www.elsevier.com/locate/procbio

Process Biochemistry 43 (2008) 206–212

Short communication

The release of hepatitis B core antigen from Escherichia coli

by batch mode bead milling

Chin Woi Ho a, Wen Siang Tan b,c, Suryani Kamarudin a,Tau Chuan Ling d, Beng Ti Tey a,c,*

a Department of Chemical & Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia,

43400 Serdang, Selangor Darul Ehsan, Malaysiab Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,

43400 Serdang, Selangor Darul Ehsan, Malaysiac Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

d Department of Process & Food Engineering, Faculty of Engineering, Universiti Putra Malaysia,

43400 Serdang, Selangor Darul Ehsan, Malaysia

Received 6 July 2007; received in revised form 19 October 2007; accepted 7 November 2007

Abstract

The performance of a batch model bead mill on the release of hepatitis B core antigen (HBcAg) from Escherichia coli was investigated in this

study. The operating parameters examined were impeller tip speed (8–14 m/s), biomass concentration [5–20% (w/v)] and bead loading [65–80%

(v/v)]. The highest yield (24.3 mg/g cell) and rate constant (0.471 l/min) of HBcAg release were achieved at impeller tip speed of 14 m/s. However,

the high-shear stress under these operating conditions caused damage of the HBcAg. The highest yield (22.7 mg/g cell) and rate constant (0.344 l/

min) of HBcAg release were observed at biomass concentration of 20% (w/v). There was no significant effect of bead loading on the performance

of bead milling being observed. In conclusion, the optimal operating condition for the release of HBcAg was at bead loading of 75% (v/v), biomass

concentration of 20% (w/v) and impeller tip speed of 10 m/s.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Bead mill; Batch mode; Cell disruption; Hepatitis B core antigen; Downstream process; Escherichia coli

1. Introduction

Liver cirrhosis and hepatocellular carcinoma (HCC) caused

by hepatitis B virus (HBV) is one of the serious global health

problems [1]. There is no effective treatment for acute hepatitis

B and it can only be prevented by vaccination. The viral core

protein also known as hepatitis B core antigen (HBcAg) is

widely used as antigen for the detection of anti-HBcAg

antibody [2–4] in serum sample. During viral hepatitis B

infection, anti-HBcAg antibodies are produced at the early

stage of infection and can persist years after the recovery from

the illness [2,5]. Indeed, anti-HBcAg antibodies are the only

detectable markers during the period when the hepatitis B

* Corresponding author at: Department of Chemical & Environmental Engi-

neering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang,

Selangor Darul Ehsan, Malaysia. Tel.: +60 3 89466289; fax: +60 3 86567120.

E-mail address: btey@eng.upm.edu.my (B.T. Tey).

1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2007.11.004

surface antigen (HBsAg) has disappeared and anti-HBcAg

antibodies indicate that HBV has not yet been eliminated. This

characteristic has made anti-HBcAg antibodies a very useful

serological marker in diagnosing HBV infections during the

screening of populations at risk for HBV infection. Further-

more, several observations have suggested that the screening

for anti-HBcAg antibodies in blood donors could reduce the

incidence of post-tranfusion hepatitis [6]. Therefore, HBcAg

has great potential in the development of diagnostic tools for

the detection of HBV. The gene encoding HBcAg was

successfully expressed in Escherichia coli (E. coli), where it

assembles into particles which are indistinguishable from the

viral capsids isolated from virally infected liver [7]. We had

successfully scaled up the production of this intracellular

recombinant HBcAg in a stirred tank bioreactor [8].

There is an increased interest in the efficient and cost effective

release of intracellular products like antibiotics, enzymes and

therapeutic drugs from the impermeable cell wall and membrane

of their host micro-organisms. Prior to the protein purification

C.W. Ho et al. / Process Biochemistry 43 (2008) 206–212 207

operations, the cell wall must be disrupted to allow the release of

these intracellular products into the surrounding medium.

Optimization of cell disruption is of economically important

because the nature of the disruption process may affect the extent

of product recovery, the ease of the subsequent purification steps,

the nature of the suspension processed and the form and quality

of the final products. In the production of recombinant proteins,

the ratio of variable recovery process costs to fermentation costs

vary from 1 to 3 for enzyme and antibiotic recovery, and the ratio

reached up to 10 for the recovery of intracellular recombinant

insulin [9,10]. Thus, continuous improvement has been made

both by adapting and refining existing cell disruption equipment

and by adding pre-treatment stages to the disruption processes

[11].

