression

A.

0

50

100

150

200

250

Early Late

GA

DD

15

3 m

RN

A e

xp

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(% r

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)

Generation

B.

0

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Early Late

GA

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3 p

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in

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Generation

C.

Early Late

GADD153

ERK

Feed addition

Generation

DMSO addition- + - + - + - +

- - + + - - + +

*

♦

*

277

Figure 6.17 XBP-1(s) mRNA was lowered in response to DMSO



A CD feed and 2% (v/v) DMSO was added to the relevant culture, as described in

Section 2.3.2. cDNA was synthesised from RNA extracted on day 9 of batch culture

(Section 2.7.1.3) and the PCR was performed using the newly synthesised cDNA and

XBP-1(s) primers (Section 2.7.2). The PCR products were electrophoresed on a 2%

(w/v) agarose gel and visualised by UV transillumination (Section 2.6.1.4). Bands were

analysed using Image J software and the ratio of spliced XBP-1 mRNA to total XBP-1

mRNA is shown. Error bars represent SEM for three biological replicates. * indicates

p<0.05 and ♦ indicates p<0.10, using independent samples t-test to compare cultures

with DMSO to the corresponding control culture without DMSO addition.



+ DMSO addition



278

Figure 6.17 XBP-1(s) mRNA was lowered in response to DMSO

0

10

20

30

40

50

60

Early Late

Ra

tio

of

spli

ced

XB

P-1

mR

NA

to to

tal

XB

P-1

mR

NA

(%)

A.

B.

Early Late

Feed addition

Generation

DMSO addition- + - + - + - +

- - + + - - + +

♦

♦ *

♦

279

Figure 6.18 Rates of glucose utilisation were increased for 51.69 cultures in the

presence of DMSO



A CD feed and 2% (v/v) DMSO was added to the relevant culture, as described in

Section 2.3.2. Supernatant samples taken on days 5, 7, 9 and 13 of batch culture were

analysed enzymatically to determine the concentration of glucose (Section 2.10.1) and

lactate (Section 2.10.2). The rates of utilisation and production were determined using

the relevant CCT values (for calculation see Section 2.11.2). Error bars represent SEM

for three biological replicates. * indicates p<0.05, and ♦ indicates p<0.10, using

independent samples t-test to compare cultures with DMSO to the corresponding




+ DMSO addition



280

Figure 6.18 Rates of glucose utilisation were increased for 51.69 cultures in the

presence of DMSO

B.

A.

0

0.5

1

1.5

2

2.5

Early Late

Ra

te o

f lg

luco

seu

tili

sati

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(d9

-d1

3)

(pM

/cell

/da

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Generation

-1

-0.5

0

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Early Late

Ra

te o

f la

cta

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ro

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(d5

-d1

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(pM

/cell

/da

y)

Generation

♦

* ♦

281

6.5 DISCUSSION

Analysis of 3.90 showed this cell line to be unstable in response to LTC (Chapter 3).

Feed (Chapter 4) and DMSO addition (Chapter 5) increased productivity for this cell

line. To ensure the changes to 3.90 were not cell line-specific another cell line 51.69

was characterised in response to LTC (Section 6.2), feed addition (Section 6.3) and

DMSO addition (Section 6.4).

6.5.1 How does 51.69 compare to 3.90 in response to LTC?

Antibody titre values for early and late generation 51.69 cultures (Figure 6.1A) were

similar to the respective 3.90 culture (Figure 3.1A). However, Qp values were greater

for 51.69 cultures by approximately 10 pg/cell/day (regardless of generation time of

culture), as 51.69 cultures were capable of achieving the same antibody titres with fewer

viable cells (Figure 6.1B) than 3.90 cultures (Figure 3.1B). Although maximal viable

cell densities were different between the two cell lines, cell cycle distribution between

51.69 (Figure 6.2) and 3.90 (Figure 3.3) was similar. As discussed in Chapter 3

alterations to cell cycle transition between early and late generation cultures could be

due to changes in cell growth, however, there is also the possibility that the differences

in cell cycle distribution between early and late generation cultures was due to

alterations in ER stress regulation. An UPR can inhibit translation of cyclin D, causing

cells to halt in G1 cell cycle phase (Brewer et al, 1999). Halting cells in G0/G1 may

provide the cell with a time window to decide whether conditions favour cell survival or

cell death (Niwa & Walter, 2000). The increase in G0/G1 for early generation cultures

may be a route to prevent these cultures from entering cell death pathways.

51.69 cultures experienced ER stress during batch culture, with expression of UPR

markers dependent on the generation time of cultures (these findings are summarised in

Figure 6.19). Late generation 51.69 cultures, on day 7 of batch culture, had increased

GADD153 mRNA compared to early generation 51.69 cultures (Figure 6.4B). Similar

findings were shown for 3.90 cultures in Section 3.4. Lower levels of ER stress for early

generation cultures would allow for increased rates of protein secretion (Malhotra et al,

2008), observed with greater antibody titres. Increased ER stress in response to LTC

could remain a possible explanation for the changes to antibody titres between early and

282

late generation cultures. Enhanced ER stress may also be the result of nutrient

limitations and changes to the metabolic activity of the cell.

Late generation 51.69 cultures had greater rates of lactate utilisation than early

generation 51.69 cultures (Figure 6.5). Similar to the rates of lactate utilisation observed

for 3.90 early and late generation cultures (Figure 3.21C). The potential linkage

between metabolic changes and LTC has been discussed previously in Section 3.6.

Further investigations of alanine production and re-utilisation for cell line 51.69 would

be needed to confirm the increased production of extracellular alanine as a marker of

poor protein expression within these cell lines.

6.5.2 How does 51.69 compare to 3.90 in response to feed addition?

Antibody titre values from cultures in the presence of feed were similar for both cell

lines, however, due to differences in cell biomass Qp (d0-d15) values in the presence of

feed were two-fold greater for 51.69 cultures (Figure 6.7) than 3.90 cultures (Figure

4.5A). As observed for 3.90 cultures feed addition had the greatest impact on Qp for

51.69 cultures during the end (decline) phase of batch culture. The addition of feed to

cultures increased productivity whilst lowering the relative expression of ATF4,

GADD34, and GADD153 mRNA (Figure 6.8), XBP-1(s) mRNA (Figure 6.10) and

GADD153 protein (Figure 6.9). The relative expression of the ER stress markers for

51.69 cultures in response to feed addition was similar to the expression observed for

3.90 cultures (Section 4.4). Explanations for the lower expression of these markers in

response to feed addition have been described previously in Section 4.6.

Addition of feed to 51.69 cultures did not prevent loss of productivity in response to

LTC, also shown for 3.90 cultures (Chapter 4). In the presence of feed late generation

51.69 cultures had lower antibody titres (Figure 6.6A) and Qp values (Figure 6.7) than

early generation 51.69 cultures. It is possible that flux through metabolic pathways was

altered in response to LTC, consequently affecting antibody synthesis and secretion.

However, further investigations, such as intracellular metabolite analysis, would be

needed to confirm metabolic changes. Ultimately investigations with cell line 51.69

show that findings observed for 3.90 in response to feed addition (Figure 4.19) were not

283

specific for this cell line as they were also apparent for 51.69. These findings are

summarised in Figure 6.20.

6.5.3 How does 51.69 compare to 3.90 in response to DMSO addition?

Initial investigation found that 2% (v/v) DMSO addition had the greatest influence on

productivity for 51.69 cultures compared to the other DMSO concentrations

investigated (data not shown). Although the addition of DMSO to 3.90 cultures

increased antibodies titres, regardless of generation time of culture or fed culture

condition (Section 5.2), addition of DMSO to 51.69 cultures did not enhance antibody

titre values for early generation 51.69 cultures (Figure 6.12), and only increased

antibody titres for late generation 51.69 cultures when added to cultures that were also

undergoing feed additions (Figure 6.13). However, due to changes in cell biomass the

fold change in Qp for 51.69 cultures in the presence of DMSO (Figure 6.14) was similar

to the fold change in Qp for 3.90 cultures with DMSO (Figure 5.3).

Other alterations characterised for 3.90 cultures in response DMSO addition were also

observed for 51.69 cultures (detailed in Figure 6.21). Expression of GADD153 (Figure

6.16) and XBP-1(s) (Figure 6.17) mRNA was less in response to DMSO. As ATF4

protein expression was not affected by DMSO addition (data not shown) lower

expression of GADD153 mRNA could be the result of less XBP-1 splicing, or

decreased activation of other UPR markers such as ATF6 (Yoshida et al, 2001;Yoshida

et al, 2003). A lower expression of stress markers in response to DMSO addition could

also be the result of metabolic changes. 51.69 cultures in the presence of DMSO

showed altered rates of glucose and lactate utilisation (Figure 6.18), similar to findings

for 3.90 cultures in response to DMSO addition (Figure 5.15 and 5.16). As previously

suggested in Section 5.6.3, less lactate production and increased glucose utilisation

during the end (decline) phase of batch culture may have provided the cultures with a

beneficial metabolic shift allowing for increased Qp values. Detailed metabolic flux

analysis would be needed to confirm metabolic changes in response to DMSO addition

(Goudar et al, 2009; Omasa et al, 2010; Quek et al, 2010).

284

6.6 SUMMARY

The response of cell line 51.69 to LTC, feed addition and DMSO addition gave similar

characteristics to those previously identified for cell line 3.90. Late generation 51.69

cultures had fewer cells in G0/G1, less GADD34 mRNA, increased GADD153 and

XBP-1(s) mRNA, and greater lactate utilisation than early generation 51.69 cultures.

Cultures in the presence of feed had lower expression of ATF4, GADD34, GADD153

and XBP-1(s) mRNA and less GADD153 protein, whilst cultures in the presence of

DMSO addition had lower expression of GADD153 and XBP-1(s) mRNA and less

GADD153 protein. Both feed and DMSO additions resulted in alterations to glucose

utilisation and lactate accumulation, but further investigations would be needed to

determine the metabolic changes as a result of LTC, feed and DMSO addition.

285

Figure 6.19 Alterations to nutrient utilisation, ER stress markers and antibody

titre for cell line 51.69 in response to LTC

Cell line 51.69 had altered characteristics in response to LTC. Characteristics of

antibody titre loss were examined for alterations to cell growth, cellular stress and

metabolism during batch culture for early and late generation cultures. The changes in

these markers were possibly related to the decrease in antibody titre values seen for late

generation cultures during the end (decline) phase of batch culture. indicates a

significant change for late generation cultures compared early generation cultures

(p<0.10 using independent samples t-test).


Day 0 3 5 7 9 11 13 15

Antibody titre

Percentage of cells in G0/G1

GADD153 mRNA*

*

Rates of lactate utilisation

Increased values

Decreased values

286

Figure 6.20 Alterations to 51.69 cultures in response to feed addition

The changes to productivity, expression of ER stress markers and nutrient utilisation in

the presence of feed. In response to feed addition the expression of ER stress markers

and lactate re-utilisation were lowered, viable cell densities, CCT and recombinant

mRNA expression remained unaltered, and antibody titre and Qp values were

increased.

Antibody titre

Qp


CCT

Recombinant mRNA

ATF4, GADD34 mRNA


XBP-1(s) mRNA

Lactate re-utilisation

Enhanced in response to feed addition

Lowered in response to feed addition

287

Figure 6.21 Alterations to 51.69 cultures in response to DMSO addition

The changes to productivity, expression of ER stress markers and nutrient utilisation in

the presence of DMSO. In response to DMSO addition viable cell densities, CCT, and

the expression of GADD153 and XBP-1(s) mRNA were less, recombinant mRNA and

ATF and GADD34 mRNA remained unaltered, and antibody titre and Qp values were

increased. Lactate accumulation was either lowered in response to DMSO addition or

rates of lactate re-utilisation were increased. ? indicates that potentially lactate

accumulation was either lowered or lactate re-utilisation was increased in response to

DMSO addition.

Qp

Glucose utilisation (decline phase)

Lactate re-utilisation ?

Recombinant mRNA

ATF4, GADD34 mRNA


CCT


XBP-1(s) mRNA

Lactate accumulation ?

Enhanced in response to DMSO addition

Lowered in response to DMSO addition

288

12

CHAPTER 6

13

OVERALL DISCUSSION

289

7. OVERALL DISCUSSION

The results in this thesis have been presented in four discrete chapters, and as such

detailed discussions have been made at the end of each Chapter. In summary, the key

findings described in this study are:

Characterisation studies on an exemplar cell line (3.90) highlighted instability,

observed with decreased final antibody titres and Qp values. The key findings of

this cell line were replicated with a second exemplar cell line (51.69).

The instability was not due to loss of recombinant gene copies or lower

expression of recombinant mRNA.

Instability was associated with decreased CCT values, increased rates of lactate

utilisation, a lower proportion of cells in G0/G1 and greater expression of

GADD153 and XBP-1 mRNA.

The exemplar cell line experienced ER stress during batch culture, defined in

terms of increased ATF4, GADD34, GADD153 and XBP-1(s) mRNA, and

increased ATF4, GADD153, BiP and PDI protein during the course of the batch

culture.

Feed additions improved antibody titres and Qp values but did not reverse the

instability observed in response to LTC.

Feed addition did not increase expression of recombinant mRNA, but did

increase antibody titre.

The improvement in recombinant protein production in response to feed addition

was associated with decreased ER stress. This was exemplified by less ATF4,

GADD34, XBP-1(s) and GADD153 mRNA, and lower GADD153 protein

expression.

The addition of feed to cultures also altered the metabolic profile of the cultures,

resulting in less glycine accumulation, and prevention of alanine or lactate re-

utilisation.

The addition of DMSO to cultures increased antibody titres and Qp values,

whilst suppressing cell growth, without an increased expression of recombinant

mRNA

Cultures with DMSO had lower 80S polysome peak areas.

290

In response to DMSO addition, expression of GADD153 and XBP-1(s) mRNA

was decreased.

DMSO addition also altered the metabolic profiles of the cultures. DMSO

addition altered rates of lactate production and increased glucose utilisation

during the end (decline) phase of batch culture.

The overall findings observed with the exemplar cell line 3.90, including

changes to antibody titre, cell cycle distribution, ER stress markers, and rates of

glucose and lactate utilisation in response to LTC, feed and DMSO addition

were similar for a second exemplar cell line, 51.69. The effects were not cell line

specific.

From the findings listed above it is apparent that the main aims stated at the start of this

project have been achieved (Section 1.11). In addition to the main aims of this project,

several questions were established, which will also be considered during this overall

discussion.

7.1 IS INSTABILITY CONNECTED TO A SPECIFIC CELLULAR EVENT?

As summarised for 3.90 and 51.69 cultures in Figures 3.25 and 6.19, respectively,

instability, in terms of a loss in recombinant protein proteins in response to LTC, was

also observed with lower CCT values, greater GADD153 and XBP-1(s) mRNA and

enhanced rates of lactate utilisation for late generation cultures. Increased rates of

lactate utilisation may provide cultures with a carbon source once glucose has become

depleted, possibly to maintain ATP production (as shown in Figure 1.6).

Studies have suggested that mitochondrial integrity declines with age (Shigenaga et al,

1994). It may be possible that mitochondrial function in my cell lines diminished in

response to LTC, resulting in lower ATP concentrations for late generation cultures (as

shown in Figure 3.24). Low mitochondrial membrane potentials have also been

associated with decreased concentrations of ATP in CHO cultures (Jeong et al, 2004).

Measuring mitochondrial membrane potentials may provide a simple method of

distinguishing between cells with different intracellular ATP concentrations.

291

The intracellular mitochondrial Ca2+

concentration has been shown to be a key regulator

of ATP production (Griffiths & Rutter, 2009). Decreased mitochondrial Ca2+

concentrations, and mitochondrial membrane potential, occurs during periods of ER

stress (Arnaudeau et al, 2002). CRT expression also increases in response to ER stress

(to aid in protein refolding, shown in Figure 1.13, Scorrano et al, 2003; Zong et al,

2003), and upon ER Ca2+

depletion (Llewellyn et al, 1996). It may be possible that in

response to LTC ER stress is greater, observed with significantly increased GADD153

(Figure 3.13C, for 3.90, and Figure 6.4B, for 51.69) and XBP-1(s) mRNA (Figure

3.17C, for 3.90). Enhanced ER stress due to mis/unfolded proteins would increase CRT

expression and lower ATP production (Oyadomari & Mori, 2003). As ATP is needed

for protein folding, less ATP could allow for greater concentrations of mis/unfolded

proteins in the ER, causing further ER stress. The cycle involving Ca2+

, ATP and

mis/unfolded proteins is shown in Figure 7.1.

