In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

In Vitro Galactosylation of Human IgGat 1 kg Scale Using RecombinantGalactosyltransferase

Dale Warnock, Xiaomei Bai, Katie Autote, Johnny Gonzales, Kyle Kinealy,Boxu Yan, Jun Qian, Tom Stevenson, David Zopf, Robert J. Bayer

Neose Technologies, Inc., 102 Witmer Road, Horsham, Pennsylvania 19044;telephone: 858-552-2700, fax: 858-452-1009; e-mail: [email protected]

Received 12 January 2005; accepted 20 June 2005

Published online 26 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20658

Abstract: The Fc effector functions of immunoglobulinG (IgG) antibodies are in part determined by structuralfeatures of carbohydrates linked to each of the pairedgamma heavy chains in the antibody constant domain(CH2). One glycoform that has been shown to beadvantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitrotreatment with glycosyltransferases can remodel hetero-geneous IgG glycoforms, enabling preparation of IgGmolecules with homogeneous glycan chains. Here wedescribe optimization of conditions for use of a solublerecombinant galactosyltransferase in vitro to remodelglycans of human serum IgG, and we demonstrate ascaled-up reaction in which >98% of neutral glycansattached to 1 kg IgG are converted to the G2 glycoform.Removal of glycosylation reagents from the product isachieved in one step by affinity chromatography onimmobilized Protein A. � 2005 Wiley Periodicals, Inc.

Keywords: glycoengineering; glycoprotein remodeling;glycosylation; glycosyltransferase; antibody; antibodyeffector function

INTRODUCTION

Antibody based immunotherapy is a rapidly developing

segment of pharmaceutical drug discovery efforts. Several

antibody based treatments for various cancers (Clynes et al.,

2000; White et al., 2001) and inflammatory diseases such as

rheumatoid arthritis (Maini et al., 1999; Rastetter et al., 2004;

Sandborn and Hanauer, 1999) are currently approved and

many more are currently in clinical and pre-clinical

development (Presta, 2002; Souriau and Hudson, 2003).

Whereas early efforts to develop therapeutic monoclonal

antibodies (mAbs) focused primarily upon maximizing

specificity of antigen binding and minimizing immunogeni-

city of the immunoglobulin G (IgG) protein, more recent

engineering of IgG mAbs has expanded to include custo-

mized up- or down-regulation of Fc effector functions

(Shriver et al., 2004).

The glycans most commonly associated with human

serum IgG (Fujii et al., 1990; Hamako et al., 1993; Raju et al.,

2000) or therapeutic mAbs (Jefferis, 2001, 2005) are

complex bi-antennary chains N-linked to Asn-297 of the g-h-chain. These branched sugar chains are situated within a

cleft formed by the paired heavy chains in the CH2 domain

such that they may undergo extensive non-covalent interac-

tions with the adjacent polypeptide (Krapp et al., 2003).

Pharmacologic properties that may be affected by modifica-

tions in IgG glycans include: (i) plasma residence time (ii)

rate of antigen clearance from the plasma, (iii) potential for

activation of the complement pathway, (iv) efficiency in

promoting cell killing via antibody-dependent cellular

cytotoxicity (ADCC), and (v) capacity to trigger inflamma-

tory responses (Dijstelbloem et al., 2001; Jefferis and Lund,

2002; Krapp et al., 2003). The precise structural basis for

glycan-related modulation of antibody efficacy in vivo

remains to be fully elucidated, but it has become clear that

binding to FcgRII and FcgRIII receptors requires the N-

linked glycan at Asp-297 in the CH2 domain (Coloma et al.,

2000; Leatherbarrow et al., 1985; Walker et al., 1989), and

that while the minimal trimannosyl-chitobiose core structure

(see Fig. 1) is sufficient to allow FcgRIIb binding (Mimura

et al., 2001), more extended glycan structures exert effects on

the Fc tertiary structure that could serve to modulate specific

FcgR–IgG interactions (Jefferis et al., 1998; Mimura et al.,

2001). The key mechanism by which glycans may affect

antibody binding to Fc receptors appears to be through

stabilization of a polypeptide conformation of the Fc domain

with increased affinity for Fc receptors (Lund et al., 1995;

Radaev and Sun, 2001; Wormald et al., 1997).

The presence or absence of galactose on IgG glycans

correlates with modified Fc effector function in some

(Kumpel et al., 1994) but not all (Boyd et al., 1995; Lund

et al., 1995; Wright and Morrison, 1998) monoclonal IgG

antibodies, suggesting that the effects observed may be, in

�2005 Wiley Periodicals, Inc.

Correspondence to: R. J. Bayer

part, antibody specific. Several investigators have assessed

Fc effector functions in vitro after enzymatic removal of

galactose from IgG with b-galactosidase. Tsuchiya et al.

(1989) found that agalacto IgG had reduced C1q and Fc

receptor binding, and Boyd et al. (1995) found that agalacto

Campath1 (monoclonal anti-CD52) had reduced cell-

mediated lysis (CML) but intact capacity to trigger ADCC.

Other reported effects of galactosylation on IgG molecules

include modification of physicochemical properties such as

conformation and surface accessibility (Dwek et al., 1995;

Ghirlando et al., 1999; Krapp et al., 2003; Mimura et al.,

2000).

It would be desirable to convert IgG molecules to a single

(>90%) defined glycoform, both to develop reagents for

studying the impact of glycosylation onFc effector functions,

and to prepare mAb drugs with a desired glycoform at scale.

Fujii et al. (1990) previously demonstrated at analytical scale

that the glycans on intact IgG molecules can function as

acceptors for glycosyltransferases. In this article,we describe

conditions for use of a recombinant glycosyltransferase to

remodel IgG glycans and demonstrate in vitro enzymatic

galactosylation of human IgG at the 1 kg scale, resulting in

nearly complete (>98%) conversion to the G2 glycoform

(Fig. 1).

MATERIALS AND METHODS

Enzymes, Antibodies, and Reagents

A gene construct encoding a truncated form of bovine b-1,4galactosyltransferase-I (NCBI protein no. AAM54035)

(GalT-1) was cloned into a recombinant expression vector

that allows high level expression and secretion from

mammalian derived cell lines (Narimatsu et al., 1986). The

gene product was overexpressed in the mouse myeloma NS0

cell line and recovered as a secreted product from NS0 cell

culture (971 U/L) carried out for 432 h in a 40 L bioreactor.

Clarified and concentrated culture supernatant was buffer

exchanged into 50 mM NaCl, 20 mM 2-(N-morpholi-

no)ethane-sulfonic acid (MES), pH 6.0, then loaded onto a

column of SP sepharose (18� 17 cm). The enzyme was

eluted with 1 M NaCl, 20 mM MES, pH 6.0, concentrated,

and buffer exchanged into 150mMNaCl, 50mMHEPES, pH

7.5 on an Amicon S10Y10 cartridge filter (Millipore Corpo-

ration, Bedford, MA), and sterile filtered. The enzymatic

activity of the purified GalT-1 was assayed by transfer of

[3H]-galactose (0.63 mM uridine 50-diphosphate (UDP)-

galactose) to N-acetylglucosamine (20 mM) in 50 mM

3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.4),

Figure 1. Typical oligosaccharide structures found in the antibody fragment that crystallizes (Fc) domain of IgG. The sugars highlighted in orange boxes are

variably present, and account for much of the variability observed in Fc glycans.A: Completely galactosylated and sialylated complex bi-antennary structure,

including ‘‘bisecting’’ N-acetyl glucosamine (GlcNAc) linked b1–4 to mannose, and ‘‘core’’ fucose linked a1–6 to the asparagine-linked GlcNAc; (B) G2oligosaccharides havegalactosemoieties onboth antennae; (C)G0 indicates that no galactose is present; (D,E)G1 structures havegalactose on either the 1–6or1–3 arm.