Bead milling has been an industrially applicable cell

disruption method due to its capability to process large volume

of biomass and relatively low-power consumption. It is

considered as the method of choice for the disruption of yeast

[12–17] and bacteria [18–21]. It has also been applied

successfully to plant and animal cell disruption [22]. Among

the different type of bead mills, Dyno mill is the most suitable

for scaling up [23]. It consists of several discs or impellers

mounted concentrically or eccentrically on a motor-driven shaft

inside a vertical or horizontal cylindrical chamber. The discs

rotating at high speed and thus generate high-shear force inside

the grinding chamber. The inclusion of small beads produces an

abrasive action to grind the cells. Under the shearing or

compaction of cells and energy transfer from beads to cells, the

microbial cells wall subsequently undergo disintegration, thus

allowing the release of intracellular proteins into the

surrounding medium [23–25]. The performance of bead

milling is influenced by a number of parameters such as

biomass concentration, agitator speed, bead loading, density

and size of bead, temperature, feed flow rate, residence time,

nature and state of the cell and size of organisms [26].

Therefore, the aim of this study was to investigate the effect of

impeller tip speed, biomass concentration and bead loading on

the performance of batch mode bead milling for the release of

HBcAg. The optimized conditions achieved can be used for the

development of an integration recovery process involving bead

milling and expanded bed adsorption chromatography.

2. Materials and methods

2.1. Cultivation of E. coli

The E. coli W3110IQ cells harbouring plasmid PR1-11E encoding trun-

cated HBcAg [7,27] was cultured in Luria Bertani (LB) broth supplemented

with 100 mg/ml ampicillin. The culture was incubated at 37 8C as previously

described by Tey et al. [28]. The cells were harvested by centrifugation at

3750 � g (Avanti JLA-16.250, USA) for 15 min at 4 8C.

2.2. Cell disruption by batch mode bead milling

The pelleted E. coli biomass was washed and resuspended in lysis buffer

[50 mM Tris–HCl (pH 8.0)] to the appropriate biomass concentration [5%,10%,

15%, and 20% (w/v)]. The biomass suspension was fed into the 600-ml glass

chamber of the Dynomill Type MultiLab (CH-4005, W.A. Bachofen, Switzer-

land) loaded with 0.3 mm Zirconia beads [bead loadings of 65%, 75% and 80%

(v/v)]. Cell disruption was carried out under several impeller tip speeds (8, 10

and 14 m/s) for 17 min. The disruption chamber was cooled by an outer jacket

circulated with ice-cooled water. The intracellular protein release generally

obeys the first order kinetics law, in which the rate of protein release is

proportional to the amount of unreleased proteins [25,26]. Samples were

collected at regular interval times and were clarified by centrifugation at

16,249 � g (Mikro 20, H. Zentrifugen, Germany) for 1 min to remove cell

debris.

2.3. Total protein analysis and quantification

The total protein released was analysed with 12% SDS-PAGE and stained

with Coomassie Brilliant Blue R-250 as described by Laemmli [29]. The

amount of total protein was measured with the Bradford assay [30] using

bovine-serum albumin as standard. The absorbance of the sample was read with

a microplate reader (Model 550, Bio-Rad, Japan). All measurements were

carried out in triplicate.

2.4. Purity and quantitation of HBcAg

Purity and quantitation of HBcAg was based on the method previously

described by Ho et al. [31] and Ng et al. [32] with slight modifications. Briefly,

the purity of HBcAg (relative quantity of HBcAg band on the SDS-PAGE gels),

which represents the relative quantity of HBcAg in the sample, was determined

by using the Quantity One1 Quantitation software (Gel Doc, Bio-Rad, USA).

The amount of HBcAg was calculated from the relative quantity of the HBcAg

against the amount of total protein obtained from the Bradford assay. All

measurements were carried out in triplicate and the data presented in Table 1

were statistically significant.

2.5. Measurement of intact cell density and viable cell concentration

The intact cell density was measured spectrophotometrically under a

wavelength of 600 nm (OD600 nm) with a visible spectrophotometer (UVmine

1240, Shidmadzu, Japan). For the measurement of viable cell concentrations, a

10-fold dilution of disrupted sample was serially diluted under sterile condi-

tions. Diluted sample (50 ml) was spread on a LB agar surface (containing

100 mg/ml ampicillin) and colonies were counted as colony forming unit (cfu)

after incubating the plates at 37 8C for 16 h. All measurements were carried out

in triplicate.

2.6. Measurement of viscosity

Sample was loaded into a viscometer cone and its viscosity was determined

with Spindle SC 14-18 (rotating at a standard speed of 60 rpm) of a viscometer

(DV-II + Viscometer, Brookfield Eng. Lab., USA). All measurements were

carried out in triplicate.