Figure 7.1 A pathway linking mitochondrial Ca2+

and ATP concentrations to

mis/unfolded proteins

The downregulation of the TCA cycle, observed with the accumulation of alanine,

glycerol and glycine, (shown for 3.90 in Figure 3.19) could also decrease mitochondrial

ATP generation (Duchen, 2000). ATP concentration not only regulates protein folding,

but also transcription, translation, protein secretion and protein degradation (Figure 1.7).

ATP is also needed for cell growth (Kondo et al, 2000), with depletion of ATP

(Izyumov et al, 2004), and NAD+ (Ha & Snyder, 1999; Virag, 2005) resulting in cell

ATP

CRT

Mt Ca2+Mis/unfolded

proteins

292

death. Lower ATP (and NADH and NAD+) concentrations for late generation cultures

by the stationary phase of batch culture (Figure 3.24) could be linked to the decrease in

CCT values, seen for both late generation 3.90 (Figure 3.2B) and 51.69 (Figure 6.1C)

cultures. Late generation cells may be more predisposed to cell death. The trypan-blue

exclusion method, used to determine viable and non-viable cells, cannot elucidate

between cells in different stages of apoptosis. Differential staining with FITC-

conjugated Annexin V or acridine orange/ethidium bromide may provide a useful

method for identifying early apoptotic cells (Bradbury et al, 2000). It is possible than in

response to LTC the apoptotic nature of the cultures increased (potentially due to

limitations in ATP and NAD+ concentrations). The possible up-regulation of early

apoptotic cells for late generation cultures may also account for enhanced GADD153

mRNA for late generation 3.90 (Figure 3.13C) and 51.69 (Figure 6.4B) cultures.

Although productivity for 3.90 and 51.69 cultures was enhanced with feed (Section 4.2

and Section 6.3) and DMSO (Section 5.2 and Section 6.2) additions, final antibody

titres and Qp values were still lower for late generation cultures than early generation

cultures. Again these late generation cultures (with feed and DMSO additions)

illustrated greater ER stress than the corresponding early generation cultures. The ER

stress phenotype for late generation cultures in the presence of feed was delayed

compared to cultures in the absence of feed. Feed addition did not prevent instability,

instead it delayed the ER stress characteristics.

The productivity of cultures, and the ability to maintain high, stable, expression during

LTC, may also be regulated by other pathways such as mammalian target of rapamycin

(mTOR). mTOR has been described as the cellular central coordinator, linking growth

factors, amino acids, energy and nutrient availability signals to cell growth, protein

synthesis, cell size, cell cycle and autophagy (Fingar & Blenis, 2004; Hay & Sonenberg,

2004). REDD1 (regulated in development and DNA damage responses 1) has been

indicated to mediate cellular responses to energy stress through the mTOR pathway in

glucose-withdrawn or ATP depleted cells, promoting dephosphorylation of S6K and

4E-BP1 (Sofer et al, 2005). It is also possible that the regulation of the mTOR pathway

becomes altered in response to LTC, resulting in late generation cultures having

decreased protein synthesis and cellular growth.

293

The loss of antibody titre in response to LTC, observed for 3.90 and 51.69, may also be

the result of other alterations, including mycoplasma contamination and alterations to

the heterogeneity of the cultures. The loss in protein production from cultures in

response to LTC could have been due the cultures developing a diverse population of

cells with differing Qp values. Methods used to detect intracellular heavy chain and

light chain proteins (Section 2.4.2) did not highlight any differences in the heterogeneity

of early or late generation cultures for both cell lines (data not shown). Both cell lines

were also routinely analysed for mycoplasma. Mycoplasma can alter cell function,

possibly resulting in lower recombinant gene expression (Eldering et al, 2004).

Mycoplasma was not detected during culture (see Appendix 6). Therefore mycoplasma

contamination did not contribute to lower protein expression in response to LTC.

7.2 HOW IS RECOMBINANT PROTEIN PRODUCTION INCREASED IN

RESPONSE TO FEED ADDITION?

The alterations to cultures in response to feed addition are summarised for 3.90 in

Figure 4.19 and for 51.69 in Figure 6.20. The addition of feeds to cultures increased

antibody titres and Qp values with alterations to the metabolic profiles of cultures,

observed with no lactate re-utilisation for 3.90 (Figure 3.21D) or 51.69 (Figure 6.5)

cultures. The activity of LDH activity has been shown to alter in CHO cells during fed-

batch culture (Ma et al, 2009). As previously mentioned in Section 1.5, decreased LDH

expression within recombinant CHO cultures has been shown to increase ATP and

antibody protein production (Jeong et al, 2006; Jeong et al, 2004). Feed addition may

have lowered LDH activity, allowing for altered glycolytic and TCA cycle fluxes,

observed with increased concentrations of ATP, NADH and NAD+, for 3.90 cultures

(Figure 4.20).

The improvement in antibody titres in response to feed addition may be due to increased

rates of protein translation. In response to amino acid feeding of CHO cells, the mTOR

target, eukaryotic initiation factor 4E-binding protein1 (4E-BP1), becomes highly

phosphorylated and dissociates from eIF4E (Proud, 2002b). Free eIF4E can then bind

other factors to assemble productive initiation complexes promoting successful

translation (Section 1.7.1). Intracellular recombinant heavy chains or light chains were

294

detected by specific conjugated antibodies and were found to decrease after feed

addition (Figure 4.11). However, as previously suggested in Chapter 4 less intracellular

protein does not necessarily signify lower rates of protein translation. Rates of specific

recombinant protein synthesis, via techniques such as pulse-chase analysis, would be

needed to confirm the translational activity of the cultures in response to feed addition.

Lower amounts of intracellular recombinant proteins in response to feed addition could

imply greater protein secretion, possibly as a result of greater protein folding. The

expression of ATF4, GADD153 and XBP-1(s) mRNA and GADD153 protein was

decreased in response to feed addition (shown in Section 4.4 for 3.90 cultures and

Section 6.3.2 for 51.69 cultures). As previously mentioned ER stress markers, ATF4

(Siu et al, 2002) and GADD153 (Bruhat et al, 2000; Bruhat et al, 1997; Carlson et al,

1993) have been shown to increase as a result of nutrient deprivation. Nutrient

deprivation, mis/unfolded proteins, as well as hypoxia and oxidative stress, all result in

a common ER stress mechanism including the phosphorylation of eIF2α and the

upregulation of ATF4. These stress responses have been collectively termed the

Integrated Stress Response (ISR, Fels et al, 2005; Harding et al, 2003; Rzymski &

Harris, 2007; Wek & Staschke, 2010).

I investigated the stress response in the parental cell line (cells which do not express the

recombinant antibody). The parental cells utilised glucose and amino acids at rates that

were similar to these observed with the exemplar cell lines (data not shown), but had

significantly lower expression of ATF4, GADD153 and XBP-1(s) mRNA during batch

culture relative to the exemplar cell lines, and interestingly had relatively low

expression of ATF4 protein (Appendix 5.2). ATF4 protein was observed for all

recombinant cultures regardless of culture condition, or generation time of culture. It is

not known if ATF4 protein production during recombinant culture was a method for

overcoming or adapting to ER stress within these cell lines, or just a consequence of

excessive protein load on the ER. ATF4 is central to the ISR, and possibly the most

sensitive indicator of ER stress and mis/unfolded proteins. As ATF4, GADD153 and

XBP-1(s) mRNA expression for the parental line was similar for cultures in the

presence and absence of feed (Appendix 5) I predict the decrease observed for these

markers in response to feed and DMSO in the exemplar cell lines were due to lower

295

protein load within the ER, by enhanced protein folding and less mis/unfolded,

potentially due to altered metabolic flux pathways.

It has been previously suggested the ER chaperone BiP is also affected by nutrient

starvation (Ledford & Leno, 1994), which may alter its ability to bind mis/unfolded

proteins. Relative expression of BiP protein was similar for all culture regardless of feed

addition (Figure 5.10A). However, the functionality of BiP may have been improved in

response to feed addition. Enhanced BiP binding for cultures supplemented with feed

may have improved protein folding, lowering ER stress in these cultures. Altered post-

translational modifications may have also aided protein folding in response to feed

addition. Although N-linked glycosylation profiles of the secreted proteins from the

exemplar cell line (3.90) were not changed in response to feed (or DMSO addition,

Figure 5.12) intracellular glycosylation patterns may have been modified under the

influence of different culture conditions. Experiments have shown that there is a link

between the adenylate energy change of cultures and their glycosylation profiles

(Kochanowski et al, 2008). Enhanced protein folding by improved BiP binding and N-

linked glycosylation patterns in response to feed addition may have led to greater

recombinant protein titres and decreased UPR. Investigating recombinant protein

folding during culture (Section 7.5) may provide evidence to support this proposal.

7.3 HOW IS RECOMBINANT PROTEIN PRODUCTION INCREASED IN

RESPONSE TO DMSO ADDITION?

The alterations to cultures in response to DMSO addition are summarised for 3.90 in

Figure 5.19 and for 51.69 in Figure 6.21. Previously Li et al, found that enzymes, such

as triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH),

and aldolase, were downregulated in response to DMSO addition, with suggestion that

DMSO altered the metabolic state of the CHO cultures (Li et al, 2006b). DMSO

additions altered the metabolic status of the exemplar cell lines, observed with greater

rates of glucose utilisation during the end (decline) phase of batch cultures, for both

3.90 (Section 5.15B) and 51.69 (Section 6.16A). Potentially the addition of DMSO

controlled the use of nutrients during culture, extending the cell culture function.

Greater metabolic activity during the decline phase of culture may have allowed for

296

enhanced ATP concentrations in response to DMSO addition. Investigations confirming

ATP concentrations for cultures with DMSO would be needed.

Initial analyses suggested that rates of protein translation were increased for cultures in

response to DMSO addition (Figure 5.17). Protein translation was measured as the rate

of incorporation of L-[4,5-3H] leucine into TCA precipitable-material. After 48 hrs

incubation protein translation was two-fold greater for cultures in the presence of

DMSO. The fold increase in protein translation for these cultures was not seen with

same fold increase in antibody titres. As L-[4,5-3H] leucine incorporation measures

global protein synthesis it is possible that the translational rates of the recombinant

protein were unaffected by DMSO addition, however, it is also likely that other

mechanisms influencing protein folding and protein secretion, affect the potentially

translational affects of DMSO addition, possibly suggesting a secretory bottleneck for

cultures with high productivity.

In response to DMSO addition ER stress markers were decreased for both 3.90 (Section

5.4) and 51.69 (Section 6.4.2), regardless of fed status of the cultures. This suggests that

cultures with DMSO were not limited by protein folding mechanisms. Mead et al, have

shown that protein turnover in high producing cell lines can limit protein production

(Mead et al, 2009). High protein turnover may have been occurred for cultures in the

presence of DMSO to replenish the amino acids utilised during protein translation. It is

also possible that high antibody titres and Qp values observed as a result of adding both

feed and DMSO to cultures was due to a lower rates of protein turnover consequent to

increased amino acid availability as a result of feed addition. Investigating rates of

protein turnover would be needed to confirm any changes as a result of different culture

conditions.

7.4 ARE THERE MARKERS TO PREDICT THE LIKELIHOOD OF

INSTABILITY IN RECOMBINANT PROTEIN PRODUCTION?

As decreased recombinant protein was observed for late generation cultures, from

generation 60, during the end (decline) phase of batch culture it is difficult to identify

markers that predict culture stability at the onset of characterisation assays.

297

Markers of ER stress, ATF4, GADD153 and XBP-1(s) mRNA were lower for

conditions which promoted enhanced protein production. As ER stress makers were

altered in response to LTC and feed additions investigating GCN2 may provide a link

with the alterations observed with ER stress markers and changes with the metabolic

activity of the cells. It may be possible to screen for recombinant cell lines based on the

IRS phenotype, and select cells exhibiting lower levels of cellular stress.

Individual experiments have shown overexpression of ATF4, GADD34 and XBP-1(s)

increased productivity of CHO cells, with the suggestion that enhanced productivity

was due to expansion of the ER and less translation attenuation due to feedback

inhibition (Ku et al, 2008; Ohya et al, 2008; Omasa et al, 2008; Tigges & Fussenegger,

2006). Isolating cells for a low ER stress-response phenotype, for example cells with

little expression of GADD153 and spliced XBP-1, may ensure the cells are not limited

at the level of translation and secretion.

7.5 FUTURE WORK

Whilst addressing the questions in this Chapter some future investigations have been

suggested. However, the main future investigations would involve further analysis of

intracellular ATP, NADH and NAD+, needed to confirm the concentrations of energy

intermediates in response to LTC, feed and DMSO addition.

Measuring rates of protein secretion and protein folding would also be advantageous to

determine if the changes in antibody titres were at the level of secretion, or if problems

in protein folding resulted in a greater degree of protein degradation. Exocytotic

secretion of proteins in CHO cells has been visualised using secreted Gaussia luciferase

as a reporter protein method of real-time bioluminescence imaging to investigate protein

trafficking in mammalian cells (Suzuki et al, 2007). As the folding within the cell is also

controlled by the oxidation state of the ER (Helenius et al, 1992) studies have also

involved imaging dynamic redox changes in mammalian cells with fluorescent protein

indicators (Dooley et al, 2004). GFP-based probes can be introduced into the cell,

together with the recombinant gene, and targeted to specific subcellular locations to

298

determine the redox state. These strategies may determine if protein folding, or the

environment needed to maintain protein folding, is altered in response to LTC, feed and

DMSO addition.

Finally as the investigation only focuses upon two cell lines, both of which produce the

same particular recombinant protein, the observations seen during LTC, and under the

influence of different feeding regimes, may not be indicative of protein expression on a

global scale. To gain a further understanding of the findings within this thesis future

work researching a „stable‟ cell line would be required. The comparison of a „stable‟

cell line, which does not have experience a loss in protein production with LTC, with an

„unstable‟ cell line, may offer more supportive data with the changes observed during

LTC, feed and DMSO addition.

As ER stress has been implicated in diabetes (Harding & Ron, 2002), cardiovascular

diseases (Vasa-Nicotera, 2004), cancer (Ma & Hendershot, 2004), immune responses

(Brewer & Hendershot, 2005) and neurodegenerative diseases (Rao & Bredesen, 2004),

hopefully a greater knowledge of the mechanisms involved within the UPR and protein

folding, and strategies to regulate protein secretion, may provide a greater deal of

understanding to ER stress in disease.

1

2

299

1

2

3

4 REFERENCES

5

6

7

8

300

Aggeler J, Kapp LN, Tseng SC, Werb Z (1982) Regulation of protein secretion in

Chinese hamster ovary cells by cell cycle position and cell density. Plasminogen

activator, procollagen fibronectin. Exp Cell Res 139(2): 275-283

Al-Fageeh MB, Smales CM (2006) Control and regulation of the cellular responses to

cold shock: the responses in yeast and mammalian systems. Biochem J 15(397): 247-

259

Al-Rubeai M, Emery AN (1990) Mechanisms and kinetics of monoclonal antibody

synthesis and secretion in synchronous and asynchronous hybridoma cell cultures. J

Biotechnol 16(1-2): 67-85

Allen MJ, Boyce JP, Trentalange MT, Treiber DL, Rasmussen B, Tillotson B, Davis R,

Reddy P (2008) Identification of novel small molecule enhancers of protein production

by cultured mammalian cells. Biotechnol Bioeng 100(6): 1193-1204

Altamirano C, Berrios J, Vergara M (2009) Galactose and lactate as nutrients in

continuous cultures of CHO cells producing rh-tPA. New Biotechnology 25(Supplement

1): S239-S239

Altamirano C, Illanes A, Becerra S, Cairó JJ, Gòdia F (2006) Considerations on the

lactate consumption by CHO cells in the presence of galactose. J Biotechnol 125(4):

547-556

Andersen DC, Krummen L (2002) Recombinant protein expression for therapeutic

applications. Curr Opin Biotechnol 13(2): 117-123

Andrulis IL, Hatfield GW, Arfin SM (1979) Asparaginyl-tRNA aminoacylation levels

and asparagine synthetase expression in cultured Chinese hamster ovary cells. J Biol

Chem 254(21): 10629-10633

Arden N, Betenbaugh MJ (2006) Regulating apoptosis in mammalian cell cultures.