832 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 92, NO. 7, DECEMBER 30, 2005

30 mM MnCl2, 0.2 mg/mL bovine serum albumin (BSA),

during a 3 min incubation at 378C. The radioactive productwas isolated through a 1 mL column of Dowex AG1X8

(Biorad, Hercules, CA) pre-washed in water, and quantified

by scintillation counting. The purity of the GalT-1 prepara-

tion was measured by scanning and integration of an sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE) gel stained with fluorescent SYPRO Tangerine

Protein Gel Stain (Ex/Em 300, 490/640 nm) fromMolecular

Probes according to the manufacturer’s instructions. A

digital image was acquired taking care not to saturate any

of the bands on the gel using a FACE Imaging System from

Glyko (300 nm transilluminator). The image was saved in

TIFF format, and the relative band densities were integrated

by using software (Scion Image for Windows, a modified

version of NIH Image).

Polyclonal antisera raised against bovine GalT-1 was

produced in rabbits. Donkey anti-rabbit horseradish perox-

idase (HRP) conjugate and West-Pico chemiluminescent

reagent were obtained from Pierce (Rockford, IL). All SDS–

PAGE gels and buffers were from Invitrogen (Carlsbad, CA).

UDP-[3H]-galactosewas a product of NewEngland Nuclear/

PerkinElmer (Boston, MA) and UDP-galactose was a

product of Boehringer Mannheim (Indianapolis, IN).

Human gamma globulin was purchased from Serologicals

Corporation (Norcross, GA). Unless otherwise specified, all

other reagent grade chemicals were purchased from Sigma

(St. Louis, MO). Chromatography resins were obtained from

Amersham Biosciences/GE Healthcare (Piscataway, NJ)

unless otherwise noted.

Remodeling Reaction Conditions

Small scale test reactions were carried out in 1.5 mL

microfuge tubes containing 50 mL reaction buffer: 20 mM

MnCl2, 10 mM UDP-galactose, 100 mM MES, pH 6.5. The

concentration of human serum IgG was 20 mg/mL unless

otherwise specified. GalT-1 activity added varied from 10 to

100 mU/mg IgG and reactions were incubated for up to 72 h

at 328C.Large scale remodelingwas performed in a final volume of

33.5 L in a 40 L, temperature controlled, stirred bioreactor

vessel (Wheaton, Inc., Millville, NJ) equipped with pH and

temperature monitors. One kilogram of lyophilized human

IgG was dissolved in 25 L buffer containing 0.1 M MES, 20

mMMnCl2, and 0.02% sodiumazide, pH6.5.UDP-galactose

was added to a concentration of 10 mM, and GalT-1 was

added to a final activity ratio of 25 mU/mg IgG. The reaction

mixture was brought to a volume of 33.5 L (30 mg/mL final

concentration of IgG) and carefully re-adjusted to pH 6.5.

Samples were removed at appropriate time points during the

reaction and stored frozen at �208C until analyzed.

rProtein A-Sepharose Fast Flow (FF) AffinityChromatography

Human IgG (30 mg/mL) remodeled with GalT-1 (48 h

reaction time) was thawed and filtered using a 0.45 mm

Acrodisc HT Tuffryn Syringe filter obtained from Pall

Corporation (AnnArbor,MI). One hundredmilligrams of the

sample was injected onto a 5 mL column of rProtein A-

Sepharose (XK16/60 column on an Akta fast protein liquid

chromatography (FPLC) system, Amersham Biosciences/

GE Healthcare) via a 5 mL sample loop. The loading and

washing buffer was 20 mM Tris, 150 mMNaCl, pH 7.2. The

elution buffer was 0.2 M glycine-HCl, pH 3.0. One-milliliter

fractions were collected and immediately neutralized with

0.1 mL 1 M Tris, pH 9.0.

Sample Preparation for Oligosaccharide Analysis

Twenty-five microliter aliquots of remodeling reaction

samples were diluted to 100 mL with phosphate buffered

saline (PBS), and then centrifuged in a microfuge at 14,000g

for 5 min to remove precipitated manganese phosphate.

Supernatants were applied to protein desalting spin columns

(Pierce) pre-equilibrated with 20 mM sodium phosphate

buffer, pH 7.0, and eluted by centrifugation at 2,000g. Eluted

samples were assayed for protein concentration, diluted to 1

mg/mL, and prepared for 2-anthranilic acid (2-AA) glycan

analysis (Anumula and Dhume, 1998). Glycan release from

100 mm of protein was achieved by incubation overnight at

378C in 50 mM sodium phosphate pH 7.2 containing 0.2%

SDS, 1% NP-40, 0.1 M b-mercaptoethanol, and 50 mU

peptide N-glycosidase F (PNGase F, Calbiochem, La Jolla,

CA). Enzyme reactions were stopped by the addition of 0.6

mL ice-cold ethanol and the resulting mixtures were

incubated on ice for 10 min, centrifuged at 14,000g for 5

min, and concentrated to dryness in a vacuum centrifuge.

Released glycans were labeled with 2-amino anthranilic acid

with a LudgerTag 2-AA glycan labeling kit (Ludger Ltd.,

Oxford, UK) according to the manufacturer’s instructions.

The 2-AA labeled glycanswere desalted on 13mmAP10MF

support pads (Millipore) to remove salts and labeling

reagents, eluted with dH2O, and lyophilized.

HPLC Analysis of 2-AA Labeled Oligosaccharides

Immediately prior to high performance liquid chromatogra-

phy (HPLC) analysis, lyophilized 2-AA labeled glycanswere

re-dissolved at approximately 0.4 mg/mL in dH2O. HPLC

analysis was performed on a Shodex Asahipak NH2P-50 4D

amino column (4.6� 150 mm, Shodex Inc., Tokyo, Japan) at

a flow rate of 0.8 mL/min and a column temperature of 408C.To analyze for neutral glycans, typically a 10 mg sample was

injected in 25 mL and eluted isocratically for 5 min with 70%

mobile phase A (2% acetic acid, 1% tetrahydrofuran in

acetonitrile) and 30% mobile phase B (5% acetic acid, 1%

tetrahydrofuran, 3% triethanolamine in water). Next the

column was eluted with a linear gradient to 50% mobile

phaseAover 60min, 50%mobile phaseA isocratically for 10

min, then a linear gradient to 5% mobile phase A for 10 min,

then re-equilibrated to 70% mobile phase A for 15 min prior

to injection of the next sample. To analyze charged

oligosaccharides, the column was eluted isocratically with

70% mobile phase A for 2.5 min, followed by a linear

WARNOCK ET AL.: GALACTOSYLATION OF HUMAN IGG 833

gradient to 5% mobile phase A for 97.5 min, and 5% mobile

phase A isocratically for 15 min, followed by column re-

equilibration to 70% mobile phase A for 15 min before

injecting the next sample. Eluted glycans were monitored by

fluorescence with excitation and emission wavelengths of

230 and 420 nm, respectively. Peak elution positions were

compared with 2-AA labeled oligosaccharide standards

purchased from Glyko/Prozyme (San Leandro, CA).