2.7. Analysis of the intactness of HBcAg particles released

The intactness of the HBcAg particles released was analysed using sucrose

density gradient ultracentrifugation as previously described by Tan et al. [7] and

Ho et al. [31].

2.8. Enzyme-linked immunosorbent assay (ELISA)

The antigenicity of the HBcAg was determined by ELISA using a micro-

plate reader (Model 550, Bio-Rad, Japan) as previously described by Ho et al.

[31,33]. All measurements were carried out in triplicate.

3. Results and discussion

The profile of total protein and HBcAg release from batch

mode bead milling is depicted in Fig. 1. Observations in Fig. 1

indicate that the amount of HBcAg in the disruptate started to

Tab

le1

Th

ep

erfo

rman

ceo

fb

atch

mo

de

bea

dm

illi

ng

op

erat

edat

var

iou

so

per

atin

gco

nd

itio

ns

for

the

dis

rup

tio

no

fE

sch

eric

hia

coli

Par

amet

erM

axim

um

amou

nt

of

tota

lp

rote

in

rele

ased

(mg/g

cell

)

Max

imu

mam

ou

nt

of

HB

cAg

rele

ased

(mg

/gce

ll)

Pu

rity

of

HB

cAg

(%)

Rat

eco

nst

ant

of

tota

lp

rote

in

rele

ase

(l/m

in)

Rat

eco

nst

ant

of

HB

cAg

rele

ase

(1/m

in)

Tem

per

atu

re

of

dis

rup

tate

(8C

)

Inta

ctce

ll

con

cen

trat

ion

(OD

600

nm

)

Via

ble

cell

con

cen

trat

ion

(�1

04

cfu

/ml)

Vis

cosi

tyo

f

dis

rup

tate

(cP

)

Imp

elle

rti

psp

eed

(m/s

)[b

iom

ass

con

cen

trat

ion

=1

0%

(w/v

);

bea

dlo

adin

g=

80

%(v

/v)]

87

5.3�

1.5

16

.9�

0.5

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.9�

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48�

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33

.0�

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.

10

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00

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C.W. Ho et al. / Process Biochemistry 43 (2008) 206–212208

decrease after 13 min of disruption and a 15% drop in amount

of HBcAg was recorded at 17 min of disruption. Thus,

disruption was ended at 17 min since further disruption might

lead to more HBcAg damage. The performance of the cell

disruption operation with respect to yield (maximum amount of

total protein and HBcAg released), purity of HBcAg released,

the kinetics of protein release and the degree of cell disruption

(intact cell and viable cell concentrations), and the temperature

and viscosity of disruptate under various operating conditions

are summarised in Table 1.

3.1. Effect of impeller tip speed on batch bead milling

The result of the present study shows that the maximum

amount of protein and HBcAg released at 8 m/s are

considerably lower than those obtained at 10 and 14 m/s

(Table 1). A further increase of impeller tip speed from 10 to

14 m/s has led to a further 40% and 22.7% increase in the rate

constant of total protein and HBcAg release, respectively. As

the impeller tip speed increases, the amount of kinetic energy

and shear force imparted from the rotating impeller to the beads

and cells increased substantially [24]. As a result, the bead–

bead and bead–cell collision rates were elevated. This increased

the rates of cell disruption and as a result more proteins were

released. A higher cell disruption rate at higher impeller tip

speed has been reported for the disruption of Saccharomyces

cerevisiae [14,16,17] and Enterobacter cloacae [9] in a bead

mill by other researchers. Indeed, the intact cell (OD600 nm) and

viable cell (cfu/ml) concentrations of the samples obtained at

the end of the cell disruption (Table 1) clearly showed that the

E. coli cells were not completely disrupted at the operation

using impeller tip speed of 8 m/s (Table 1). Small-size cells like

E. coli generally require higher impeller tip speed for effective

disruption. Indeed, Kula and Schutte [34] have recommended

that an impeller tip speed of 8 m/s for disruption of larger size

of cells such as yeast cells and tip speed higher than 10 m/s

should be used for disruption of bacteria.

A drastic increase of temperature from 24 to 65 8C was

recorded after 12 min of disruption at the impeller tip speed of

14 m/s and the temperature remained constant until the end of

operation. However, such temperature rise did not occur during

the operation at 8 and 10 m/s (Table 1). Higher impeller tip

speed required higher energy input [25] and this energy input

eventually dissipated as heat into cell suspension [15]. At

impeller tip speed of 14 m/s, the heat dissipation rate was

higher than the rate of heat transfer from the disruption chamber

to the cooling jacket due to the limitation of heat transfer area

provided. Excessive temperature rise was also reported by

Woodrow and Quirk [19] when a Dyno-Mill type KDL was

operated at tip speed higher than 15 m/s.