Cytotechnology 50(1): 77-92

Arnaudeau S, Frieden M, Nakamura K, Castelbou C, Michalak M, Demaurex N (2002)

Calreticulin Differentially Modulates Calcium Uptake and Release in the Endoplasmic

Reticulum and Mitochondria. J Biol Chem 277(48): 46696-46705

Asami Y, Nagano H, Ikematsu S, Murasugi A (2000) An Approach to the Removal of

Yeast Specific O-Linked Oligo-Mannoses from Human Midkine Expressed in Pichia

pastoris Using Site-Specific Mutagenesis. J Biol Chem 128(5): 823-826

Ashe MP, De Long SK, Sachs AB (2000) Glucose Depletion Rapidly Inhibits

Translation Initiation in Yeast. Mol Biol Cell 11(3): 833-848

Averous J, Bruhat A, Jousse Cl, Carraro Vr, Thiel G, Fafournoux P (2004) Induction of

CHOP Expression by Amino Acid Limitation Requires Both ATF4 Expression and

ATF2 Phosphorylation. J Biol Chem 279(7): 5288-5297

Bacsi SG, Wejksnora PJ (1986) Effect of increase in ploidy on the activation of

nucleolar organizer regions in Chinese hamster ovary (CHO) cells. Exp Cell Res 165(1):

283-289

301

Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP (2008) The impact of

microRNAs on protein output. Nature 455(7209): 64-71

Balow JP, Weissman JD, Kearse KP (1995) Unique Expression of Major

Histocompatibility Complex Class I Proteins in the Absence of Glucose Trimming and

Calnexin Association. J Biol Chem 270(48): 29025-29029

Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin

Biotechnol 10(5): 411-421

Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in

Escherichia coli. Nat Biotech 22(11): 1399-1408

Banik GG, Tood P, Kompala DS (1996) Foreign protein expression from S phase

specific promoters in continuous cultures of recombinant CHO cells. Cytotechnology

22: 179-184

Barnes LM, Bentley CM, Dickson AJ (2001) Characterization of the stability of

recombinant protein production in the GS-NS0 expression system. Biotechnol Bioeng

73(4): 261-270

Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production from

recombinant mammalian cells. Biotechnol Bioeng 81(6): 631-639

Bartel DP (2004) MicroRNAs - Genomics, Biogenesis, Mechanism, and Function. Cell

116: 281-297

Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):

215-233

Bassik MC, Scorrano L, Oakes SA, Pozzan T, Korsmeyer SJ (2004) Phosphorylation of

BCL-2 regulates ER Ca2+ homeostasis and apoptosis. EMBO J 23(5): 1207-1216

Bates S, Parry D, Bonetta L, Vousden K, Dickson C, Peters G (1994) Absence of cyclin

D/cdk complexes in cells lacking functional retinoblastoma protein. Oncogene 9(6):

1633-1640

Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992)

High-level expression of a recombinant antibody from myeloma cells using a glutamine

synthetase gene as an amplifiable selectable marker. Biotechnology 10(2): 169-175

Beck A, Wurch T, Bailly C, Corvaia N (2010) Strategies and challenges for the next

generation of therapeutic antibodies. Nat Rev Immunol 10(5): 345-352

Benton T, Chen T, McEntee M, Fox B, King D, Crombie R, Thomas TC, Bebbington C

(2002) The use of UCOE vectors in combination with a preadapted serum free,

suspension cell line allows for rapid production of large quantities of protein.


Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate

ribonuclease in the initiation step of RNA interference. Nature 409(6818): 363-366

302

Bertolotti A, Ron D (2001) Alterations in an IRE1-RNA complex in the mammalian

unfolded protein response. J Cell Sci 114(17): 3207-3212

Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D (2000) Dynamic

interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell

Biol 2(6): 326-332

Bi JX, Shuttleworth J, Al-Rubeai M (2004) Uncoupling of cell growth and proliferation

results in enhancement of productivity in p21CIP1-arrested CHO cells. Biotechnol

Bioeng 85(7): 741-749

Bibila TA, Ranucci CS, Glazomitsky K, Buckland BC, Aunins JG (1994) Monoclonal

antibody process development using medium concentrates. Biotechnol Prog 10(1): 87-

96

Birch J, Bebbington, CR., Field, R., Renner, G., Brand, H. & Finney, H. (1993) The

production of recombinant antibodies using the glutamine synthetase (GS) system., Vol.

5, Dordrecht: Kluwer Academic Publishers.

Bole DG, Hendershot LM, Kearney JF (1986) Posttranslational association of

immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting

and secreting hybridomas. J Cell Biol 102(5): 1558-1566

Bonarius HPJ, Özemre A, Timmerarends B, Skrabal P, Tramper J, Schmid G, Heinzle E

(2001) Metabolic-flux analysis of continuously cultured hybridoma cells using mass

spectrometry in combination with 13C-lactate nuclear magnetic resonance spectroscopy

and metabolite balancing. Biotechnol Bioeng 74(6): 528-538

Bordone L, Motta MC, Picard F, Robinson A, Jhala US, Apfeld J, McDonagh T,

Lemieux M, McBurney M, Szilvasi A, Easlon EJ, Lin S-J, Guarente L. (2006) Sirt1

Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells. PLoS Biol 4(2):

e31

Borth N, Mattanovich D, Kunert R, Katinger H (2005) Effect of Increased Expression

of Protein Disulfide Isomerase and Heavy Chain Binding Protein on Antibody Secretion

in a Recombinant CHO Cell Line. Biotechnology Progress 21(1): 106-111

Bradbury DA, Simmons TD, Slater KJ, Crouch SPM (2000) Measurement of the

ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell

viability, necrosis and apoptosis. Journal of Immunological Methods 240(1-2): 79-92

Brewer JW, Diehl JA (2000) PERK mediates cell-cycle exit during the mammalian

unfolded protein response. PNAS 97(23): 12625-12630

Brewer JW, Hendershot LM (2005) Building an antibody factory: a job for the unfolded

protein response. Nat Immunol 6(1): 23-29

Brewer JW, Hendershot LM, Sherr CJ, Diehl JA (1999) Mammalian unfolded protein

response inhibits cyclin D1 translation and cell-cycle progression. PNAS USA 96(15):

8505-8510

303

Brostrom CO, Brostrom MA (1998) Regulation of translational initiation during cellular

responses. PNAS 58: 79-125

Brown M, Renner G, Field, RP, Hassell T. (1992) Process development for the

production of recombinant antibodies using the glutamine synthetase (GS) system.

Cytotechnology 9: 231-236

Bruhat A, Cherasse Y, Maurin A-C, Breitwieser W, Parry L, Deval C, Jones N, Jousse

C, Fafournoux P (2007) ATF2 is required for amino acid-regulated transcription by

orchestrating specific histone acetylation. Nucl Acids Res 35(4): 1312-1321

Bruhat A, Jousse C, Carraro V, Reimold AM, Ferrara M, Fafournoux P (2000) Amino

Acids Control Mammalian Gene Transcription: Activating Transcription Factor 2 Is

Essential for the Amino Acid Responsiveness of the CHOP Promoter. Mol Cell Biol

20(19): 7192-7204

Bruhat A, Jousse C, Wang XZ, Ron D, Ferrara M, Fafournoux P (1997) Amino acid

limitation induces expression of CHOP, a CCAAT/enhancer binding protein-related

gene, at both transcriptional and post-transcriptional levels. J Biol Chem 272(28):

17588-17593

Brush MH, Weiser DC, Shenolikar S (2003) Growth Arrest and DNA Damage-

Inducible Protein GADD34 Targets Protein Phosphatase 1α to the Endoplasmic

Reticulum and Promotes Dephosphorylation of the α Subunit of Eukaryotic Translation

Initiation Factor 2. Mol Cell Biol 23(4): 1292-1303

Bulleid NJ, Freedman R, B. (1988) Defective co-translational formation of disulphide

bonds in protein disulphide-isomerase-deficient microsomes. Nature 335: 649-651

Cabibbo A, Pagani M, Fabbri M, Rocchi M, Farmery MR, Bulleid NJ, Sitia R (2000)

ERO1-L, a Human Protein That Favors Disulfide Bond Formation in the Endoplasmic

Reticulum. J Biol Chem 275(7): 4827-4833

Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D

(2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing

the XBP-1 mRNA. Nature 415(6867): 92-96

Carlson SG, Fawcett TW, Bartlett JD, Bernier M, Holbrook NJ (1993) Regulation of the

C/EBP-related gene gadd153 by glucose deprivation. Mol Cell Biol 13(8): 4736-4744

Carrera AC (2004) TOR signaling in mammals. J Cell Sci 117(20): 4615-4616

Carvalhal AV, Marcelino I, Carrondo MJT (2003) Metabolic changes during cell

growth inhibition by p27 overexpression. Applied Microbiology and Biotechnology

63(2): 164-173

Cenci S, Sitia R (2007) Managing and exploiting stress in the antibody factory. FEBS

letters 581(19): 3652-3657

Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant

proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13(4):

329-332

304

Chai H, Al-Rubeai M, Chua K, L., Oh S, W.,L., Yap M, G.,S. (1996) Insect cell line

dependent gene expression of recombinant human tumor necrosis factor-β. Enzyme

Microb Technol 18: 126-132

Chaveroux C, Lambert-Langlais S, Cherasse Y, Averous J, Parry L, Carraro V, Jousse

C, Maurin A-C, Bruhat A, Fafournoux P (2010) Molecular mechanisms involved in the

adaptation to amino acid limitation in mammals. Biochimie 92(7): 736-745

Chaya M, Soon Hye P, Joo Young C, Gyun Min L (2007) Effect of doxycycline-

regulated protein disulfide isomerase expression on the specific productivity of

recombinant CHO cells: Thrombopoietin and antibody. Biotechnol Bioeng 98(3): 611-

615

Chaya M, Yeon-Gu K, Jane K, Gyun Min L (2008) Assessment of cell engineering

strategies for improved therapeutic protein production in CHO cells. Biotechnology

Journal 3(5): 624-630

Chen BP, Wolfgang CD, Hai T (1996) Analysis of ATF3, a transcription factor induced

by physiological stresses and modulated by gadd153/Chop10. Mol Cell Biol 16(3):

1157-1168

Cherasse C, Chaveroux C, Jousse AC, Maurin V, Carraro L, Parry P, Bruhat A (2008)

Role of repressor JDP2 in the amino acid-regulated transcription of CHOP. Febs Lett

582 1537-154

Chen H, Pan Y-X, Dudenhausen EE, Kilberg MS (2004) Amino Acid Deprivation

Induces the Transcription Rate of the Human Asparagine Synthetase Gene through a

Timed Program of Expression and Promoter Binding of Nutrient-responsive Basic

Region/Leucine Zipper Transcription Factors as Well as Localized Histone Acetylation.

J Biol Chem 279(49): 50829-50839

Chirino AJ, Mire-Sluis A (2004) Characterizing biological products and assessing

comparability following manufacturing changes. Nat Biotech 22(11): 1383-1391

Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins

using Escherichia coli. Applied Microbiology and Biotechnology 64(5): 625-635

Choi Y, Lee D, Kim I, Kim H, Park H, Choe T, Kim I-H (2007) Enhancement of

erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate

addition. Biotechnology and Bioprocess Engineering 12(1): 60-72

Chong WPK, Reddy SG, Yusufi FNK, Lee D-Y, Wong NSC, Heng CK, Yap MGS, Ho

YS (2010) Metabolomics-driven approach for the improvement of Chinese hamster

ovary cell growth: Overexpression of malate dehydrogenase II. J Biotechnol 147(2):

116-121

Chusainow J, Yang YS, Yeo J, H. M. , Toh PC, Asvadi P, Wong N, S. C. , Yap M, G.

S. (2009) A study of monoclonal antibody-producing CHO cell lines: What makes a

stable high producer? Biotechnology and Bioengineering 102(4): 1182-1196

305

Cost GJ, Freyvert Y, Vafiadis A, Santiago Y, Miller JC, Rebar E, Collingwood T, N,

Snowden A, Gregory PD (2009) BAK and BAX deletion using zinc-finger nucleases

yields apoptosis-resistant CHO cells. Biotechnol Bioeng 105(2): 330-340

Cox JS, Shamu CE, Walter P (1993) Transcriptional induction of genes encoding

endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell

73(6): 1197-1206

Cox JS, Walter P (1996) A Novel Mechanism for Regulating Activity of a Transcription

Factor That Controls the Unfolded Protein Response Cell 87(3): 391-404

Crucell (2010) http://crucell.com/

Cudna RE, Dickson AJ (2003) Endoplasmic reticulum signaling as a determinant of

recombinant protein expression. Biotechnol Bioeng 81(1): 56-65

Cumming DA (1991) Glycosylation of recombinant protein therapeutics: control and

functional implications. Glycobiology 1(2): 115-130

Dai Y, Grant S (2003) Cyclin-dependent kinase inhibitors. Current Opinion in

Pharmacology 3(4): 362-370

Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of

antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science

6(5): 219-226

Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos F (1996) Cytometry of cyclin

proteins. Cytometry 25(1): 1-13

Darzynkiewicz Z, Sharpless T, Staiano-Coico L, Melamed MR. (1980a)

Subcompartments of the G1 phase of cell cycle detected by flow cytometry.

Darzynkiewicz Z, Traganos F, Melamed M, R. (1980b) New cell cycle compartments

identified by multiparameter flow cytometry. Cytometry 1(2): 98-108

Davies SL, James DC (2009) Engineering Mammalian Cells for Recombinant

Monoclonal Antibody Production. In Cell Line Development, pp 153-173.

Davis T, Wickham T, McKenna K, Granados R, Shuler M, Wood H (1993)

Comparative recombinant protein production of eight insect cell lines. In Vitro Cellular

& Developmental Biology - Animal 29(5): 388-390

Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and

higher organisms. Biotechnology Advances 27(3): 297-306

Demeshkina N, Hirokawa G, Kaji A, Kaji H. (2007) Novel activity of eukaryotic

translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with

GTP. Oxford University Press.