MALDI-TOF analysis was performed on the 2AA-labeled

glycan samples after mixing (1:1) with a 2,5-dihydroxyben-

zoic acid solution (10 mg/mL), and then spotting and drying

on a target. An Applied Biosystems DE-Pro MALDI-TOF

mass spectrometer (Foster City, CA) was operated in linear/

negative-ion mode to analyze the samples. Glycan structures

were assigned based on the observed mass-to-charge ratios

and literature precedence (Fujii et al., 1990; Raju et al., 2000).

LC/MS/MS Analysis of SDS–PAGE FractionatedProteins

Coomassie blue stained gel bands were cut out with a clean

razor blade and placed in low-binding 0.5 mL tubes (CLP,

San Diego, CA). The gel pieces were destained by repeated

extraction with 50% acetonitrile containing 50 mM ammo-

nium carbonate for 30 min at 378C, evaporated to dryness byvacuum centrifugation, and rehydrated for 10 min in 10 mL5% acetonitrile, 50 mM ammonium carbonate, 0.1 mg/mL

modified trypsin (Promega, Madison WI). The volume was

then brought to 75 mL with 5% acetonitrile, 50 mM

ammonium carbonate, and the sample incubated at 378Covernight.Acetic acid (10mLof a 10% solution)was added to

stop the digests, and then samples were reduced to a volume

of approximately 10 mL by vacuum centrifugation.

Liquid chromatography electrospray ionization mass

spectrometry (LC/ESI/MS) analysis was performed using a

Haisil 300 reversed-phase C18 column (0.3� 150 mm,

Higgins Analytical, Inc., Mountain View, CA) on an Agilent

1100 system (Palo Alto, CA) coupled with an LCQ Deca XP

MAX (ThermoElectron, Waltham, MA). The column was

eluted with 98%mobile phase A (0.1% formic acid in water)

for 5 min, followed by a linear gradient to 40%mobile phase

B (0.1% formic acid in acetonitrile) over 65min at a flow rate

of 8 mL/min. After each full MS scan from m/z 400 to m/z

2,000 of the column effluent, MS/MS spectra of the twomost

intense MS peaks were recorded. Chromatograms were

analyzed against the April 2004 NCBI human or bovine non-

redundant protein databases with the program SEQUEST

using Bioworks 3.1 (ThermoElectron). SEQUEST cross-

correlation (Xcorr) values and Dcorr values were used to filter

all peptide matches. Acceptable matches had Dcorr values

�0.1 and Xcorr values �1.7 (þ1 ions) or �2.0 (þ2 ions) or

�3.0 (þ3 ions) (Link et al., 1999).

HPLC Analysis of Nucleotides and NucleotideSugars

Ten-microliter samples containingUDP-galactose, UDP, and

uridine 50-monophosphate (UMP) were analyzed by HPLC

on a Vydac 302IC4.6 Ion-Chromatography Column (4.6�250mm,GraceVydac,Hesperia, CA) at a flow rate of 2.0mL/

min and maintained at 288C. The column was eluted as

follows: isocratic elution with 100%mobile phase A (25mM

NaH2PO4/Na2HPO4, 1:1 molar ratio, pH 2.8) for 2 min,

followed by elution with a linear gradient to 100% mobile

phase B (125 mM NaH2PO4/Na2HPO4, 1:1 molar ratio,

adjusted to pH 2.9 with acetic acid) over a period of 17 min.

Eluted nucleotides were detected by absorption at 260 nm.

The method was standardized using a sample matrix

containing bovine gamma globulin at 30 mg/mL and shown

to give accurate and reproducible results under these

conditions.

Protein Determination by Absorbance and BioRadDC Protein Assay

Fractions from rProtein A Sepharose FF affinity chromato-

graphy were diluted into UV clear bottom 96-well plates and

the absorbance read at 280 nm on a Molecular Devices

Spectramax 384 Plus microplate reader (Sunnyvale, CA).

Affinity chromatography fractions and remodeled samples

were quantified with BioRad DC protein determination

reagents in 96-well plate assay format according to the

manufacturer’s instructions. A standard curve of BSA was

included on each plate, and the absorbance was measured at

750 nm.

SDS–PAGE and Western Analysis

Protein fractions from remodeling reactions and rProtein A-

Sepharose FF affinity chromatography were resolved by

SDS–PAGE on 4%–12% NuPAGE gels in MOPS or MES

running buffer. Protein patterns were visualized with Simply

Blue colloidal Coomassie stain (Invitrogen) according to the

manufacturer’s instructions.

For Western blot analysis, proteins were transferred to

0.4 mm nitrocellulose paper using the Novex transfer

blotting module (Invitrogen). Transfer uniformity was

verified by the transfer efficiency of SeeBlue Plus2

prestained markers (Invitrogen). Blots were blocked in

TBS-T20 (20 mM Tris, pH 7.2, 150 mM NaCl, 0.05%

Tween-20) with 5% dry milk for 30 min. The blots were then

placed in TBS-T20 with 5% dry milk containing rabbit anti-

galactosyltransferase anti-sera and incubated at 48C over-

night with rocking. Blots were washed four times for 5 min

with TBS-T20 then incubated for 1 h at room temp with

gentle shaking in a 1:20,000 dilution of donkey anti-rabbit

HRP-conjugated antibody in TBS-SuperBlock (Pierce).

Blots were washed five times for 5 min with TBS-T20 before

incubation with West Pico Chemiluminescent reagent

(Pierce), as per the kit instructions. Blots were exposed on

Bio Max film (Kodak, Rochester, NY). After scanning the

film on a flatbed scanner, uniform reduction of background

was applied to electronic images with Photoshop1 software

(Adobe, San Jose, CA).


RESULTS

Galactosyltransferase Purification

In total, 32,700Uof purifiedGalT-1with a specific activity of

6 U/mg was recovered in 3.5 L (84% overall yield). The

purity of the final preparation determined by scanning and

integrating an SDS–PAGE gel stained with Sypro Tangerine

was 60%. Sixteen percent of the preparation was serum

albumin, with the balance (24%) being accounted for by a

number of low-abundance bands on the gel.

In Vitro Remodeling of Human IgG Glycans WithRecombinant GalT-1 Enzyme

In order to demonstrate the practicality and scalability of in

vitro enzymatic galactosylation, we undertook the large scale

enzymatic remodeling of the glycans on IgG isolated from

human serum using a recombinant form of the bovine

galactosyltransterase GalT-1. The N-glycans of human IgG

(Fujii et al., 1990; Kornfeld et al., 1971) consist primarily of

neutral bi-antennary complex oligosaccharides. The neutral

bi-antennary structures with zero, one or two terminal b-1,4linked galactosyl residues (Fig. 1) are termed, respectively,

G0, G1, and G2 glycans (Wormald et al., 1997).

The N-glycan profile for commercial pooled human serum

IgG used for the galactosylation experiments reported here

was measured by reversed phase-liquid chromatography

(RP-HPLC) of 2-AA-derivatized glycans released by

PNGase F (Fig. 2). Determinations of total N-glycans species

[mean (�SD) from five separate analyses] revealed that

75.1% (�6.5%) were neutral species, whereas 17.2%

(�3.1%) were singly charged and 7.7% (�3.4%) doubly

charged. The distribution of neutral glycan species calculated

from six separate HPLC glycan analyses of the starting IgG

stock was as follows: G0, 33.2% (�1.8%); G1, 46.7%

(�0.6%); G2, 20.1% (�2.3%).

Incubation of human serum IgG (25 mg/mL) with GalT-1

converted the structurally heterogeneous neutral glycans to

>98%G2 structureswithin 48 h (Fig. 2, Table I). The enzyme

is known to selectively catalyze addition of galactose inb1–4linkage to terminal N-acetyl glucosamine (GlcNAc) on

complexN-linked oligosaccharides, utilizing the high energy

donor UDP-galactose (Khatra et al., 1974). UDP is released

during the reaction. By varying the enzyme/substrate activity

ratio and time of incubation, we determined that conversion

of G0 to G1 occurs rapidly, even at the lowest activity ratio of

GalT-1 tested (10 mU/mg IgG), but conversion of G1 to G2

occurs more slowly, reaching levels¼ 98% after 48 h at

activity ratios of¼ 25 mU GalT-1/mg IgG. Conversion to

100% G2 was achieved only after prolonged incubation (72

h) at a much higher activity ratio (75 mU/mg).

Optimization of Substrate Concentration

To further optimize conditions for large scale IgG glycan

remodeling we carried out galactosylation at increasing

concentrations of IgG. In preliminary experiments, we

observed less than 80% G2 associated with IgG incubated

at 1 mg/mL for 48 h with 50 mU GalT-1/mg IgG. Increasing

the concentration of IgG improved the rate of conversion to

G2 even after decreasing the activity ratio to 25 mU GalT-1/

mg IgG: at 20 mg/mL IgG, neutral glycans were converted to

93%G2 after 24 h (Fig. 2, Table II); at 30mg/mL IgG, 97%of

neutral glycans were converted to G2 after 24 h. At IgG

concentrations¼ 30 mg/mL, conversion to G2 is approxi-

mately 99% in 48 h (Table II). The activity of GalT-1

incubated at 328C for 48 h in the same buffer used for the

remodeling reaction remained constant (data not shown).

Large Scale Remodeling and Glycan AnalysisUsing the Optimized Reaction Conditions

Based on results from smaller scale test reactions (Fig. 2, and

Tables I and II), we chose to remodel 1 kg IgG in a reaction

volume of 33.5 L containing 30 mg/mL IgG, 25 mU GalT-1

Figure 2. Reversed phase-high performance liquid chromatography (RP-

HPLC) analysis of 2-anthranilic acid (2-AA) derivatized glycans from

human IgGbefore and after conversionwith galactosyltransferase-I (GalT-1)

to the G2 glycoform. Human IgG was reacted with GalT-1 and UDP-Gal for

(A) 0 h, (B) 24 h, and (C) 48 h. Glycans eluting at approximately 59 min

terminate in sialic acid on one branch (�1 charge) while those eluting at

approximately 78min terminate in sialic acid on both branches (�2 charges).

The starred peak represents an artifact of glycan preparation.


per milligram IgG, and 10 mM UDP-galactose (final

concentrations). The reaction was carried out for 48 h in a

continuously monitored 40 L bioreactor at 328C, pH 6.5 with

constant stirring. Glycan analysis of aliquots at intervals

(Figs. 3 and 4 showed complete conversion of G0 structures

to G1 within 4 h and nearly complete (98.5%) conversion to

G2 structures after 48 h.

Protein A-Sepharose ChromatographyFractionation of Remodeled IgG

To demonstrate removal of remodeling reagents from the

large scale reaction mixture, an aliquot removed at 48 h was

applied to a column of rProtein A-Sepharose and the column

washed with Tris buffer at neutral pH. Most of the human

serum IgG molecules bound to the affinity matrix, whereas

GalT-1 and UDP-galactose and its breakdown products

passed through the column (Fig. 5A). SDS–PAGE analysis

of fractions eluted at neutral pH and fractions eluted at pH 3

(Fig. 5B) showed that neutral flow-through fractions

contained only a trace of IgG plus other protein contaminants

from the original serum IgGpreparation. Themost prominent

contaminating protein bands visualized on SDS–PAGE

gels (marked in Fig. 5B) were excised, digested with trypsin,

and identified by LC/MS/MS as (a) recombinant GalT-1

(approximately 40 kDa), (b) heavy chains of human IgG3

and IgA, immunoglobulins with weak affinity for Protein A

(approximately 55 kDa), (c) human serum albumin

Table I. Impact of galactosyltransferase-I (GalT-1)/IgG ratio on percent G0, G1, and G2 glycans of human

serum IgG after various periods of time.

Time (h) Glycoform

Percent of neutral glycansb

Control

Activity ratio (mU GalT-1/mg IgG)a

10 17 25 50 75

24 G0 33.2 (�1.8) — — — — —

G1 46.7 (�0.6) 28.7 11.9 6.9 2.4 1.2

G2 20.1 (�2.3) 71.3 88.1 93.1 97.6 98.8

48 G0 — — — — —

G1 12.7 4.1 2.0 0.6 0.8

G2 87.3 95.9 98.0 99.4 99.2

72 G0 — — — — —

G1 7.3 2.3 1.7 0.6 —

G2 92.7 97.7 98.3 99.4 100

aReactions were carried out at a concentration of human serum IgG of 20 mg/mL plus GalT-1 at the indicatedactivity ratio.

bPercentages of glycans before (control) and after galactosylation with GalT-1, determined by integration ofRP-HPLC chromatograms of 2-AA labeled glycans analyzed under conditions to resolve neutral glycan structures(see Materials and Methods). Control values are reported as mean (�SD) calculated from six separate analyses.

—, indicates the structure was not detected.

Table II. Impact of increasing IgG concentrations on percent G0, G1,

and G2 glycans after galactosylation by GalT-1 for 24 and 48 h.

Time (h)

Percent of total N-glycansa

Glycoform

IgG concentration (mg/mL)b

Control 20 25 30 35

24 G0 33.2 (�1.8)a — — — —

G1 46.7 (�0.6) 7.2 5.1 3.1 3.5

G2 20.1 (�2.3) 92.8 94.9 96.9 96.5

48 G0 — — — —

G1 2.6 1.4 0.8 1.8

G2 97.4 98.6 99.2 98.7

aPercentages of glycans before (control) and after galactosylation withGalT-1 were determined by integration of RP-HPLC chromatograms of 2-AA labeled glycans analyzed under conditions to resolve neutral glycanstructures (see Materials andMethods). Control values are reported as mean(�SD) calculated from six separate analyses.

bReactionswere carriedout at the indicated concentrations of humanserum IgG in the presence of 25 mU GalT-1/mg IgG (see Materials andMethods).

—, indicates the structure was not detected.