Derouazi M, Martinet D, Besuchet Schmutz N, Flaction R, Wicht M, Bertschinger M,

Hacker DL, Beckmann JS, Wurm FM (2006) Genetic characterization of CHO

http://crucell.com/

306

production host DG44 and derivative recombinant cell lines. Biochemical and

Biophysical Research Communications 340(4): 1069-1077

deZengotita VM, Miller WM, Aunins JG, Zhou W (2000) Phosphate feeding improves

high-cell-concentration NS0 myeloma culture performance for monoclonal antibody

production. Biotechnol Bioeng 69(5): 566-576

Ding W-X, Ni H-M, Gao W, Yoshimori T, Stolz DB, Ron D, Yin X-M (2007) Linking

of Autophagy to Ubiquitin-Proteasome System Is Important for the Regulation of

Endoplasmic Reticulum Stress and Cell Viability. Am J Pathol 171(2): 513-524

Dinnis DM, James DC (2005) Engineering mammalian cell factories for improved

recombinant monoclonal antibody production: lessons from nature? Biotechnol Bioeng

91(2): 180-189

Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes

Dev 17(4): 438-442

Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY (2004)

Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein

Indicators. J Biol Chem 279(21): 22284-22293

Dorner AJ, Wasley LC, Kaufman RJ. (1992) Overexpression of GRP78 mitigates stress

induction of glucose regulated proteins and blocks secretion of selective proteins in

Chinese hamster ovary cells. EMBO J 11(4) 1563-1571

Duchen MR (2000) Mitochondria and calcium: from cell signalling to cell death. The

Journal of Physiology 529(1): 57-68

Dutton RL, Scharer J, Moo Young M (2006) Cell cycle phase dependent productivity of

a recombinant chinese hamster ovary cell line. Cytotechnology 52: 55-69

Dyson N (1998) The regulation of E2F by pRB-family proteins. Genes Dev 12(15):

2245-2262

Eldering JA, Felten C, Veilleux CA, Potts BJ (2004) Development of a PCR method for

mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of

recombinant therapeutic proteins. Biologicals 32(4): 183-193

Ellgaard L, Helenius A (2001) ER quality control: towards an understanding at the

molecular level. Current Opinion in Cell Biology 13(4): 431-437

Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev

Mol Cell Biol 4(3): 181-191

Ellis GK, Bone HG, Chlebowski R, Paul D, Spadafora S, Smith J, Fan M, Jun S (2008)

Randomized Trial of Denosumab in Patients Receiving Adjuvant Aromatase Inhibitors

for Nonmetastatic Breast Cancer. J Clin Oncol 26(30): 4875-4882

Fallaux FJ, Bout A, van der Velde I, van den Wollenberg DJM, Hehir KM, Keegan J,

Auger C, Cramer SJ, van Ormondt H, van der Eb AJ, Valerio D, Hoeben RC (1998)

New Helper Cells and Matched Early Region 1-Deleted Adenovirus Vectors Prevent

307

Generation of Replication-Competent Adenoviruses. Human Gene Therapy 9(13):

1909-1917

Fang S, Ferrone M, Yang C, Jensen JP, Tiwari S, Weissman AM (2001) The tumor

autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in

degradation from the endoplasmic reticulum. PNAS USA 98(25): 14422-14427

Fann CH, Guirgis F, Chen G, Lao MS, Piret JM (2000) Limitations to the amplification

and stability of human tissue-type plasminogen activator expression by Chinese hamster

ovary cells. Biotechnol Bioeng 69(2): 204-212

Fawcett TW, Martindale JL, Guyton KZ, Hai T, Holbrook NJ (1999) Complexes

containing activating transcription factor (ATF)/cAMP-responsive-element-binding

protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF

composite site to regulate Gadd153 expression during the stress response. Biochem J

339 ( Pt 1): 135-141

Fels D, Bi M, Naczki C, Koritzinsky M, Wouters BG, Koumenis C (2005) Activation of

the Integrated Stress Response (ISR) is required for adaptation of tumor cells to hypoxic

stress and contributes to tumor growth. AACR Meeting Abstracts 2005(1): 884-a-

Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-

transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9(2):

102-114

Fingar DC, Blenis J (2004) Target of rapamycin (TOR): an integrator of nutrient and

growth factor signals and coordinator of cell growth and cell cycle progression.

Oncogene 23(18): 3151-3171

Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis J (2004) mTOR

Controls Cell Cycle Progression through Its Cell Growth Effectors S6K1 and 4E-

BP1/Eukaryotic Translation Initiation Factor 4E. Mol Cell Biol 24(1): 200-216

Fiore M, Degrassi F (1999) Dimethyl Sulfoxide Restores Contact Inhibition-Induced

Growth Arrest and Inhibits Cell Density-Dependent Apoptosis in Hamster Cells. Exp

Cell Res 251(1): 102-110

Fiore M, Zanier R, Degrassi F (2002) Reversible G(1) arrest by dimethyl sulfoxide as a

new method to synchronize Chinese hamster cells. Mutagenesis 17(5): 419-424

Frand AR, Kaiser CA (1999) Ero1p oxidizes protein disulfide isomerase in a pathway

for disulfide bond formation in the endoplasmic reticulum. Mol Cell 4(4): 469-477

Fraser CS, Doudna JA (2007) Structural and mechanistic insights into hepatitis C viral

translation initiation. Nat Rev Micro 5(1): 29-38

Frickel EM, Riek R, Jelesarov I, Helenius A, Wuthrich K, Ellgaard L (2002) TROSY-

NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain. PNAS

USA 99(4): 1954-1959

Galfre G, Milstein C (1981) Preparation of monoclonal antibodies: strategies and

procedures. Methods Enzymol 73(Pt B): 3-46

308

Gammell P, Barron N, Kumar N, Clynes M (2007) Initial identification of low

temperature and culture stage induction of miRNA expression in suspension CHO-K1

cells. J Biotechnol 130(3): 213-218

Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal

antibody reactive with a human nuclear antigen associated with cell proliferation.

International Journal of Cancer 31(1): 13-20

Gerngross TU (2004) Advances in the production of human therapeutic proteins in

yeasts and filamentous fungi. Nat Biotech 22(11): 1409-1414

Gingras A-C, Raught B, Sonenberg N (1999) eIF4 INITIATION FACTORS: Effectors

of mRNA Recruitment to Ribosomes and Regulators of Translation. Annual Review of

Biochemistry 68(1): 913-963

Goldberg AL (2003) Protein degradation and protection against misfolded or damaged

proteins. Nature 426(6968): 895-899

Gong SS, Guerrini L, Basilico C (1991) Regulation of asparagine synthetase gene

expression by amino acid starvation. Mol Cell Biol 11(12): 6059-6066

Goudar C, Biener R, Boisart C, Heidemann R, Piret J, de Graaf A, Konstantinov K

(2009) Metabolic flux analysis of CHO cells in perfusion culture by metabolite

balancing and 2D [13C, 1H] COSY NMR spectroscopy. Metabolic Engineering 12(2):

138-149

Gregory CF, Jan P, Mark TF, James ER. (1991) Peptide-binding specificity of the

molecular chaperone BiP. Nature Publishing Group.

Griffiths EJ, Rutter GA (2009) Mitochondrial calcium as a key regulator of

mitochondrial ATP production in mammalian cells. Biochimica et Biophysica Acta

(BBA) - Bioenergetics 1787(11): 1324-1333

Gu MB, Tood P, Kompala DS (1996) Cell cycle analysis of foreign gene (b-

galactosidase) expression in recombinant mouse cells under the control of mouse

mammary tumor virus promoter. Biotechnol Bioeng 50: 229-237

Guerrini L, Gong SS, Mangasarian K, Basilico C (1993) Cis- and trans-acting elements

involved in amino acid regulation of asparagine synthetase gene expression. Mol Cell

Biol 13(6): 3202-3212

ICH Guidelines (1996) Quality of Biotechnological Products: Analysis of the

expression construct in cells used for the production of r-DNA derived from protein

products.

Imani S-I (2009) The NAD World: A new systemic regulatory network for metabolism

and aging - Sirt1, systemic NAD biosynthesis, and their importance. Cell Biochemistry

and Biophysics 53(2): 65-74

Ha HC, Snyder SH (1999) Poly(ADP-ribose) polymerase is a mediator of necrotic cell

death by ATP depletion. PNSA USA 96(24): 13978-13982

309

Haas IG, Wabl M (1983) Immunoglobulin heavy chain binding protein. Nature

306(5941): 387-389

Hamilton AJ, Baulcombe DC (1999) A Species of Small Antisense RNA in

Posttranscriptional Gene Silencing in Plants. Science 286(5441): 950-952

Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P,

Stadheim TA, Li H, Choi B-K, Hopkins D, Wischnewski H, Roser J, Mitchell T,

Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (2006) Humanization of Yeast to

Produce Complex Terminally Sialylated Glycoproteins. Science 313(5792): 1441-1443

Hammond C, Braakman I, Helenius A (1994) Role of N-linked oligosaccharide

recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.

Proceedings of the National Academy of Sciences of the United States of America 91(3):

913-917

Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease

mediates post-transcriptional gene silencing in Drosophila cells. Nature 404(6775):

293-296

Hansen R, Dickson AJ, Goodacre R, Stephens GM, Sellick CA (2010) Rapid

characterisation of N-linked glycans from secreted and gel-purified monoclonal

antibodies using MALDI-ToF mass spectroscopy. Biotechol and Bioengin: DOI:

10.1002/bit22879

Harding HP, Calfon M, Urano F, Novoa I, Ron D (2002) Transcriptional and

translational control in the Mammalian unfolded protein response. Annu Rev Cell Dev

Biol 18: 575-599

Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000a) Regulated

translation initiation controls stress-induced gene expression in mammalian cells. Mol

Cell 6(5): 1099-1108

Harding HP, Ron D (2002) Endoplasmic Reticulum Stress and the Development of

Diabetes. Diabetes 51(suppl 3): S455-S461

Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D (2000b) Perk is essential for

translational regulation and cell survival during the unfolded protein response. Mol Cell

5(5): 897-904

Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an

endoplasmic-reticulum-resident kinase. Nature 397(6716): 271-274

Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B,

Paules R, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D (2003) An Integrated

Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress.

Molecular Cell 11(3): 619-633

Hashimoto Y, Zhang C, Kawauchi J, Imoto I, Adachi MT, Inazawa J, Amagasa T, Hai

T, Kitajima S (2002) An alternatively spliced isoform of transcriptional repressor ATF3

and its induction by stress stimuli. Nucl Acids Res 30(11): 2398-2406

310

Hay N, Sonenberg N. (2004) Upstream and downstream of mTOR. Genes & Dev 18:

1926-1945

Hayes NVL, Smales CM, Klappa P (2010) Protein disulfide isomerase does not control

recombinant IgG4 productivity in mammalian cell lines. Biotechnol Bioeng 105(4):

770-779

Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange P, Tong JM, Bull

AT. (1992) Glucose-limited chemostat culture of chinese hamster ovary cells producing

recombinant human interferon-gamma. Biotechnol Bioengin 39(3): 327-335

Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Bull AT

(1991) Chinese hamster ovary cell growth and interferon production kinetics in stirred

batch culture. Applied Microbiology and Biotechnology 34(5): 559-564

Haze K, Yoshida H, Yanagi H, Yura T, Mori K (1999) Mammalian Transcription

Factor ATF6 Is Synthesized as a Transmembrane Protein and Activated by Proteolysis

in Response to Endoplasmic Reticulum Stress. Mol Biol Cell 10(11): 3787-3799

Helenius A, Aebi M (2001) Intracellular Functions of N-Linked Glycans. Science

291(5512): 2364-2369

Helenius A, Marquardt T, Braakman I (1992) The endoplasmic reticulum as a protein-

folding compartment. Trends in Cell Biology 2(8): 227-231

Hendershot L, Bole D, Kohler G, Kearney JF (1987) Assembly and secretion of heavy

chains that do not associate posttranslationally with immunoglobulin heavy chain-

binding protein. J Cell Biol 104(3): 761-767

Hendershot LM (2004) The ER function BiP is a master regulator of ER function. Mt

Sinai J Med 71(5): 289-297

Hetz C, Bernasconi P, Fisher J, Lee A-H, Bassik MC, Antonsson B, Brandt GS,

Iwakoshi NN, Schinzel A, Glimcher LH, Korsmeyer SJ (2006) Proapoptotic BAX and

BAK Modulate the Unfolded Protein Response by a Direct Interaction with IRE1α.

Science 312(5773): 572-576

Hinnebusch AG (2005) Translation regulation of GCN4 and the generation amino acid

control of yeast. Annual Review of Microbiology 59: 407-450

Hochegger H, Takeda S, Hunt T (2008) Cyclin-dependent kinases and cell-cycle

transitions: does one fit all? Nat Rev Mol Cell Biol 9(11): 910-916

Holland M, Yagi H, Takahashi N, Kato K, Savage COS, Goodall DM, Jefferis R (2006)

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera

of patients with ANCA-associated systemic vasculitis. Biochimica et Biophysica Acta

(BBA) 1760(4): 669-677

Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM (2003) Domain antibodies:

proteins for therapy. Trends in Biotechnology 21(11): 484-490

311

Hong L, Schroth GP, Matthews HR, Yau P, Bradbury EM (1993) Studies of the DNA

binding properties of histone H4 amino terminus. Thermal denaturation studies reveal

that acetylation markedly reduces the binding constant of the H4 "tail" to DNA. J Biol

Chem 268(1): 305-314

Hosokawa N, Wada I, Hasegawa K, Yorihuzi T, Tremblay LO, Herscovics A, Nagata K

(2001) A novel ER alpha-mannosidase-like protein accelerates ER-associated

degradation. EMBO Rep 2(5): 415-422

Houdebine L-M (2009) Production of pharmaceutical proteins by transgenic animals.

Comparative Immunology, Microbiology and Infectious Diseases 32(2): 107-121

Huang EP, Marquis CP, Gray PP (2004) Process development for a recombinant

Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified

metallothionein expression system. Biotechnol Bioeng 88(4): 437-450

Huang Y, Li Y, Wang YG, Gu X, Wang Y, Shen BF (2007) An efficient and targeted

gene integration system for high-level antibody expression. Journal of Immunological

Methods 322(1-2): 28-39

Hutson RG, Kitoh T, Moraga Amador DA, Cosic S, Schuster SM, Kilberg MS (1997)

Amino acid control of asparagine synthetase: relation to asparaginase resistance in

human leukemia cells. Am J Physiol 272: C1691-C1699

Hwang SO, Lee GM (2008) Nutrient deprivation induces autophagy as well as

apoptosis in Chinese hamster ovary cell culture. Biotechnology and Bioengineering

99(3): 678-685

Irani N, Beccaria AJ, Wagner R (2002) Expression of recombinant cytoplasmic yeast

pyruvate carboxylase for the improvement of the production of human erythropoietin by

recombinant BHK-21 cells. J Biotechnol 93(3): 269-282

Izyumov DS, Avetisyan AV, Pletjushkina OY, Sakharov DV, Wirtz KW, Chernyak BV,

Skulachev VP (2004) "Wages of Fear": transient threefold decrease in intracellular ATP

level imposes apoptosis. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1658(1-

2): 141-147

Jayapal KR, Wlaschin KF, Hu WS, Yap MGS (2007) Recombinant protein therapeutics

from CHO cells - 20 years and counting. Chem Eng Prog 103(10): 40-47

Jefferis R (2005) Glycosylation of Recombinant Antibody Therapeutics. Biotechnology

Progress 21(1): 11-16

Jefferis R (2007) Antibody therapeutics. Expert Opinion on Biological Therapy 7(9):

1401-1413

Jefferis R (2009a) Glycosylation as a strategy to improve antibody-based therapeutics.

Nat Rev Drug Discov 8(3): 226-234

Jefferis R (2009b) Recombinant antibody therapeutics: the impact of glycosylation on

mechanisms of action. Trends in Pharmacological Sciences 30(7): 356-362

312

Jenkins N (2007) Modifications of therapeutic proteins: challenges and prospects.


Jenkins N, Parekh RB, James DC (1996) Getting the glycosylation right: Implications

for the biotechnology industry. Nat Biotech 14(8): 975-981

Jeong D-W, Cho I, Kim T, Bae G, Kim I-H, Kim I (2006) Effects of lactate

dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on

cellular metabolism. Molecular and Cellular Biochemistry 284(1): 1-8

Jeong D-W, Kim T-S, Cho IT, Kim IY (2004) Modification of glycolysis affects cell

sensitivity to apoptosis induced by oxidative stress and mediated by mitochondria.

Biochemical and Biophysical Research Communications 313(4): 984-991

Jiang Z, Huang Y, Sharfstein ST (2006) Regulation of recombinant monoclonal

antibody production in chinese hamster ovary cells: a comparative study of gene copy

number, mRNA level, and protein expression. Biotechnology progress 22(1): 313-318

Jiang Z, Sharfstein S, T. (2008) Sodium butyrate stimulates monoclonal antibody over-

expression in CHO cells by improving gene accessibility. Biotechnology and

Bioengineering 100(1): 189-194

Jin C, Ugai H, Song J, Murata T, Sun K, Horikoshi M, Yokoyama KK (2001)

Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-2. Febs

Lett 489(1): 34-41

Jones PL, Wolffe AP (1999) Relationships between chromatin organization and DNA

methylation in determining gene expression. Semin Cancer Biol 9(5): 339-347

Jun SC, Kim MS, Hong HJ, Lee GM (2006) Limitations to the Development of

Humanized Antibody Producing Chinese Hamster Ovary Cells Using Glutamine

Synthetase-Mediated Gene Amplification. Biotechnology Progress 22(3): 770-780

Kanehara K, Kawaguchi S, Ng DTW (2007) The EDEM and Yos9p families of lectin-

like ERAD factors. Seminars in Cell & Developmental Biology 18(6): 743-750

Kang S-W, Hegde RS (2008) Lighting Up the Stressed ER. Cell 135(5): 787-789

Kao F-T, Puck TT (1967) Genetics of somatic mamalian cells IV. Genetics 55(3): 513-

524

Kao FT, Puck TT (1969) Genetics of somatic mammalian cells. IX. Quantitation of

mutagenesis by physical and chemical agents. Journal of Cellular Physiology 74(3):

245-257

Karupiah G, Chaudhri G (2004) IMMUNOLOGY, INFECTION, AND IMMUNITY.