Figure 3. Time course for galactosylation of the neutral glycans of human

serum IgG by recombinant GalT-1 at the kilogram scale. Aliquots were

removed from the bioreactor vessel at the indicated time points and N-

glycans released by peptide N-glycosidase F (PNGase F) were analyzed by

RP-HPLC as 2-AA derivatives (see Materials and Methods).


(approximately 65 kDa) and (d) a-2 macroglobulin (approxi-

mately 160 kDa) (data not shown).

Western blot analysis with anti-GalT-1 comparing the

enzyme reaction mixture before and after Protein A

chromatography demonstrated removal of at least 90% of

GalT-1 (Fig. 5C) and other non-IgG contaminants (Fig. 5B)

during this single step affinity purification. Analysis of the

same fractions by ion-exchange chromatography demon-

strated a 310-fold reduction in concentration of total

nucleotides. The concentration of UDP-galactose, UDP,

and UMP, and uridine was 9.31 mM in the remodeling

reaction, 10.5 mM in the Protein A Sepharose flow-through,

and 0.03 mM in the Protein A Sepharose low pH eluate. The

primary species present after the remodeling reaction was

uridine, presumably due to the presence of a phosphatase in

the polyclonal antibody preparation, which may have aided

in relieving any possible inhibition by UDP, a known

inhibitor of the galactosyltransferase.

Glycan Analysis of Remodeled IgG

N-glycan analysis of the commercial human serum IgG

starting material used in this study showed bi-antennary,

mainly core fucosylated, complex G0, G1, and G2 chains,

plus minor amounts of mono- and di-sialylated species, and a

small fraction of neutral and charged glycans with bisecting

GlcNAc (Figs. 2A and 4A). The profile of N-glycans

obtained after the 48 h remodeling reaction at the 1 kg scale

(Figs. 2B,C and 4B,C) showed bi-antennary complex

structures terminating in galactose on both antennae plus

minor amounts of monosialylated glycan structures termi-

nated with galactose on the non-sialylated antenna. Matrix-

assisted laser desorption ionization time-of-flight mass

spectrometry (MALDI-TOF MS) analysis of samples from

intermediate time points during the scaled-up reaction

confirmed rapid loss of G0 structures, then gradual comple-

tion of galactosylation on all available acceptor sites (data not

shown). A minor fraction of neutral oligosaccharides with a

molecular weight consistent with the presence of bisecting

GlcNAc were converted to glycoforms containing no more

than two galactose residues, both presumed to be linked as

follows: Galb1–4GlcNAcb1–2Mana1-.Analysis of N-glycans of remodeled IgG before and after

fractionation on Protein A showed no significant change in

percentages of neutral glycans, but showed an apparent drop

in the percentage of singly sialylated glycans from 22% to

18% and doubly sialylated glycans from 12% to 6% of the

total (Table III). This decrease in charged glycans, also

observed after Protein A fractionation of unremodeled IgG

starting material (data not shown), was shown to be due to

removal of sialylated glycoprotein contaminants during

chromatography rather than desialylation of IgG during the

remodeling reaction or a change in affinity for Protein A

induced in a subpopulation of IgGmolecules by galactosyla-

tion (data not shown). MALDI analysis of the unfractionated

reaction mixture after remodeling of IgG for 48 h identified

triantennary charged glycans (Fig. 5B), known to occur on

rGalT-1 (data not shown), which remained detectable in trace

amounts in the Protein A-purified IgG product (Fig. 5C).

DISCUSSION

It is widely recognized that modifying the structures of N-

glycans linked to the CH2 domain in the Fc region of certain

Figure 4. Analysis of 2-AA derivatized oligosaccharides from human

serum IgG after GalT-1 conversion to the G2 glycoform by matrix-assisted

laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF

MS) in linear/negative-ion mode. Spectra of 2-AA labeled glycans from (A)the starting pool of polyclonal human serum IgG, (B) 48-h GalT-1-

remodeled IgG reaction mixture, and (C) rProtein A affinity purified

remodeled IgG. Glycan structures are represented by the symbols; ~,

fucose; *, mannose; &, GlcNAc; *, galactose, and *, sialic acid. An

asterisk above a peak indicates a sodium adduct, and an X represents a peak

that is not identified.No attemptwasmade to fully characterize or distinguish

among isobaric structures.


IgG molecules may modulate binding interactions with

FcgRII and FcgRIII receptors (Jefferis and Lund, 2002;

Radaev and Sun, 2001; Tsuchiya et al., 1989) and with

activators of the complement cascade (Boyd et al., 1995).

The N-linked glycans of human IgG consist primarily of

neutral bi-antennary complex oligosaccharides, often a1–6fucosylated on the asparagine-linked core GlcNAc, with a

minor fraction containing bisecting GlcNAc (Fujii et al.,

1990; Kornfeld et al., 1971) (see Fig. 1 for structures).

Increased specific activities of some IgG molecules in

Figure 5. Analysis of chromatography fractions from rProtein A purification of GalT-1-remodeled human serum IgG. Panel A: Protein concentration traceduring fractionation of GalT-1-remodeled human serum IgG on Protein A-Sepharose. pH 3.0 glycine buffer elution was begun at fraction number 19. Panel B:SDS–PAGE analysis of fractions from Protein A-Sepharose chromatography of remodeled IgG. Equivalent volumes of each fraction were resolved on 4%–

12%NuPAGE gels with 3-(N-morpholino)propanesulfonic acid (MOPS) running buffer. Lanemarked ‘‘load’’ represents the IgG remodeling reactionmixture;

lanesmarked 5–10were flow-through fractions, fromwhich protein bandsmarked (a)–(d) were isolated and sequenced as described inMaterials andMethods.

Lanes marked 24–30 represent fractions eluted at pH 3.0. Panel C: Western blot analysis to detect GalT-1 in fractions from the Protein A Sepharose

chromatography fractionation of remodeled IgG. Approximately 3 mg of the remodeled IgG (lane 1 and 3) and Protein A purified remodeled IgG (lane 2 and 4)were resolved by NuPAGE gel electrophoresis. Lanes labeled Std, 1, and 2 were stained with colloidal Coomassie blue and imaged. Equivalent samples plus

prestainedmolecular weight markers (lane Std) were resolved in parallel on the same gel, blotted to nitrocellulose and probedwith antibody specific for GalT-1

as described in Materials and Methods. Lanes 3 and 4 were aligned based on the prestained molecular weight markers.


triggering Fc receptor mediated ADCC in vitro has been

associated with both the presence of bisecting GlcNAc

(Davies et al., 2001; Rademacher et al., 1986; Umana et al.,

1999) and the absence of core fucose (Shields et al., 2002;

Shinkawa et al., 2003). The neutral bi-antennary sugar chains

containing zero, one, or two galactosyl units are commonly

termed, respectively, G0, G1, and G2 glycans (Dwek et al.,

1995; Wormald et al., 1997) (see Fig. 1). That the relative

abundance of these glycoforms of IgG may be associated

with human disease was described by Parekh et al. (1985)

who observed hypo-galactosylated IgG in patients with

rheumatoid arthritis and osteoarthritis. Malhotra et al. (1995)

found that aggregated agalacto-IgG may activate the

complement pathway through interaction with the man-

nose-binding protein and suggested the potential importance

of this pathway in the genesis of auto-immune diseases.

Placental transport and rescue of antibody from catabolism

are hypothesized to occur via a transcytosis process that

requires binding to the FcgRn (Martin et al., 2001). Although

agalacto-antibody binds efficiently to the FcgRn in vitro,

some speculation and contradictory evidence exists as to IgG

glycan involvement on FcgRn binding in vivo, particularly inplacental transport where more highly galactosylated IgGs

appear to be preferentially transported to the fetal circulation

(Jefferis et al., 1998; Williams et al., 1995).

In the present work, we have explored the possibility of

preparative in vitro conversion of IgG molecules to

homogeneousG2 glycoforms by treatment with recombinant

bovine b1–4 galactosyltransferase (GalT-1, EC 2.4.1.22)

plus the sugar nucleotide donor, UDP-galactose. The

acceptor specificity and reaction products of this enzyme

have been studied extensively (Khatra et al., 1974), and at

analytical scale the enzyme previously has been shown to act

on intact IgG, adding galactose in b1–4 linkage to terminal

GlcNAc residues on both arms of the G0 N-linked bi-

antennary chain (Fujii et al., 1990; Raju et al., 2001). The

human serum IgG starting material used for the present study

contained approximately 33% G0, 47% G1, and 20% G2, a

distribution of neutral glycans that is undergalactosylated

compared with the values, 19% G0, 34% G1, 47% G2,

reported by Routier et al. (1998), for purified human IgG, but

similar to the values, 28%G0, 46%G1, 26%G2, reported for

human IgG purchased from a commercial source (Raju et al.,

2000).

We found that the rate of conversion ofG0 toG1was rapid,

proceeding essentially to completion within 4 h in a reaction

mixture containing IgG at 30 mg/mL and GalT-1 at 25 mU/

mg IgG (Fig. 3). In the same reaction mixture, conversion of

G1 to G2 proceeded more slowly, decreasing dramatically

after 18 h as the concentration of G1 acceptor diminished to

<5% of the starting value. As expected, we observed that the

rate of transfer of galactose increases as the activity ratio (U

GalT-1/mg IG) is increased (Table I), and that galactosylation

can be carried outmore efficiently at higher concentrations of

IgG up to 30mg/mL (Table II). The latter results suggest that

at concentrations of IgG less than 30 mg/mL (187 mM), the

acceptor substrate is below that required for enzyme

saturation. For rat liver GalT-1 the apparent Km for G0 is

0.13 mM, and the Km values are 0.43 and 6.28 mM,

respectively, for a1,3 and a1,6 branched G1 acceptors as

isolated oligosaccharides (Paquet et al., 1984). It has been

reported that the corresponding glycan structures within

native IgG are less accessible to galactosyltransferase (Fujii

et al., 1990; White et al., 1997).

In a production setting, heterogeneity of glycosylation of a

recombinant glycoprotein may be the result of incomplete

Golgi processing in cells due to changing metabolic states

during fermentation and/or the presence of glycosidases

released into the cell culture medium (Goochee et al., 1991;

Jenkins et al., 1996). Recombinant mAbs expressed in

mammalian cell lines exhibit G0, G1, and G2 glycoforms

(Raju et al., 2000), but percentage distributions may vary

depending upon multiple factors, including the species from

which the host expressor cell line was derived and details of

the cell culture conditions (Hills et al., 2001; Kumpel et al.,

1994; Raju et al., 2000; Jefferis, 2005). Thus, it can be a

Table III. Total N-glycan profiles of starting human IgG and of galactosylated IgG before and after purification

on Protein A.

Percent of total glycans

Neutral glycans Charged glycans: number of sialic acids

G0 G1 G2 1 2 3 4

Starting IgG 20.2a 31.2 15.9 21.0 11.7 — —

IgG/GalT-1 reactionmixb — — 65.3 22.6 12.1 — —

IgG/GalT-1 pH 3 eluate — — 75.5 18.5 6.0 — —

IgG/GalT-1 flow-through [26.0] 26.6 45.9 1.5 —

aPercentages of total glycans were determined by integration peaks from single RP-HPLC chromatogramsof 2-AA labeled derivatives analyzed under conditions to optimally resolve charged glycans (see Materials andMethods).

bIgG (1 kg/33.5 L)was incubatedwith 25mUGalT-1/mg IgG for 48 h. An aliquot of the reactionwas applied toProtein A-Sepharose, washed with Tris-buffered saline at pH 7.2, and eluted with 0.2 M glycine-HCl, pH 3.0 (seeMaterials and Methods).

—-, indicates the structure was not detected.


significant challenge to achieve uniform batch-to-batch

glycosylation for a recombinant IgG product. Attempts have

beenmade to engineer glycosylation pathways so as to enrich

for specific glycoforms in mAbs secreted by a variety of

expression systems, including cell lines from mammalian

species (Davies et al., 2001), yeast (Bobrowicz et al., 2004;

Vervecken et al., 2004), and plants (Ko et al., 2003; Shriver

et al., 2004; Tekoah et al., 2004).

An emerging alternative is the use of enzymes post-

production to remodel glycan chains, a process made pos-

sible by high-level overexpression of glycosyltransferases in

soluble form (Malissard et al., 2000; Perugino et al., 2004).

We have demonstrated previously the preparative in vitro

remodeling of glycan chains on a therapeutic glycoprotein at

the 10 g scale, including enzymatic sialylation and fucosyla-

tion of complex biantennary N-glycans to homogeneous

sialyl Lewis X (sLex)-active glycoforms (Thomas et al.,

2004). The present work demonstrates the feasibility of using

recombinant GalT-1 to achieve complete (>98%) galacto-

sylation of IgG under mild conditions within 48 h at pilot

production (1 kg) scale. Using Protein A affinity chromato-

graphy, we showed that it is feasible to purify fully galacto-

sylated IgG from the enzyme reactionmixture containing the

glycosylating reagents GalT-1 and UDP-galactose. To

demonstrate scale-up of the galactosylation reaction, we

used a relatively inexpensive and easily available commer-

cial preparation of pooled human IgG containing <5%

contaminating human serum proteins. The fact that this IgG

preparation undoubtedly contains multiple IgG subtypes and

allotypes provides evidence that in vitro enzymatic galacto-

sylation as described here is likely to be robust in its

applicability to multiple forms of the human IgG molecule,

including mAbs.

ABBREVIATIONS

2-AA 2-anthranilic acid

ADCC antigen dependent cellular cytotoxicity

b4GalT-1 b4-galactosyltransferase-IBSA bovine serum albumin

CH2 antibody constant domain

CML cell-mediated lysis

Fc antibody fragment that crystallizes

FF fast flow

FPLC fast protein liquid chromatography

GlcNAc N-acetyl glucosamine

HPLC high performance liquid chromatography

HRP horseradish peroxidase

IgG immunoglobulin G

LC/ESI/MS liquid chromatography electrospray ionization mass

spectrometry

mAb monoclonal antibody

MALDI-TOF MS matrix-assisted laser desorption ionization

time-of-flight mass spectrometry

MES (N-morpholino)ethane-sulfonic acid

MOPS 3-(N-morpholino)propanesulfonic acid

MS mass spectrometry

NSO murine cell line (Lonza Biologics)

PBS phosphate buffered saline

PNGase F peptide N-glycosidase F

RP-HPLC reversed phase liquid chromatography

SDS sodium dodecyl sulfate

SDS–PAGE sodium dodecyl sulfate–polyacrylamide

gel electrophoresis

sLex sialyl Lewis X

TBS tris buffered saline

TBS-T20 20mMTris, pH 7.2, 150mMNaCl, 0.05%Tween-20

UDP uridine 50-diphosphateUMP uridine 50-monophosphate

References

Anumula KR, Dhume ST. 1998. High resolution and high sensitivity

methods for oligosaccharide mapping and characterization by normal

phase high performance liquid chromatography following derivatization

with highly fluorescent anthranilic acid. Glycobiology 8(7):685–694.