Immunol Cell Biol 82(6): 651-651

Kaufman RJ (1990) Vectors used for expression in mammalian cells. Methods Enzymol

185: 487-511

313

Kaufman RJ (2002) Orchestrating the unfolded protein response in health and disease. J

Clin Invest 110(10): 1389-1398

Kaufman RJ, Scheuner D, Schroder M, Shen X, Lee K, Liu CY, Arnold SM (2002) The

unfolded protein response in nutrient sensing and differentiation. Nat Rev Mol Cell Biol

3(6): 411-421

Keating MJ, Dritselis A, Yasothan U, Kirkpatrick P (2010) Ofatumumab. Nat Rev Drug

Discov 9(2): 101-102

Kennard ML, Goosney DL, Monteith D, Zhang L, Moffat M, Fischer D, Mott J (2009)

The generation of stable, high MAb expressing CHO cell lines based on the artificial

chromosome expression (ACE) technology. Biotechnol Bioeng 104(3): 540-553

Keränen S, Penttilä M (1995) Production of recombinant proteins in the filamentous

fungus Trichoderma reesei. Curr Opin Biotechnol 6(5): 534-537

Kim JM, Kim JS, Park DH, Kang HS, Yoon J, Baek K, Yoon Y (2004) Improved

recombinant gene expression in CHO cells using matrix attachment regions. J

Biotechnol 107(2): 95-105

Kim NS, Kim SJ, Lee GM (1998) Clonal variability within dihydrofolate reductase-

mediated gene amplified Chinese hamster ovary cells: stability in the absence of

selective pressure. Biotechnol Bioeng 60(6): 679-688

Kim NS, Lee GM (2000) Overexpression of bcl-2 inhibits sodium butyrate-induced

apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody

production. Biotechnol Bioengin 71(3): 184-193

Kim R, Emi M, Tanabe K, Murakami S (2006) Role of the unfolded protein response in

cell death. Apoptosis 11(1): 5-13

Kim S, Lee G (2007) Down-regulation of lactate dehydrogenase-A by siRNAs for

reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.

Applied Microbiology and Biotechnology 74(1): 152-159

Kim SJ, Lee GM (1999) Cytogenetic analysis of chimeric antibody-producing CHO

cells in the course of dihydrofolate reductase-mediated gene amplification and their

stability in the absence of selective pressure. Biotechnol Bioengin 64(6): 741-749

Kim Y-G, Kim JY, Mohan C, Lee GM (2009) Effect of Bcl-x overexpression on

apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient-

deprived condition. Biotechnol Bioengin 103(4): 757-766

Kimball SR, Jefferson LS (2004) Molecular mechanisms through which amino acids

mediate signaling through the mammalian target of rapamycin. Current Opinion in

Clinical Nutrition & Metabolic Care 7(1): 39-44

Kitagawa M, Higashi H, Jung HK, Suzuki-Takahashi I, Ikeda M, Tamai K, Kato J,

Segawa K, Yoshida E, Nishimura S, Taya Y. (1996) The consensus motif for

314

phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin

A/E-Cdk2.

Kito M, Itami S, Fukano Y, Yamana K, Shibui, Shibui T (2002) Construction of

engineered CHO strains for high-level production of recombinant proteins. Applied

Microbiology and Biotechnology 60(4): 442-448

Kochanowski N, Blanchard F, Cacan R, Chirat F, Guedon E, Marc A, Goergen JL

(2008) Influence of intracellular nucleotide and nucleotide sugar contents on

recombinant interferon-γ glycosylation during batch and fed-batch cultures of CHO

cells. Biotechnol Bioeng 100(4): 721-733

Kondo M, Yamaoka T, Honda S, Miwa Y, Katashima R, Moritani M, Yoshimoto K,

Hayashi Y, Itakura M (2000) The Rate of Cell Growth Is Regulated by Purine

Biosynthesis via ATP Production and G1 to S Phase Transition. J Biochem 128(1): 57-

64

Kornberg RD, Lorch Y (1999) Twenty-Five Years of the Nucleosome, Fundamental

Particle of the Eukaryote Chromosome. Cell 98(3): 285-294

Kost TA, Condreay JP (1999) Recombinant baculoviruses as expression vectors for

insect and mammalian cells. Curr Opin Biotechnol 10(5): 428-433

Kouzarides T (2000) Acetylation: a regulatory modification to rival phosphorylation?

Embo J 19(6): 1176-1179

Kromenaker SJ, Srienc F (1991) Cell-cycle-dependent protein accumulation by

producer and nonproducer murine hybridome cell lines: A population analysis

Biotechnol Bioeng 28: 665-677

Ku SC, Ng DT, Yap MG, Chao SH (2008) Effects of overexpression of X-box binding

protein 1 on recombinant protein production in Chinese hamster ovary and NS0

myeloma cells. Biotechnol Bioeng 99(1): 155-164

Kubbies M, Stockinger H (1990) Cell cycle-dependent DHFR and t-PA production in

cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry. Exp

Cell Res 188(2): 267-271

Kutz D, Burg M (1998) Evolution of osmotic stress signaling via MAP kinase cascades.

J Exp Biol 201(22): 3015-3021

Kuwae S, Ohda T, Tamashima H, Miki H, Kobayashi K (2005) Development of a fed-

batch culture process for enhanced production of recombinant human antithrombin by

Chinese hamster ovary cells. J Biosci Bioeng 100(5): 502-510

Kuystermans D, Al-Rubeai M (2009) cMyc increases cell number through uncoupling

of cell division from cell size in CHO cells. BMC Biotechnology 9(1): 76

Larrick JW, Thomas DW (2001) Producing proteins in transgenic plants and animals.

Curr Opin Biotechnol 12(4): 411-418

315

Lavric V, Ofieru ID, Woinaroschy A (2005) A sensitivity analysis of the fed-batch

animal-cell bioreactor with respect to some control parameters. Biotechnol Appl

Biochem 41(Pt 1): 29-35

Ledford BE, Leno GH (1994) ADP-ribosylation of the molecular chaperone

GRP78/BiP. Molecular and Cellular Biochemistry 138(1): 141-148

Lee A-H, Iwakoshi NN, Glimcher LH (2003) XBP-1 Regulates a Subset of

Endoplasmic Reticulum Resident Chaperone Genes in the Unfolded Protein Response.

Mol Cell Biol 23(21): 7448-7459

Lee AS (1987) Coordinated regulation of a set of genes by glucose and calcium

ionophores in mammalian cells. Trends in Biochemical Sciences 12: 20-23

Lee AS (2001) The glucose-regulated proteins: stress induction and clinical

applications. Trends in Biochemical Sciences 26(8): 504-510

Lee C, J, Gargi S, Joni T, Robert WH (2009a) A clone screening method using mRNA

levels to determine specific productivity and product quality for monoclonal antibodies.

Biotechnology and Bioengineering 102(4): 1107-1118

Lee K, Tirasophon W, Shen X, Michalak M, Prywes R, Okada T, Yoshida H, Mori K,

Kaufman RJ (2002) IRE1-mediated unconventional mRNA splicing and S2P-mediated

ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response.

Genes Dev 16(4): 452-466

Lee Y-Y, Cevallos RC, Jan E (2009b) An Upstream Open Reading Frame Regulates

Translation of GADD34 during Cellular Stresses That Induce eIF2α Phosphorylation.

Journal of Biological Chemistry 284(11): 6661-6673

Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, Bobrowicz P, Choi B-K,

Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR,

Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Wildt S,

Gerngross TU (2006a) Optimization of humanized IgGs in glycoengineered Pichia

pastoris. Nat Biotech 24(2): 210-215

Li J, Holbrook NJ (2004) Elevated gadd153/chop expression and enhanced c-Jun N-

terminal protein kinase activation sensitizes aged cells to ER stress. Experimental

Gerontology 39(5): 735-744

Li J, Huang Z, Sun X, Yang P, Zhang Y (2006b) Understanding the enhanced effect of

dimethyl sulfoxide on hepatitis B surface antigen expression in the culture of Chinese

hamster ovary cells on the basis of proteome analysis. Enzyme and Microbial

Technology 38(3-4): 372-380

Li J, Ni M, Lee B, Barron E, Hinton DR, Lee AS (2008) The unfolded protein response

regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced

autophagy in mammalian cells. Cell Death Differ 15(9): 1460-1471

Li J, Sun X, Zhang Y (2006c) Improvement of hepatitis B surface antigen expression by

dimethyl sulfoxide in the culture of recombinant Chinese hamster ovary cells. Process


316

Lim A, Reed-Bogan A, Harmon BJ (2008) Glycosylation profiling of a therapeutic

recombinant monoclonal antibody with two N-linked glycosylation sites using liquid

chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer.

Analytical Biochemistry 375(2): 163-172

Lim SF, Chuan KH, Liu S, Loh SOH, Chung BYF, Ong CC, Song Z (2006) RNAi

suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells.

Metabolic Engineering 8(6): 509-522

Liu C-H, Chen L-H (2007a) Enhanced recombinant M-CSF production in CHO cells by

glycerol addition: model and validation. Cytotechnology 54(2): 89-96

Liu C-H, Chen L-H (2007b) Promotion of recombinant macrophage colony stimulating

factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells.

Journal of Bioscience and Bioengineering 103(1): 45-49

Liu C-H, Chu IM, Hwang S-M (2001) Enhanced expression of various exogenous genes

in recombinant Chinese hamster ovary cells in presence of dimethyl sulfoxide.

Biotechnology Letters 23(20): 1641-1645

Liu CY, Schroder M, Kaufman RJ (2000) Ligand-independent dimerization activates

the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum.

J Biol Chem 275(32): 24881-24885

Llewellyn DH, Kendall JM, Sheikh FN, Campbell AK (1996) Induction of calreticulin

expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and

inhibition of N-linked glycosylation. Biochem J 318(2): 555-560

Lloyd DR, Al-Rubeai M (1999) Cell cycle, New York: John Wiley and Sons.

Lloyd DR, Holmes P, Jackson L, P., Emery AN, Mohamed AR (2000) Relationship

between cell size, cell cycle and specific recombinant protein productivity.

Cytotechnology 34: 59-70

Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL,

Darnell J (2004) Molecular Cell Biology, 5 edn. New York: WH Freeman.

Lonberg N (2005) Human antibodies from transgenic animals. Nat Biotech 23(9): 1117-

1125

Lonberg N (2008) Fully human antibodies from transgenic mouse and phage display

platforms. Current Opinion in Immunology 20(4): 450-459

Lonza (2010) http://www.lonza.com/

Lu PD, Harding HP, Ron D (2004) Translation reinitiation at alternative open reading

frames regulates gene expression in an integrated stress response. The Journal of Cell

Biology 167(1): 27-33

Lu S, Sun X, Zhang Y (2005) Insight into metabolism of CHO cells at low glucose

concentration on the basis of the determination of intracellular metabolites. Process


http://www.lonza.com/

317

Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal

structure of the nucleosome core particle at 2.8A resolution. Nature 389(6648): 251-260

Ma, Ningning., Ellet J, Okediadi C, Hermes P, McCormick E, Casnocha S (2009) A

single nutrient feed supports both chemically defined NS0 and CHO fed-batch

processes: Improved productivity and lactate metabolism. Biotechnology Progress

25(5): 1353-1363

Ma Y, Brewer JW, Alan Diehl J, Hendershot LM (2002) Two Distinct Stress Signaling

Pathways Converge Upon the CHOP Promoter During the Mammalian Unfolded

Protein Response. Journal of Molecular Biology 318(5): 1351-1365

Ma Y, Hendershot LM (2003) Delineation of a Negative Feedback Regulatory Loop

That Controls Protein Translation during Endoplasmic Reticulum Stress. Journal of

Biological Chemistry 278(37): 34864-34873

Ma Y, Hendershot LM (2004) The role of the unfolded protein response in tumour

development: friend or foe? Nat Rev Cancer 4(12): 966-977

Ma Z, Yi X, Zhang Y (2008) Enhanced intracellular accumulation of recombinant

HBsAg in CHO cells by dimethyl sulfoxide. Process Biochemistry 43(6): 690-695

Majors B, S., Betenbaugh M, J. , Pederson N, E. , Chiang G, G. (2009) Mcl-1

overexpression leads to higher viabilities and increased production of humanized

monoclonal antibody in Chinese hamster ovary cells. Biotechnology Progress 25(4):

1161-1168

Malhotra J, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe S, Kaufman R (2008)

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. PNAS

105(47): 18525-18530

Marchant R, Al-Fageeh M, Underhill M, Racher A, Smales C (2008) Metabolic Rates,

Growth Phase, and mRNA Levels Influence Cell-Specific Antibody Production Levels

from In Vitro-Cultured Mammalian Cells at Sub-Physiological Temperatures.

Molecular Biotechnology 39(1): 69-77

Masciarelli S, Sitia R (2008) Building and operating an antibody factory: Redox control

during B to plasma cell terminal differentiation. Biochimica et Biophysica Acta (BBA) -

Molecular Cell Research 1783(4): 578-588

Mastrangelo AJ, Hardwick JM, Zou S, Betenbaugh MJ (2000) Part II. Overexpression

of bcl-2 family members enhances survival of mammalian cells in response to various

culture insults. Biotechnol Bioeng 67(5): 555-564

Mayer M, Kies U, Kammermeier R, Buchner J (2000) BiP and PDI Cooperate in the

Oxidative Folding of Antibodiesin Vitro. Journal of Biological Chemistry 275(38):

29421-29425

Mazur X, Fussenegger M, Renner WA, Bailey JE (1998) Higher productivity of

growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase

inhibitor p27. Biotechnol Progress 14(5);705-13

318

McClung MR, Lewiecki EM, Cohen SB, Bolognese MA, Woodson GC, Moffett AH,

Peacock M, Miller PD, Lederman SN, Chesnut CH, Lain D, Kivitz AJ, Holloway DL,

Zhang C, Peterson MC, Bekker PJ, the AMGBLSG (2006) Denosumab in

Postmenopausal Women with Low Bone Mineral Density. N Engl J Med 354(8): 821-

831

McCullough KD, Martindale JL, Klotz L-O, Aw T-Y, Holbrook NJ (2001) Gadd153

Sensitizes Cells to Endoplasmic Reticulum Stress by Down-Regulating Bcl2 and

Perturbing the Cellular Redox State. Mol Cell Biol 21(4): 1249-1259

McManus MT, Sharp PA (2002) Gene silencing in mammals by small interfering

RNAs. Nat Rev Genet 3(10): 737-747

Mead EJ, Chiverton LM, Smales CM, von der Haar T (2009) Identification of the

limitations on recombinant gene expression in CHO cell lines with varying luciferase

production rates. Biotechnology and Bioengineering 102(6): 1593-1602

Meents H, Enenkel B, Eppenberger HM, Werner RG, Fussenegger M (2002) Impact of

coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-

x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in

suspension and serum-free media. Biotechnol Bioeng 80(6): 706-716

Meister A (1976) [Enzymology and function of the gamma-glutamyl moiety (author's

transl)]. Seikagaku 48(3): 155-166

Merksamer PI, Papa FR (2010) The UPR and cell fate at a glance. J Cell Sci 123(7):

1003-1006

Merksamer PI, Trusina A, Papa FR (2008) Real-Time Redox Measurements during

Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions. Cell

135(5): 933-947

Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: the long road to destruction.

Nat Cell Biol 7(8): 766-772

Meyerson M, Harlow E (1994) Identification of G1 kinase activity for cdk6, a novel

cyclin D partner. Mol Cell Biol 14(3): 2077-2086

Mezghrani A, Fassio A, Benham A, Simmen T, Braakman I, Sitia R (2001)

Manipulation of oxidative protein folding and PDI redox state in mammalian cells.

EMBO J 20(22): 6288-6296

Mohan C, Lee GM (2010) Effect of inducible co-overexpression of protein disulfide

isomerase and endoplasmic reticulum oxidoreductase on the specific antibody

productivity of recombinant Chinese hamster ovary cells. Biotechnology and


Mohan C, Park SH, Chung JY, Lee GM (2007) Effect of doxycycline-regulated protein

disulfide isomerase expression on the specific productivity of recombinant CHO cells:

Thrombopoietin and antibody. Biotechnology and Bioengineering 98(3): 611-615

Morgan D, O. (2007) The Cell Cycle: Principles of Control: New Science Press.

319

Mori K, Ogawa N, Kawahara T, Yanagi H, Yura T (2000) mRNA splicing-mediated C-

terminal replacement of transcription factor Hac1p is required for efficient activation of

the unfolded protein response. PNAS USA 97(9): 4660-4665

Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y (2002a) An

Endoplasmic Reticulum Stress-specific Caspase Cascade in Apoptosis. Journal of

Biological Chemistry 277(37): 34287-34294

Nadanaka S, Yoshida H, Sato R, Mori K (2006) Analysis of ATF6 Activation in Site-2

Protease-deficient Chinese Hamster Ovary Cells. Cell Structure and Function 31(2):

109-116

Nakagawa T, Yuan J (2000) Cross-Talk between Two Cysteine Protease Families:

Activation of Caspase-12 by Calpain in Apoptosis. J Cell Biol 150(4): 887-894

Nam JH, Zhang F, Ermonval M, Linhardt RJ, Sharfstein ST (2008) The effects of

culture conditions on the glycosylation of secreted human placental alkaline

phosphatase produced in Chinese hamster ovary cells. Biotechnol Bioeng 100(6): 1178-

1192

Nelson AL, Reichert JM (2009) Development trends for therapeutic antibody

fragments. Nat Biotech 27(4): 331-337

Nishitoh H, Saitoh M, Mochida Y, Takeda K, Nakano H, Rothe M, Miyazono K, Ichijo

H (1998) ASK1 Is Essential for JNK/SAPK Activation by TRAF2. Molecular Cell 2(3):

389-395

Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J (1997) A Glycine-

rich RNA-binding Protein Mediating Cold-inducible Suppression of Mammalian Cell

Growth. The Journal of Cell Biology 137(4): 899-908

Niwa M, Walter P (2000) Pausing to decide. PNAS USA 97(23): 12396-12397

Novoa I, Zeng H, Harding HP, Ron D (2001) Feedback Inhibition of the Unfolded

Protein Response by GADD34-Mediated Dephosphorylation of eIF2α. J Cell Biol

153(5): 1011-1022

Nurse P (2000) A Long Twentieth Century of the Cell Cycle and Beyond. Cell 100(1):

71-78

Oberdorf J, Carlson EJ, Skach WR (2001) Redundancy of Mammalian Proteasome Î²

Subunit Function during Endoplasmic Reticulum Associated Degradation Biochemistry

40(44): 13397-13405

Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H (2005) TRB3, a novel ER stress-

inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death.

EMBO J 24(6): 1243-1255

Ohya T, Hayashi T, Kiyama E, Nishii H, Miki H, Kobayashi K, Honda K, Omasa T,

Ohtake H (2008) Improved production of recombinant human antithrombin III in

Chinese hamster ovary cells by ATF4 overexpression. Biotechnol Bioeng 100(2): 317-

324

320

Okada T, Yoshida H, Akazawa R, Negishi M, Mori K (2002) Distinct roles of

activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein

kinase-like endoplasmic reticulum kinase (PERK) in transcription during the

mammalian unfolded protein response. Biochem J 366(2): 585-594

Olivari S, Cali T, Salo KEH, Paganetti P, Ruddock LW, Molinari M (2006) EDEM1

regulates ER-associated degradation by accelerating de-mannosylation of folding-

defective polypeptides and by inhibiting their covalent aggregation. Biochemical and

Biophysical Research Communications 349(4): 1278-1284

Omasa T, Furuichi K, Iemura T, Katakura Y, Kishimoto M, Suga K-i (2009) Enhanced

antibody production following intermediate addition based on flux analysis in

mammalian cell continuous culture. Bioprocess and Biosystems Engineering 33(1):

117-125

Omasa T, Onitsuka M, Kim W-D (2010) Cell Engineering and Cultivation of Chinese

Hamster Ovary (CHO) Cells. Current Pharmaceutical Biotechnology 11: 233-240

Omasa T, Takami T, Ohya T, Kiyama E, Hayashi T, Nishii H, Miki H, Kobayashi K,

Honda K, Ohtake H (2008) Overexpression of GADD34 Enhances Production of

Recombinant Human Antithrombin III in Chinese Hamster Ovary Cells. Journal of

Bioscience and Bioengineering 106(6): 568-573

Otero JH, Lizák B, Hendershot LM (2009) Life and death of a BiP substrate. Seminars

in Cell & Developmental Biology 21(5): 472-478

Ouwens DM, de Ruiter ND, van der Zon GCM, Carter AP, Schouten J, van der Burgt

C, Kooistra K, Bos JL, Maassen JA, van Dam H (2002) Growth factors can activate

ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK

pathway and of Thr69 through RalGDS-Src-p38. EMBO J 21(14): 3782-3793

Oyadomari S, Mori M (2003) Roles of CHOP//GADD153 in endoplasmic reticulum

stress. Cell Death Differ 11(4): 381-389

Pagani M, Fabbri M, Benedetti C, Fassio A, Pilati S, Bulleid NJ, Cabibbo A, Sitia R

(2000) Endoplasmic Reticulum Oxidoreductin 1-LÎ² (ERO1-LÎ²), a Human Gene

Induced in the Course of the Unfolded Protein Response. Journal of Biological

Chemistry 275(31): 23685-23692

Page MJ (1988) Expression of Foreign Genes in Mammalian Cells. In New Nucleic

Acid Techniques, pp 371-384.

Pan Y, Chen H, Siu F, Kilberg MS (2003) Amino Acid Deprivation and Endoplasmic

Reticulum Stress Induce Expression of Multiple Activating Transcription Factor-3

mRNA Species That, When Overexpressed in HepG2 Cells, Modulate Transcription by

the Human Asparagine Synthetase Promoter. Journal of Biological Chemistry 278(40):

38402-38412

Pau MG, Ophorst C, Koldijk MH, Schouten G, Mehtali M, Uytdehaag F (2001) The

human cell line PER.C6 provides a new manufacturing system for the production of

influenza vaccines. Vaccine 19(17-19): 2716-2721

321

Pazin MJ, Kadonaga JT (1997) What's up and down with histone deacetylation and

transcription? Cell 89(3): 325-328

Peng R-W, Fussenegger M (2009a) Engineering the Secretory Pathway in Mammalian

Cells. In Cell Line Development, Al-Rubeai M, Hauser H, Betenbaugh M, Fussenegger

M, Jenkins N, Merten O-W (eds), Vol. 6, pp 233-248. Springer Netherlands

Peng R-W, Fussenegger M (2009b) Molecular engineering of exocytic vesicle traffic

enhances the productivity of Chinese hamster ovary cells. Biotechnology and


Peng R-W, Guetg C, Tigges M, Fussenegger M (2010) The vesicle-trafficking protein

munc18b increases the secretory capacity of mammalian cells. Metabolic Engineering

12(1): 18-25

Perlmutter DH (2002) Chemical Chaperones: A Pharmacological Strategy for Disorders

of Protein Folding and Trafficking. Pediatric Research 52(6): 832-836

Petch D, Butler M (1996) The effect of alternative carbohydrates on the growth and

antibody production of a murine hybridoma. Applied Biochemistry and Biotechnology

59(1): 93-104

Petiot A, Ogier-Denis E, Blommaart EFC, Meijer AJ, Codogno P (2000) Distinct

Classes of Phosphatidylinositol 3-Kinases Are Involved in Signaling Pathways That

Control Macroautophagy in HT-29 Cells. Journal of Biological Chemistry 275(2): 992-

998

Ponzio G, Loubat A, Rochet N, Turchi L, Rezzonico R, Farahi Far D, Vjekoslav D,

Rossi B (1998) Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin

D2 inhibition and p21[CIP1] induction. Oncogene 17(3): 1159-1166

Potter M, Boyce CR (1962) Induction of plasma-cell neoplasms in strain BALB/c mice

with mineral oil and mineral oil adjuvants. Nature 193: 1086-1087

Pouysségur J, Shiu RPC, Pastan I (1977) Induction of two transformation-sensitive

membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or

glucose deprivation. Cell 11(4): 941-947

Prostko CR, Brostrom MA, Brostrom CO (1993) Reversible phosphorylation of

eukaryotic initiation factor 2α in response to endoplasmic reticular signaling. Molecular

and Cellular Biochemistry 127(1): 255-265

Prostko CR, Brostrom MA, Malara EM, Brostrom CO (1992) Phosphorylation of

eukaryotic initiation factor (eIF) 2 alpha and inhibition of eIF-2B in GH3 pituitary cells

by perturbants of early protein processing that induce GRP78. Journal of Biological

Chemistry 267(24): 16751-16754

Proud CG (2002a) Control of the translational machinery in mammalian cells. Eur J

Biochem 269(22): 5337

Proud CG (2002b) Regulation of mammalian translation factors by nutrients. Eur J

Biochem 269(22): 5338-5349

322

Puck TT, Cieciura SJ, Robinson A (1958) Genetics of somatic mammalian cells. III.

Long-term cultivation of euploid cells from human and animal subjects. J Exp Med

108(6): 945-956

Quek L-E, Dietmair S, Krömer JO, Nielsen LK (2010) Metabolic flux analysis in

mammalian cell culture. Metabolic Engineering 12(2): 161-171

Raju TS, Briggs JB, Borge SM, Jones AJS (2000) Species-specific variation in

glycosylation of IgG: evidence for the species-specific sialylation and branch-specific

galactosylation and importance for engineering recombinant glycoprotein therapeutics.

Glycobiology 10(5): 477-486

Rao RV, Bredesen DE (2004) Misfolded proteins, endoplasmic reticulum stress and

neurodegeneration. Current Opinion in Cell Biology 16(6): 653-662

Ravikumar B, Berger Z, Vacher C, O'Kane CJ, Rubinsztein DC (2006) Rapamycin pre-

treatment protects against apoptosis. Hum Mol Genet 15(7): 1209-1216

Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC (2005) Monoclonal antibody

successes in the clinic. Nat Biotechnol 23(9): 1073-1078

Rodriguez J, Spearman M, Huzel N, Butler M (2005) Enhanced Production of

Monomeric Interferon-beta by CHO Cells through the Control of Culture Conditions.

Biotechnology Progress 21(1): 22-30

Römisch K (2004) A Cure for Traffic Jams: Small Molecule Chaperones in the

Endoplasmic Reticulum. Traffic 5(11): 815-820

Ron D, Habener JF (1992) CHOP, a novel developmentally regulated nuclear protein

that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-

negative inhibitor of gene transcription. Genes & Dev 6(3): 439-453

Ross J (1995) mRNA stability in mammalian cells. Microbiol Rev 59(3): 423-450

Roth RA, Pierce SB (1987) In vivo cross-linking of protein disulfide isomerase to

immunoglobulins. Biochemistry 26(14): 4179-4182

Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA (2001) Glycosylation and the

Immune System. Science 291(5512): 2370-2376

Ruddock LW, Molinari M (2006) N-glycan processing in ER quality control. J Cell Sci

119(21): 4373-4380

Rüegsegger U, Leber JH, Walter P (2001) Block of HAC1 mRNA Translation by Long-

Range Base Pairing Is Released by Cytoplasmic Splicing upon Induction of the

Unfolded Protein Response. Cell 107(1): 103-114

Rutkowski DT, Kaufman RJ (2004) A trip to the ER: coping with stress. Trends Cell

Biol 14(1): 20-28

323

Rutkowski DT, Ott CM, Polansky JR, Lingappa VR (2003) Signal Sequences Initiate

the Pathway of Maturation in the Endoplasmic Reticulum Lumen. Journal of Biological

Chemistry 278(32): 30365-30372

Rzymski T, Harris AL (2007) The Unfolded Protein Response and Integrated Stress

Response to Anoxia. Clinical Cancer Research 13(9): 2537-2540

Schlatter S, Stansfield S, H, Dinnis DM, Racher AJ, Birch JR, James DC (2005) On the

Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody

Production by CHO Cells. Biotechnology Progress 21(1): 122-133

Schulze A, Standera S, Buerger E, Kikkert M, van Voorden S, Wiertz E, Koning F,

Kloetzel P-M, Seeger M (2005) The Ubiquitin-domain Protein HERP forms a Complex

with Components of the Endoplasmic Reticulum Associated Degradation Pathway.

Journal of Molecular Biology 354(5): 1021-1027

Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer

SJ (2003) BAX and BAK Regulation of Endoplasmic Reticulum Ca2+: A Control Point

for Apoptosis. Science 300(5616): 135-139

Scriven P, Brown N, Pockley A, Wyld L (2007) The unfolded protein response and

cancer: a brighter future unfolding? Journal of Molecular Medicine 85(4): 331-341

Sellick C, Knight D, Croxford A, Maqsood A, Stephens G, Goodacre R, Dickson A

(2010) Evaluation of extraction processes for intracellular metabolite profiling of

mammalian cells: matching extraction approaches to cell type and metabolite targets.

Metabolomics 6(3): 427-438

Sellick CA, Hansen R, Maqsood AR, Dunn WB, Stephens GM, Goodacre R, Dickson

AJ (2008) Effective Quenching Processes for Physiologically Valid Metabolite

Profiling of Suspension Cultured Mammalian Cells. Analytical Chemistry 81(1): 174-

183

Shen J, Chen X, Hendershot L, Prywes R (2002) ER Stress Regulation of ATF6

Localization by Dissociation of BiP/GRP78 Binding and Unmasking of Golgi

Localization Signals. Developmental Cell 3(1): 99-111

Shen J, Prywes R (2004) Dependence of Site-2 Protease Cleavage of ATF6 on Prior

Site-1 Protease Digestion Is Determined by the Size of the Luminal Domain of ATF6.

Journal of Biological Chemistry 279(41): 43046-43051

Shen X, Ellis RE, Lee K, Liu CY, Yang K, Solomon A, Yoshida H, Morimoto R,

Kurnit DM, Mori K, Kaufman RJ (2001) Complementary signaling pathways regulate

the unfolded protein response and are required for C. elegans development. Cell 107(7):

893-903

Shenton D, Smirnova JB, Selley JN, Carroll K, Hubbard SJ, Pavitt GD, Ashe MP, Grant

CM (2006) Global Translational Responses to Oxidative Stress Impact upon Multiple

Levels of Protein Synthesis. Journal of Biological Chemistry 281(39): 29011-29021

324

Shi Y VK, Sood R, An J, Liang J, Stramm L, Wek RC. (1998) Identification and

characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK,

involved in translational control. Mol Cell Biol 18(12): 7499-7509

Shigenaga MK, Hagen TM, Ames BN (1994) Oxidative damage and mitochondrial

decay in aging. PNAS USA 91(23): 10771-10778

Shukla AA, Thömmes J (2010) Recent advances in large-scale production of

monoclonal antibodies and related proteins. Trends in Biotechnology 28(5): 253-261

Sidrauski C, Walter P (1997) The Transmembrane Kinase Ire1p Is a Site-Specific

Endonuclease That Initiates mRNA Splicing in the Unfolded Protein Response. Cell

90(6): 1031-1039

Simone C, Roberto S (2007) Managing and exploiting stress in the antibody factory.

FEBS letters 581(19): 3652-3657

Singh N, Anand S (1994) Cell death by apoptosis. Indian J Exp Biol 32(12): 843-847

Sitton G, Srienc F (2008) Growth dynamics of mammalian cells monitored with

automated cell cycle staining and flow cytometry. Cytometry Part A 73A(6): 538-545

Siu F, Bain PJ, LeBlanc-Chaffin R, Chen H, Kilberg MS (2002) ATF4 Is a Mediator of

the Nutrient-sensing Response Pathway That Activates the Human Asparagine

Synthetase Gene. Journal of Biological Chemistry 277(27): 24120-24127

Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ,

Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murine

myeloma cell lines with varying recombinant monoclonal antibody production rate.