Bobrowicz P, Davidson RC, Li H, Potgieter TI, Nett JH, Hamilton SR,

StadheimTA,MieleRG,BobrowiczB,Mitchell T. 2004. Engineering of

an artificial glycosylation pathway blocked in core oligosaccharide

assembly in theyeastPichia pastoris: Productionof complex humanized

glycoproteins with terminal galactose. Glycobiology 14(9):757–766.

Boyd PN, Lines AC, Patel AK. 1995. The effect of the removal of sialic acid,

galactose, and total carbohydrate on the functional activity of

Campath-1H. Mol Immunol 32(17–18):1311–1318.

ClynesRA, Towers TL, Presta LG, Ravetch JV. 2000. Inhibitory Fc receptors

modulate in vivo cytoxicity against tumor targets. Nat Med 6(4):

443–446.

ColomaMJ,CliftA,WimsL,MorrisonSL. 2000.The role of carbohydrate in

the assembly and function of polymeric IgG. Mol Immunol

37(18):1081–1090.

Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M. 2001.

Expression of GnTIII in a recombinant anti-CD20 CHO production cell

line: Expression of antibodies with altered glycoforms leads to an

increase in ADCC through higher affinity for FC gamma RIII.

Biotechnol Bioeng 74(4):288–294.

DijstelbloemHM, van deWinkel JG, Kallenberg CG. 2001. Inflammation in

autoimmunity: Receptors for IgG revisited. Trends Immunol

22(9):510–516.

Dwek RA, Lellouch AC, Wormald MR. 1995. Glycobiology: ‘The function

of sugar in the IgG molecule’. J Anat 187(Pt 2):279–292.

Fujii S, Nishiura T, Nishikawa A, Miura R, Taniguchi N. 1990. Structural

heterogeneity of sugar chains in immunoglobulin G. Conformation of

immunoglobulin G molecule and substrate specificities of glycosyl-

transferases. J Biol Chem 265(11):6009–6018.

Ghirlando R, Lund J, Goodall M, Jefferis R. 1999. Glycosylation of human

IgG-Fc: Influences on structure revealed by differential scanningmicro-

calorimetry. Immunol Lett 68(1):47–52.

Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR. 1991. The

oligosaccharides of glycoproteins: Bioprocess factors affecting oligo-

saccharide structure and their effect on glycoprotein properties.

Biotechnology (NY) 9(12):1347–1355.

Hamako J, Matsui T, Ozeki Y, Mizuochi T, Titani K. 1993. Comparative

studies of asparagine-linked sugar chains of immunoglobulin G from

elevenmammalian species. CompBiochemPhysiol B 106(4):949–954.

Hills AE, Patel A, Boyd P, James DC. 2001. Metabolic control of

recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Biotechnol Bioeng 75(2):239–251.

Jefferis R. 2001. Glycosylation of human IgG antibodies: Relevance to

therapeutic applications. BioPharm International 14(9):19–27.

Jefferis R. 2005. Glycosylation of recombinant antibody therapeutics.

Biotechnol Prog 21(1):11–16.

Jefferis R, Lund J. 2002. Interaction sites on human IgG-Fc for FcgammaR:

Current models. Immunol Lett 82(1–2):57–65.

Jefferis R, Lund J, Pound JD. 1998. IgG-Fc-mediated effector functions:

Molecular definition of interaction sites for effector ligands and the role

of glycosylation. Immunol Rev 163:59–76.


Jenkins N, Parekh RB, James DC. 1996. Getting the glycosylation right:

Implications for the biotechnology industry. Nat Biotechnol 14(8):975–

981.

Khatra BS, Herries DG, Brew K. 1974. Some kinetic properties of human-

milk galactosyl transferase. Eur J Biochem 44(2):537–560.

Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA,

Rupprecht C,DietzscholdB,GolovkinM,KoprowskiH. 2003. Function

and glycosylation of plant-derived antiviral monoclonal antibody. Proc

Natl Acad Sci USA 100(13):8013–8018.

Kornfeld R, Keller J, Baenziger J, Kornfeld S. 1971. The structure of the

glycopeptide of human gamma G myeloma proteins. J Biol Chem

246(10):3259–3268.

Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P. 2003. Structural

analysis of human IgG-Fc glycoforms reveals a correlation between

glycosylation and structural integrity. J Mol Biol 325(5):979–989.

Kumpel BM, Rademacher TW, Rook GA, Williams PJ, Wilson IB. 1994.

Galactosylation of human IgG monoclonal anti-D produced by EBV-

transformedB-lymphoblastoid cell lines is dependent on culturemethod

and affects Fc receptor-mediated functional activity. Hum Antibodies

Hybridomas 5(3–4):143–151.

Leatherbarrow RJ, Rademacher TW, Dwek RA, Woof JM, Clark A, Burton

DR, Richardson N, Feinstein A. 1985. Effector functions of a

monoclonal aglycosylated mouse IgG2a: Binding and activation of

complement component C1 and interaction with human monocyte Fc

receptor. Mol Immunol 22(4):407–415.

Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM,

Yates JR III. 1999. Direct analysis of protein complexes using mass

spectrometry. Nat Biotechnol 17(7):676–682.

Lund J, Takahashi N, Pound JD, Goodall M, Nakagawa H, Jefferis R. 1995.

Oligosaccharide–protein interactions in IgG can modulate recognition

by Fc gamma receptors. FASEB J 9(1):115–119.

Maini R, St. Clair EW, Breedveld F, Furst D, Kalden J, WeismanM, Smolen

J, Emery P, Harriman G, Feldmann M, Lipsky P. 1999. Infliximab

(chimeric anti-tumour necrosis factor alpha monoclonal antibody)

versus placebo in rheumatoid arthritis patients receiving concomitant

methotrexate: A randomised phase III trial. ATTRACT Study Group.

Lancet 354(9194):1932–1939.

Malhotra R,WormaldMR, Rudd PM, Fischer PB, DwekRA, SimRB. 1995.

Glycosylation changes of IgG associated with rheumatoid arthritis can

activate complement via the mannose-binding protein. Nat Med

1(3):237–243.

Malissard M, Zeng S, Berger EG. 2000. Expression of functional soluble

forms of human beta-1, 4-galactosyltransferase I, alpha-2,6-sialyltrans-

ferase, and alpha-1, 3-fucosyltransferase VI in the methylotrophic yeast

Pichia pastoris. Biochem Biophys Res Commun 267(1):169–173.

MartinWL,West AP, Jr., Gan L, Bjorkman PJ. 2001. Crystal structure at 2.8

A of an FcRn/heterodimeric Fc complex: Mechanism of pH-dependent

binding. Mol Cell 7(4):867–877.

Mimura Y, Church S, Ghirlando R, Ashton PR, Dong S, Goodall M, Lund J,

Jefferis R. 2000. The influence of glycosylation on the thermal stability

and effector function expression of human IgG1-Fc: Properties of a

series of truncated glycoforms. Mol Immunol 37(12–13):697–706.

Mimura Y, Sondermann P, Ghirlando R, Lund J, Young SP, Goodall M,

Jefferis R. 2001. Role of oligosaccharide residues of IgG1-Fc in Fc

gamma RIIb binding. J Biol Chem 276(49):45539–45547.

Narimatsu H, Sinha S, Brew K, Okayama H, Qasba PK. 1986. Cloning and

sequencing of cDNA of bovine N-acetylglucosamine (beta 1–

4)galactosyltransferase. Proc Natl Acad Sci USA 83(13):4720–4724.

Paquet MR, Narasimhan S, Schachter H, Moscarello MA. 1984. Branch

specificity of purified rat liver Golgi UDP-galactose: N-acetylglucosa-

mine beta-1,4-galactosyltransferase. Preferential transfer of of galactose

on theGlcNAc beta 1,2-Man alpha 1,3-branch of a complex biantennary

Asn-linked oligosaccharide. J Biol Chem 259(8):4716–4721.

Parekh RB, Dwek RA, Sutton BJ, Fernandes DL, Leung A, Stanworth D,

Rademacher TW, Mizuochi T, Taniguchi T, Matsuta K, Takeuchi P,

Nagano Y, Miyamoto T, Kobata A. 1985. Association of rheumatoid

arthritis and primary osteoarthritis with changes in the glycosylation

pattern of total serum IgG. Nature 316(6027):452–457.

Perugino G, Trincone A, Rossi M, Moracci M. 2004. Oligosaccharide

synthesis by glycosynthases. Trends Biotechnol 22(1):31–37.

Presta LG. 2002. Engineering antibodies for therapy. Curr PharmBiotechnol

3(3):237–256.

Radaev S, Sun PD. 2001. Recognition of IgG by Fcgamma receptor. The role

of Fc glycosylation and the binding of peptide inhibitors. J Biol Chem

276(19):16478–16483.

Rademacher TW, Homans SW, Parekh RB, Dwek RA. 1986. Immunoglo-

bulin G as a glycoprotein. Biochem Soc Symp 51:131–148.

Raju TS, Briggs JB, Borge SM, Jones AJ. 2000. Species-specific variation in

glycosylation of IgG: Evidence for the species-specific sialylation and

branch-specific galactosylation and importance for engineering recom-

binant glycoprotein therapeutics. Glycobiology 10(5):477–486.

Raju TS, Briggs JB, Chamow SM, Winkler ME, Jones AJ. 2001.

Glycoengineering of therapeutic glycoproteins: In vitro galactosylation

and sialylation of glycoproteins with terminalN-acetylglucosamine and

galactose residues. Biochemistry 40(30):8868–8876.

Rastetter W, Molina A, White CA. 2004. Rituximab: Expanding role in

therapy for lymphomas and autoimmune diseases. Annu Rev Med

55:477–503.

Routier FH, Hounsell EF, Rudd PM, Takahashi N, BondA, Hay FC,AlaviA,

Axford JS, Jefferis R. 1998. Quantitation of the oligosaccharides

of human serum IgG from patients with rheumatoid arthritis: A

critical evaluation of different methods. J Immunol Methods

213(2):113–130.

Sandborn WJ, Hanauer SB. 1999. Antitumor necrosis factor therapy for

inflammatory bowel disease: A review of agents, pharmacology, clinical

results, and safety. Inflamm Bowel Dis 5(2):119–133.

Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH,

Presta LG. 2002. Lack of fucose on human IgG1 N-linked oligosacchar-

ide improves binding to human Fcgamma RIII and antibody-dependent

cellular toxicity. J Biol Chem 277(30):26733–26740.

Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada

M, Uchida K, Anazawa H, SatohM, Yamasaki M. 2003. The absence of

fucose but not the presence of galactose or bisecting N-acetylglucosa-

mine of human IgG1 complex-type oligosaccharides shows the critical

role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem

278(5):3466–3473.

Shriver Z, Raguram S, Sasisekharan R. 2004. Glycomics: A pathway to a

class of new and improved therapeutics. Nat Rev Drug Discov

3(10):863–873.

Souriau C, Hudson PJ. 2003. Recombinant antibodies for cancer diagnosis

and therapy. Expert Opin Biol Ther 3(2):305–318.

Tekoah Y, Ko K, Koprowski H, Harvey DJ, Wormald MR, Dwek RA, Rudd

PM. 2004. Controlled glycosylation of therapeutic antibodies in plants.

Arch Biochem Biophys 426(2):266–278.

Thomas LJ, Panneerselvam K, Beattie DT, Picard MD, Xu B, Rittershaus

CW, Marsh HC, Jr., Hammond RA, Qian J, Stevenson T. 2004.

Production of a complement inhibitor possessing sialyl Lewis X

moieties by in vitro glycosylation technology. Glycobiology

14(10):883–893.

TsuchiyaN,EndoT,MatsutaK,YoshinoyaS,AikawaT,KosugeE, Takeuchi

F, Miyamoto T, Kobata A. 1989. Effects of galactose depletion from

oligosaccharide chains on immunological activities of human IgG. J

Rheumatol 16(3):285–290.

Umana P, Jean-Mairet J,MoudryR,AmstutzH, Bailey JE. 1999. Engineered

glycoforms of an antineuroblastoma IgG1 with optimized antibody-

dependent cellular cytotoxic activity. Nat Biotechnol 17(2):176–180.

Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K,

Contreras R. 2004. In vivo synthesis of mammalian-like, hybrid-typeN-

glycans in Pichia pastoris. Appl Environ Microbiol 70(5):2639–2646.

Walker MR, Lund J, Thompson KM, Jefferis R. 1989. Aglycosylation of

human IgG1 and IgG3monoclonal antibodies can eliminate recognition

by human cells expressing Fc gammaRI and/or Fc gammaRII receptors.

Biochem J 259(2):347–353.

White KD, Cummings RD, Waxman FJ. 1997. Ig N-glycan orientation can

influence interactions with the complement system. J Immunol

158(1):426–435.


White CA,Weaver RL, Grillo-Lopez AJ. 2001. Antibody-targeted immuno-

therapy for treatment of malignancy. Annu Rev Med 52:125–145.

Williams PJ, Arkwright PD, Rudd P, Scragg IG, Edge CJ, Wormald MR,

Rademacher TW. 1995. Short communication: Selective placental

transport of maternal IgG to the fetus. Placenta 16(8):749–756.

WormaldMR,Rudd PM,HarveyDJ, Chang SC, Scragg IG,DwekRA. 1997.

Variations in oligosaccharide–protein interactions in immunoglobulin

G determine the site-specific glycosylation profiles and modulate

the dynamic motion of the Fc oligosaccharides. Biochemistry

36(6):1370–1380.

Wright A, Morrison SL. 1998. Effect of C2-associated carbohydrate

structure on Ig effector function: Studies with chimeric mouse-human

IgG1 antibodies in glycosylation mutants of Chinese hamster ovary

cells. J Immunol 160(7):3393–3402.


In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

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