Biotechnol Bioeng 88(4): 474-488

Sofer A, Lei K, Johannessen CM, Ellisen LW (2005) Regulation of mTOR and Cell

Growth in Response to Energy Stress by REDD1. Mol Cell Biol 25(14): 5834-5845

Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes:

mechanisms and biological targets. Cell 136(4): 731-745

Steinmeyer DE, McCormick EL (2008) The art of antibody process development. Drug

Discovery Today 13(13-14): 613-618

Stratling WH (1976) Stimulation of transcription on chromatin by polar organic

compounds. Nucl Acids Res 3(5): 1203-1214

Struhl K (1998) Histone acetylation and transcriptional regulatory mechanisms. Genes

Dev 12(5): 599-606

Sunley K, Butler M (2010) Strategies for the enhancement of recombinant protein

production from mammalian cells by growth arrest. Biotechnology Advances 28(3):

385-394

325

Suzuki T, Usuda S, Ichinose H, Inouye S (2007) Real-time bioluminescence imaging of

a protein secretory pathway in living mammalian cells using Gaussia luciferase. FEBS

letters 581(24): 4551-4556

Swartz JR (2001) Advances in Escherichia coli production of therapeutic proteins. Curr

Opin Biotechnol 12(2): 195-201

Tachibana H, Taniguchi K, Ushio Y, Teruya K, Osada K, Murakami H (1994) Changes

of monosaccharide availability of human hybridoma lead to alteration of biological

properties of human monoclonal antibody. Cytotechnology 16(3): 151-157

Talvas J, Obled A, Fafournoux P, Mordier S (2006) Regulation of Protein Synthesis by

Leucine Starvation Involves Distinct Mechanisms in Mouse C2C12 Myoblasts and

Myotubes. J Nutr 136(6): 1466-1471

Tan HK, Lee MM, Yap MG, Wang DI (2008) Overexpression of cold-inducible RNA-

binding protein increases interferon-gamma production in Chinese-hamster ovary cells.

Biotechnol Appl Biochem 49(4): 247-257

Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000) Influence of bcl-2 on

cell death during the cultivation of a Chinese hamster ovary cell line expressing a

chimeric antibody. Biotechnol Bioeng 68(1): 31-43

Thomas J, Ayling A, Baneyx F (1997) Molecular chaperones, folding catalysts, and the

recovery of active recombinant proteins fromE. coli. Applied Biochemistry and

Biotechnology 66(3): 197-238

Tigges M, Fussenegger M (2006) Xbp1-based engineering of secretory capacity

enhances the productivity of Chinese hamster ovary cells. Metab Eng

Tirasophon W, Welihinda AA, Kaufman RJ (1998) A stress response pathway from the

endoplasmic reticulum to the nucleus requires a novel bifunctional protein

kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev 12(12): 1812-1824

Tsao YS, Cardoso AG, Condon RGG, Voloch M, Lio P, Lagos JC, Kearns BG, Liu Z

(2005) Monitoring Chinese hamster ovary cell culture by the analysis of glucose and

lactate metabolism. J Biotechnol 118(3): 316-327

Tu BP, Ho-Schleyer SC, Travers KJ, Weissman JS (2000) Biochemical Basis of

Oxidative Protein Folding in the Endoplasmic Reticulum. Science 290(5496): 1571-

1574

Tu BP, Weissman JS (2004) Oxidative protein folding in eukaryotes. The Journal of

Cell Biology 164(3): 341-346

Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA (1999) Targeted mRNA

degradation by double-stranded RNA in vitro. Genes Dev 13(24): 3191-3197

Underdown BJ, Schiff JM (1986) Immunoglobulin A: Strategic Defense Initiative at the

Mucosal Surface. Annual Review of Immunology 4(1): 389-417

326

Underhill MF, Coley C, Birch JR, Findlay A, Kallmeier R, Proud CG, James DC (2003)

Engineering mRNA Translation Initiation to Enhance Transient Gene Expression in

Chinese Hamster Ovary Cells. Biotechnology Progress 19(1): 121-129

Urlaub G, Chasin LA. (1980) Isolation of Chinese hamster cell mutants deficient in

dihydrofolate reductase activity. PNAS USA 77(7) 4216-4220

Urlaub G, Kas E, Carothers AM, Chasin LA (1983) Deletion of the diploid

dihydrofolate reductase locus from cultured mammalian cells. Cell 33(2): 405-412

Vabulas RM, Hartl FU (2005) Protein Synthesis upon Acute Nutrient Restriction Relies

on Proteasome Function. Science 310(5756): 1960-1963

Van der Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg

Effect: The Metabolic Requirements of Cell Proliferation. Science 324(5930): 1029-

1033

Vanhove M, Usherwood YK, Hendershot LM (2001) Unassembled Ig Heavy Chains Do

Not Cycle from BiP In Vivo but Require Light Chains to Trigger Their Release.

Immunity 15: 105-114

Vasa-Nicotera M (2004) The new kid on the block: the unfolded protein response in the

pathogenesis of atherosclerosis. Cell Death Differ 11(S1): S10-S11

Verma R, Boleti E, George AJT (1998) Antibody engineering: Comparison of bacterial,

yeast, insect and mammalian expression systems. Journal of Immunological Methods

216(1-2): 165-181

Virag L (2005) Structure and Function of Poly(ADP-ribose) Polymerase-1: Role in

Oxidative Stress-Related Pathologies. Current Vascular Pharmacology 3: 209-214

Volarevic S, Stewart MJ, Ledermann B, Zilberman F, Terracciano L, Montini E,

Grompe M, Kozma SC, Thomas G (2000) Proliferation, But Not Growth, Blocked by

Conditional Deletion of 40S Ribosomal Protein S6. Science 288(5473): 2045-2047

Wai Lam WL, Liang D, Joseph L, Collette C, Susan C-C, Yan W, Marcio V (2003)

Improvement of Monoclonal Antibody Production in Hybridoma Cells by Dimethyl

Sulfoxide. Biotechnology Progress 19(1): 158-162

Wang XZ, Harding HP, Zhang Y, Jolicoeur EM, Kuroda M, Ron D (1998) Cloning of

mammalian Ire1 reveals diversity in the ER stress responses. EMBO J 17(19): 5708-

5717

Wang XZ, Lawson B, Brewer JW, Zinszner H, Sanjay A, Mi LJ, Boorstein R, Kreibich

G, Hendershot LM, Ron D (1996) Signals from the stressed endoplasmic reticulum

induce C/EBP-homologous protein (CHOP/GADD153). Mol Cell Biol 16(8): 4273-

4280

Wek RC, Staschke KA (2010) How do tumours adapt to nutrient stress. EMBO J

29(12): 1946-1947

327

Welihinda AA, Kaufman RJ (1996) The unfolded protein response pathway in

Saccharomyces cerevisiae. Oligomerization and trans-phosphorylation of Ire1p (Ern1p)

are required for kinase activation. J Biol Chem 271(30): 18181-18187

Werner RG, Noe W, Kopp K, Schluter M (1998) Appropriate mammalian expression

systems for biopharmaceuticals. Arzneimittelforschung 48(8): 870-880

Wianny F, Zernicka-Goetz M (2000) Specific interference with gene function by

double-stranded RNA in early mouse development. Nat Cell Biol 2(2): 70-75

Wickham TJ, Davis T, Granados RR, Shuler ML, Wood HA (1992) Screening of Insect

Cell Lines for the Production of Recombinant Proteins and Infectious Virus in the

Baculovirus Expression System. Biotechnology Progress 8(5): 391-396

Wickner W, Schekman R (2008) Membrane fusion. Nat Struct Mol Biol 15(n7): 658-

664

Wong DC, Wong KT, Nissom PM, Heng CK, Yap MG (2006) Targeting early

apoptotic genes in batch and fed-batch CHO cell cultures. Biotechnol Bioeng 95(3):

350-361

Wong DCF, Wong KTK, Goh LT, Heng CK, Yap MGS (2005) Impact of dynamic

online fed-batch strategies on metabolism, productivity and N-glycosylation quality in

CHO cell cultures. Biotechnoland Bioeng 89(2): 164-177

Woof JM, Kerr MA (2004) IgA function; variations on a theme. Immunology 113(2):

175-177

Woof JM, Burton DR (2004) Human antibody-Fc receptor interactions illuminated by

crystal structures. Nat Rev Immunol 4(2): 89-99

Wurm FM (2004) Production of recombinant protein therapeutics in cultivated

mammalian cells. Nat Biotechnol 22(11): 1393-1398

Wurm FM, Gwinn KA, Kingston RE (1986) Inducible overproduction of the mouse c-

myc protein in mammalian cells. PNAS USA 83(15): 5414-5418

Yamamoto K, Ichijo H, Korsmeyer SJ (1999) BCL-2 Is Phosphorylated and Inactivated

by an ASK1/Jun N-Terminal Protein Kinase Pathway Normally Activated at G2/M. Mol

Cell Biol 19(12): 8469-8478

Yang T, Sauve A (2006) NAD metabolism and sirtuins: Metabolic regulation of protein

deacetylation in stress and toxicity. The AAPS Journal 8(4): E632-E643

Yoneda T, Imaizumi K, Oono K, Yui D, Gomi F, Katayama T, Tohyama M (2001)

Activation of Caspase-12, an Endoplastic Reticulum (ER) Resident Caspase, through

Tumor Necrosis Factor Receptor-associated Factor 2-dependent Mechanism in

Response to the ER Stress. J Biol Chem 276(17): 13935-13940

Yoon SK, Hong JK, Choo SH, Song JY, Park HW, Lee GM (2006) Adaptation of

Chinese hamster ovary cells to low culture temperature: Cell growth and recombinant

protein production. J Biotechnol 122(4): 463-472

328

Yoon SK, Song JY, Lee GM (2003) Effect of low culture temperature on specific

productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster

ovary cells. Biotechnology and Bioengineering 82(3): 289-298

Yoshida H, Haze K, Yanagi H, Yura T, Mori K (1998) Identification of the cis-Acting

Endoplasmic Reticulum Stress Response Element Responsible for Transcriptional

Induction of Mammalian Glucose-regulated Proteins. INVOLVEMENT OF BASIC

LEUCINE ZIPPER TRANSCRIPTION FACTORS. J Biol Chem 273(50): 33741-

33749

Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K, Mori K (2003) A time-

dependent phase shift in the mammalian unfolded protein response. Dev Cell 4(2): 265-

271

Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced

by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active

transcription factor. Cell 107(7): 881-891

Yoshida H, Yoshizawa T, Shibasaki F, Shoji S, Kanazawa I (2002) Chemical

Chaperones Reduce Aggregate Formation and Cell Death Caused by the Truncated

Machado-Joseph Disease Gene Product with an Expanded Polyglutamine Stretch.

Neurobiology of Disease 10(2): 88-99

Yoshikawa T, Nakanishi F, Ogura Y, Oi D, Omasa T, Katakura Y, Kishimoto M, Suga

K (2000) Amplified gene location in chromosomal DNA affected recombinant protein

production and stability of amplified genes. Biotechnol Prog 16(5): 710-715

Zahn-Zabal M, Kobr M, Girod PA, Imhof M, Chatellard P, de Jesus M, Wurm F,

Mermod N (2001) Development of stable cell lines for production or regulated

expression using matrix attachment regions. J Biotechnol 87(1): 29-42

Zhang J-X, Braakman I, Matlack KES, Helenius A (1997) Quality Control in the

Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of

Glycoproteins with C-Terminal Truncations. Mol Biol Cell 8(10): 1943-1954

Zhou J, Sharfstein ST (2008) Sodium butyrate stimulates monoclonal antibody over-

expression in CHO cells by improving gene accessibility. Biotechnol Bioengin 100(1):

189-194

Zong W-X, Li C, Hatzivassiliou G, Lindsten T, Yu Q-C, Yuan J, Thompson CB (2003)

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol

162(1): 59-69

329

9

10

APPENDICES

330

APPENDIX 1 – MATERIALS, CHEMICALS AND EQUIPMENT

BACTERIAL CELLS

Professor. A. J. Dickson, University of Manchester, UK

E. coli DH5α strain (genotype F-, φ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR,

recA1, endA1, hsdR17(rk-, mk+), phoA, supE44, λ-, thi-1, gyrA96, relA1)

PLASMIDS

Professor. A. J. Dickson, University of Manchester, UK

p100-D9

MedImmune, Cambridge, UK

Recombinant IgG

MEDIA AND SUPPLEMENTS

Invitrogen, UK

Chemically-defined (CD) CHO media -glutamine, -hypoxanthine, -thymidine

Lonza

L-Glutamine

MedImmune, Cambridge, UK

CD feed

Sigma-Aldrich, UK

Dimethyl sulfoxide (DMSO)

CHEMICALS AND SOLVENTS

Acros Organics, NV

Methoxyamine hydrochloride

Amersham Bioscience, UK

L-[4,5-3H] leucine

NCS tissue solubiliser

BD Transduction Laboratories, UK

Mouse anti-pan ERK antibody (610123)

BDH Chemicals Ltd., U.K.

Acetaldehyde

Ammonium acetate

Calcium chloride

Citric acid

Disodium hydrogen phosphate

Ethylenediaminetetra-acetic acid (EDTA)

331

Glucose

Glycine

HEPES

Hydrochloric acid

Orthoboric acid

Orthophosphoric acid

Paraformaldehyde

Perchloric acid

Peroxidase

Potassium chloride

Sodium acetate

Sodium carbonate

Sodium chloride

Sodium deoxycholate

Sodium dodecyl sulphate (SDS)

Sodium hydrogen orthophosphate

Sodium hydroxide

Sodium phosphate

Sulphuric acid

Trichloroacetic acid (TCA)

Tris(hydroxymethyl) methylamine (Tris)

Bioline, UK

1kb DNA ladder (DNA marker)

Bio-Rad Laboraties, Ltd., UK

Bromophenol blue

Precision Plus Protein All Blue Standards 250 kD

Bio-Rad Laboraties Ltd., UK

Bio-Rad protein reagent

Burdick & Jackson, USA

Methanol (for metabolite extraction)

Dako

Anti-mouse Ig-HRP

Eurofins MWG Operon, Germany

Custom made oligonucleotides

Eurogentec Ltd., UK

qPCR Mastermix SYBR® Green 1

Fisher Scientific, UK

Acetic acid

Acetronitrile (ACN)

Chloroform

Ethanol

Glacial acetic acid

Glycerol

332

Isopropanol

Methanol

Perchloric acid

Potassium acetate

Trifluroacetic acid (TFA)

Fluka

NAD+

GE Healthcare, UK

[α32

P] dATP (specific activity 3000Ci/mmol, concentration 10mCi/ml)

Gibco BRL, UK

Colcemid

Invitrogen, UK

ProLong® antifade reagent

TRIzol® reagent

SlowFade® antifade reagent

Jackson Immuno Research Laboratories, USA.

AffiniPure goat anti-human IgG (109-005-098)

Allophycocyan-conjugate AffiniPure (Fab‟)2 Fragment anti-human Fcγ

(109-136-170)

Texas Red® dye-conjugated AffiniPure anti-rabbit IgG (711-075-152)

Melford Laboratories, Ltd., UK

Agar

Agarose

Phenyl-methyl sulfonyl fluoride

Tryptone

Yeast extract

National Diagnostics, UK.

Ecoscint scintillation liquid

Protogel solution

Roche Applied Science, UK

dATP (Li Salt)

dCTP (Li Salt)

dGTP (Li Salt)

dTTP (Li Salt)

PNGase F

Proteinase K

Restriction endonucleases

Taq DNA polymerase

Santa Cruz Biotechnology, CA

Polyclonal rabbit anti-GADD153 antibody (sc-793)

Polyclonal goat anti-GRP78 antibody (sc-1050)

333

Polyclonal rabbit anti-CREB2 antibody (sc-200)

Sigma-Aldrich, UK

2-mercaptoethanol

3,3‟,5,5‟-tetramethylbensidine tablets

4‟,6-diamino-2-phenylindole (DAPI)

4α-aminophenazone

Albumin bovine (BSA)

Ammonium bicarbonate (AMBIC)

Ammonium persulphate

Ampicillin

Anti-rabbit Ig-HRP

Aprotinin

Bromophenol blue

Calf thymus DNA

Cycloheximide

Diethyl polycarbonate (DEPC)

Dimethyl sulfoxide (DMSO)

Diphenylamine

Dithiothreitol (DTT)

Ethidium Bromide

Glass-acid washed beads

Glucose oxidase

Hydrogen peroxide

Hydrazine

Isoamyl alcohol

Lactate

Lactate dehydrogenase (LDH)

Leupeptin

L-methionine sulphoximine (MSX)

Magnesium acetate

Myristic acid d 27

N-lauroyl sarcosine

N,N,N‟,N‟-tetramethylethylenediamine (TEMED)

Paraformaldehyde

Phenol

Phosphate buffered saline tablets

Poly-L-lysine

Ponceau stain

Potassium

Propidium Iodide (PI)

Pyridine

RNase A

Sephadex G-50

Sodium bicarbonate

Sodium citrate

Sodium fluoride

Sodium orthovanadate

Sucrose (high grade 99.5% GC)

Trisodium phosphate

334

Triton x100

Trypan blue

Tween-20

Southern Biotechnology

Goat anti-human lamda FITC conjugate (2070-02)

The Binding Site, U.K

Sheep anti-human lambda (peroxidise conjugate)

ThermoScientific, USA

N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) with 1%

trimethylchorosilane (TMCS)

Defatted milk [MarvelTM

] can be bought from most supermarkets and PDI was kindly

donated from Professor Neil Bulleid.

KITS

Bioline, UK

cDNA Synthesis Kit (Bio-65025)

BioVision

NAD/NADH Quantification Kit (K337-100)

GE Healthcare, UK

ECL western detection kit (RPN2106)

Ab SpinTrap columns (28-4083-47)

Lonza Biologics

MycoAlert® Detection Kit (LT07-118)

Millipore Corporation, USA

MicroCon ultrafiltration unit (478)

Roche Ltd., UK

Nick Translation Kit (10976776001)

Random Primed DNA Labelling Kit (11004760001)

ATP Bioluminescence Assay Kit CLS II (1169969500)

Sigma-Aldrich, UK

DNase I kit (AMPD1-1KT)

Qiagen Ltd., UK

Plasmid Midi Kit (12143)

Qiaex II Gel Extraction Kit (20021)

335

APPARATUS

All general and disposable glassware and plasticware were obtained from standard

suppliers. Specialised equipment was purchased from the following companies

Amersham International Plc., UK

Nylon membrane (Hybond-N)

Agilent Technologies

7890A GC System

5975C Inert XL MSD with Triple-Axis Detector

ChemStation

BDH Chemicals Ltd., UK

Haemacytometer (Improved Neubauer)

Beckman Coulter Inc., USA

CyAn ADP flow cytometer, with Summit 4.3 software

J2-21 centrifuge with JA-20 rotor

L8-70 centrifuge with SW41 rotor

Bio-Rad Laboraties Ltd., UK

Chromo4 thermal cycler

Model GS-700 imaging densitometer

Mini-gel II Slab System

Thick Filter paper

Trans-blot semi- dry transfer cell

MJ white 96 well plate for real-time PCR

Clear plastic caps for MJ white plates

Bruker, Ltd.

UltraFLEX MALDI-ToF mass spectrometer and software

Dynex Technologies Inc., UK

Dias System Plate reader

Gibco BRL, UK

Horizontal electrophoresis gel tank

Glycan Corp, USA

HyperCarb solid phase TopTips column

Gonotec GmbH, Germany

Osmomat030 cryoscopic osmometer

Kodak, USA

M35-M X-OMAT film processor

X-ray film (Biomax MR-1)

X-ray film cassettes & intensifying paper

336

Labcaire systems Ltd., UK

Recirculating class II microbiological safety cabinet

Labsystems Oy

Multiskan RC plate reader and Ascent software

LaserBio Labs, France

2,5 dihydroxybenzoic acid (DHB) matrix

LTE Scientific Ltd., UK

Series 250 Autoclave

Molecular Devices

MetaVue Software

Millipore, UK

ElixTM

water purification system

Mini-instruments Ltd., UK

Series 900 mini radioactive monitor

MJ Research, UK

Opticon Monitor real-time PCR software version 2.03.6

New Brunswick Scientific Ltd., UK

Innova 4000 shaking incubator

NIH

Image J software

NIST

AMDIS

Olympus Ltd., UK

Olympus BX51 upright microscope

Packard, UK

Tri Carb 2100TR liquid scintillation analyser

Photometrics

Coolsnap HQ camera

Scientific Laboratory Supplies, UK

13mm round glass coverslips

Scintillation vials

Scleicher & Schuell, Germany

Nitrocellulose membrane

337

Sefton Scientific, USA

9/16 x 3½ inch polyallomer tube

Spectronics Corporation

SpectroLinker XL-1000 UV crosslinker

SPSS, Inc

Analytical software

Techne, UK

Thermal cycler

Hybridiser HB-1D hybridisation oven

Teledyne Isco, Inc., USA

Isco-UA-6 UV/Vis detector

Thermo Fisher Scientific

Cryovials

GC vials

NanoDrop® 1000, UV/Vis spectrophotometer

Nunc-immuno plates (maxisorp F96)

HETO VR MAXI vacuum centrifguge attached to a HETO CT/DW 60E cooling

trap

Turner Designs, USA

TD20/20 luminometer

Ultraviolet Products, USA

UV transilluminator

Verity Software House

ModFit LTTM

Vickers Instruments, UK

Light microscope

VWR International

Erlenmeyer flasks

Whatman Biosystems Ltd., UK

3mm filter paper

Zeiss, Germany

Widefield Axiovision microscope and Axiovision software

338

APPENDIX 2 – RELATIVE CONCENTRATION OF AMINO ACIDS

Figure A2.1 The relative concentrations of amino acids during batch culture




(Section 2.3.2), and a medium control sample, were spiked with the internal standard

myristic acid d27 and lyophilised. Chemical derivatization was performed in two stages,

with methyloxyamine hydrochloride in pyridine, before the addition of MSTFA and

TMCS (Section 2.10.3.1). All samples were analysed using GC-MS analysis, within 24

hr of derivatization. Raw data processing was performed using ChemStation and

AMDIS (Section 2.10.3.2). A, shows the relative percentage of isoleucine B, shows the

relative percentage leucine, C, shows the relative percentage of valine, D, shows the

relative percentage methionine, E, shows the relative percentage of proline, F, shows

the relative percentage tyrosine, and G, shows the relative percentage threonine. All

values were normalised to the internal standard, myristic acid d27. Error bars represent

SD for two biological replicates.

Annotation of the batch cultures in Figure A2.1

Early generation cultures


339

Figure A2.1 The relative concentrations of amino acids during batch culture

A.

B.

D.

C.

0

10

20

30

40

50

Medium 5 7 9 13

Rela

tiv

e iso

leu

cin

e

(%)

Day

0

20

40

60

80

Medium 5 7 9 13

Rela

tiv

e leu

cin

e

(%)

Day

0

10

20

30

40

50

Medium 5 7 9 13

Rela

tiv

e v

ali

ne

(%)

Day

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Medium 5 7 9 13

Rela

tiv

e m

eth

ion

ine

(%)

Day

340

E.

F.

G.

0

2

4

6

8

10

12

Medium 5 7 9 13

Rela

tiv

e th

reo

nin

e

(%)

Day

0

20

40

60

80

100

Medium 5 7 9 13

Rela

tiv

e p

ro

lin

e

(%)

Day

0

5

10

15

20

Medium 5 7 9 13

Rela

tiv

e ty

ro

sin

e

(%)

Day

341

APPENDIX 3 – OSMOLALITY MEASURMENTS

Table A3.1 Osmolality measurements in response to feed and DMSO addition

Cell culture supernatants, taken from day 7 of batch culture, were measured for

osmolality. The osmolality of cultures was measured using an automatic micro-

osmometer (Section 2.3.6). Water (zero mOsmole) and a 300 mOsmole standard

solution (Minimum Essential Medium, containing Earle’s salts and L-glutamine) were

used to calibrate for the measurement of samples.

Generation Day Culture condition Osm/kg

Early 3 no addition 0.318

Early 3 + feed addition 0.343

Early 7 no addition 0.308

Early 7 + feed addition 0.449

Early 7 + DMSO addition 0.573

Late 3 no addition 0.319

Late 3 + feed addition 0.352

Late 7 no addition 0.311

Late 7 + feed addition 0.452

Late 7 + DMSO addition 0.58

342

APPENDIX 4 – INVESTIGATING CHEMICAL ADDITIONS

Figure A4.1 Preliminary investigation of different chemical additions to improve

recombinant protein production

Cell Line 3.90 was subject to long-term culture in suspension using MSX supplemented

CD-CHO media. Batch growth analysis was created from 0.2x106 cells/ml, and

maintained at 37oC, 140rpm with a manual supply of 5% CO2 in air. Cultures were

supplemented with different chemical addition on day 5 of batch culture. These

additions included 0.4M glycerol, 1M glycerol, 2% (v/v) DMSO, 5% (v/v) DMSO,

0.05M sorbitol or 0.5M sorbitol. Cells were cultured under these conditions until

viability was ≤ 50%. Antibody titres were measured by ELISA (Section 2.5.1), and

viable cell densities were determined by light microscopy and trypan blue exclusion

(Section 2.3.3). This figure shows A. antibody titres, and B. viable cell densities, for the

different chemical additions.

Annotation of the generation batch cultures in Figure A4.1

Unfed

0.4M Glycerol

1M Glycerol

2% (v/v) DMSO

5% (v/v) DMSO

0.05M Sorbitol

0.5M Sorbitol

343

Figure A4.1 Preliminary investigation of different chemical additions to improve

recombinant protein production

B.

A.

0

400

800

1200

1600

0 3 5 7 9 11 13 15

An

tib

od

y t

itre

(mg

/L)

Day

0

2

4

6

0 5 10 15

Via

ble

cell

s

(x1

06

cell

s/m

l)

Day

344

Table A4.1 Analysis of different DMSO additions on cell growth and final antibody

titres


culture analysis was performed in shake flasks for early generation (≤ 40 generations)

and late (≥ 60 generation) cultures. Batch cultures were created at 0.2x106 cells/ml, and

maintained at 37oC, 140 rpm and with a manual supply of 5% CO2 in air. DMSO

addition was added to early and late generation cultures at concentrations of 2% (v/v)

on day 5 of batch culture or 1% (v/v) on day 0 of batch culture. Antibody titres were


microscopy and trypan blue exclusion (Section 2.3.3) from samples taken during batch

culture (Section 2.3.2). The table shows final antibody titres, maximal viable cell

densities and overall CCT values. For determination of CCT see Section 2.11.2. The

average value is shown ± SEM for three biological replicates. Each biological replicate

value is an average from duplicate technical repeats.

2% (v/v) DMSO addition d5

control 2% (v/v) DMSO control 2% (v/v) DMSO

Maximal viable cell densities (x106 cells/ml) 5.8 ± 0.2 4.5 ± 0.3 5.4 ± 0.2 4.7 ± 0.2

Overall cumulative cell time (x106 x day/L) 46 ± 1.8 42 ± 2.7 43 ± 0.8 41 ± 2.3

Final antibody titre (mg/L) 941 ± 20 1377 ± 38 695 ± 10 1005 ± 17

1% (v/v) DMSO addition d0

control 1% (v/v) DMSO control 1% (v/v) DMSO

Maximal viable cell densities (x106 cells/ml) 5.8 ± 0.2 3 ± 0.5 5.4 ± 0.2 2.9 ± 0.3

Overall cumulative cell time (x106 x day/L) 46 ± 1.8 29 ± 2.4 43 ± 0.8 29 ± 1.7

Final antibody titre (mg/L) 941 ± 20 1073 ± 88 695 ± 10 848 ± 11

Early Late

Early Late

345

APPENDIX 5 – INVESTIGATING EXPRESSION OF UPR MARKERS FOR

THE PARENTAL CELL LINE

Figure A5.1 Parental cells have lower GADD153 and XBP-1(s) mRNA than

recombinant CHO cultures

Cell Line 3.90 and parental cells were subject to subject to culture using MSX and L-

glutamine supplemented CD-CHO media, respectively. Batch growth analysis was

created from 0.2x106 cells/ml, and maintained at 37

oC, 140rpm with a manual supply of

5% CO2 in air. mRNA levels were compared using q-RTPCR from samples taken on day


GADD153. Samples were normalised using mRNA β-Actin primers. (A) The relative

expression of GADD153mRNA is shown for the recombinant and parental cell line.

A PCR was also performed using parental cDNA (Section 2.7.1.3) and XBP-1(s)

primers (Section 2.7.2), from samples taken on days 3, 7, and 9 during batch culture.

The PCR products were electrophoresed on a 2% (w/v) agarose gel and visualised by

UV transillumination (Section 2.6.1.4). The product bands were analysed using Image J

software. The quantified ratio of spliced XBP-1mRNA to total XBP-1mRNA (B) and the

product bands (C) is shown. Error bars represent SEM for three biological replicates. *

indicates p<0.05, using independent samples t-test to compare parental cultures to the

recombinant 3.90 culture.


3.90 early generation, no addition

Parental no addition

Parental with feed addition

346

Figure A5.1 Parental cells have lower GADD153 and XBP-1(s) mRNA than

recombinant CHO cultures

A.

B.

C.

0

50

100

150

200

GA

DD

15

3 m

RN

A e

xp

ress

ion

(% r

ela

tiv

e t

o s

tan

da

rd

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

3 7 9 Positive

XBP-1(U)

XBP-1(S)

0

5

10

15

20

25

30

35

40

3 7 9

Am

ou

nt

of

spli

ced

XB

P-1

mR

NA

to to

tal

XB

P-1

(%)

Day

*

*

347

Figure A5.2 ATF4 is significantly lower for the parental cell line

Cell Line 3.90 and parental cells were subject to subject to culture using MSX and L-

glutamine supplemented CD-CHO media, respectively. Batch growth analysis was

created from 0.2x106 cells/ml, and maintained at 37

oC, 140rpm with a manual supply of

5% CO2 in air. mRNA levels were compared using q-RTPCR from samples taken on day


ATF4. Samples were normalised using mRNA β-Actin primers. (A) The relative

expression of ATF4 mRNA is shown for the recombinant and parental cell line. ATF4

protein was also investigated via immunofluoresence (Section 2.94) and western

blotting (Section 2.5.3). Protein was extracted on day 9 of batch culture (detailed in

Section 2.5.3.1). 60µg of protein was separated by SDS-PAGE (Section 2.5.3.2),

transferred then detected using anti-rabbit polyclonal ATF4 antibody (Section 2.5.3.3).

Membranes were stripped and re-probed using an anti-mouse pan-ERK antibody

(Section 2.5.3.4). Bands were analysed using Image J software and calculated relative

to ERK (Section 2.5.3.5). Cells on day 9 of cultures were permeabilised, fixed and

incubated with ATF4, Texas Red®, and DAPI separately as described in Section 2.9.4.

Images were collected using an Olympus BX51 upright microscope using a coolsnap ES

camera through Metavue software, and analysed using Image J Software. The relative

protein intensities for western blot and immunofluorescence analyses are shown in B

and C, respectively. Typical examples of a recombinant cell line (D(i)), and a parental

cell line (D(ii)) expressing ATF4 are shown. Error bars represent SEM for three

biological replicates. * indicates p<0.05, using independent samples t-test to compare

parental cultures to the recombinant 3.90 culture. White scale bars = 10μm.


3.90 early generation, no addition

Parental no addition

Parental with feed addition

348

Figure A5.2 ATF4 is significantly lower for the parental cell line

A.

B.

0

50

100

150

200

250

300

AT

F4

mR

NA

ex

press

ion

(% r

ela

tiv

e t

o s

tan

da

rd

an

d

β-A

cti

nm

RN

A e

xp

ress

ion

)

0

20

40

60

80

100

120

140

3.90 Early 3.90 Late Parental

AT

F4

pro

tein

in

ten

sity

(rela

tiv

e to

sta

nd

ard

ER

K)

Cell line/GenerationC.

0

20

40

60

80

100

120

3.90 Early 3.90 Late Parental

AT

F4

mic

ro

sco

py

qu

an

tifi

ca

tio

n

Cell line/Generation

D.

(i) (ii)

* *

*

*

349

APPENDIX 6 - MYCOPLASMA TESTING

Table A6.1 Mycoplasma is not detected during batch culture

Early (≤ 40 generations) and late (≥ 60 generation) 3.90 and 51.69 cell cultures were

routinely tested for the presence of mycoplasma during LTC, using a MycoAlert®

Detection Kit (Section 2.3.7), according to manufacturer’s instructions.

Early Late Negative Positive

Cell line

3.90 0.32 ± 0.02 0.36 ± 0.04 0.61 ± 0.10 76.25 ± 7.43

Cell line

51 0.28 ± 0.05 0.31 ± 0.08 0.61 ± 0.11 76.25 ± 7.44

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