The large amount of dissipated heat at 14 m/s has caused the

denaturation and precipitation of host proteins as well as

deactivation of various host proteases, whilst the heat-stable

HBcAg was unaffected. Based on the SDS polyacrylamide gel

depicted in Fig. 2, a significant reduction in contaminant

protein bands (especially from 20 to 35 kDa) was observed

from the sample derived from 14 m/s compared to that of 10

Fig. 2. The SDS-PAGE of disruptates derived from various impeller tip speeds,

constant biomass concentration [(10% (w/v)] and constant bead loading [80%

(v/v)]. An equal volume of 0.5 ml of disruptate contained between 3.8 and

4.2 mg of total protein was loaded onto the polyacrylamide gel. Lane M:

molecular mass markers; lane 1: disruptate derived from 8 m/s; lane 2:

disruptate derived from 10 m/s; lane 3: disruptate derived from 14 m/s. Sig-

nificant reduction in the amount of contaminant bands of the SDS-PAGE

derived from impeller tip speed of 14 m/s indicates the denaturation and

precipitation of host proteins.

Fig. 1. A typical (a) total protein (~) and (b) HBcAg (*) profile of a batch

mode bead milling. The bead mill was operated at 10% (w/v) E. coli biomass at

impeller tip speed of 10 m/s and bead loading of 80% (v/v). The data represents

the mean � S.D. of triplicate measurements.

C.W. Ho et al. / Process Biochemistry 43 (2008) 206–212 209

and 8 m/s. Indeed, HBcAg was reported to be relatively stable

at temperature as high as 65 8C [32,35]. Ohori et al. [36] have

demonstrated that the antigenicity of HBcAg was still active

after 4 h incubation at 56 8C. Meanwhile, Naito et al. [37]

reported that the yield of HBcAg increased by approximately

50% after the E. coli disruptate was heated for 30 min at 65 8C.

The heat precipitation of host proteins may cause in slight

reduction in viscosity of disruptate (Table 1) as well as

circumvent the interaction of heat-stable HBcAg and E. coli

host proteins, which consequently increased the purity of

HBcAg.

The samples derived from 10 to 14 m/s were further

analysed with sucrose density gradient ultracentrifugation and

ELISA to examine the formation of HBcAg particles and its

antigenicity. The sucrose gradient profiles of the sample derived

from 14 m/s tip speed shows that the HBcAg was almost

uniformly distributed in fractions 5 to 24 (Fig. 3a). In a sucrose

gradient fractionation, heavy molecules are collected in earlier

fractions whilst the later fractions contained mainly lower

molecular weight particles. The uniform pattern indicates that

the HBcAg particles in the samples derived from 14 m/s tip

speed comprise aggregated (migrated to the bottom of the

gradient), ruptured, partially or fully formed core particles

(collected in the mid fractions) and denatured or unformed

HBcAg subunits (collected in the last fractions). In contrast, the

profile of sucrose gradient ultracentrifugation for 10 m/s tip

speed sample clearly shows the formation of intact core

particles in the sample (Fig. 3b). The HBcAg particles formed

were found in abundance (�90%) and migrated to form a bell-

shaped peak (fractions 9–14).

The result of ELISA test shows that the antigenicity of

HBcAg in the 14 m/s tip speed sample was affected (Fig. 4).

The A405 nm reading for 1000 ng of HBcAg for this sample after

1 h incubation with substrate was three-fold lower than that of

sample derived from 10 m/s after 20 min incubation with the

substrate. This is not surprising since a portion of the samples

derived from 14 m/s operation are comprised of ruptured,

partially formed core particles and denatured or unformed

HBcAg subunits as indicated by the sucrose gradient profile

(Fig. 3a). On the contrary, a strong reactivity signal was

detected in the sample derived from 10 m/s tip speed. The anti-

HBcAg monoclonal antibody could detect as low as 50 ng

(0.5 ng/mL) of HBcAg, confirming that HBcAg prepared by

10 m/s tip speed was functionally active and can be potentially

used as a diagnostic reagent for the detection of anti-HBcAg

antibodies.

3.2. Effect of initial cell biomass concentration on batch

bead milling

The maximum amount of both total protein and HBcAg

released was increased as the biomass concentration increased

from 5% to 20% (w/v) (Table 1). The bead milling operation

with 20% (w/v) biomass concentration has released a total of

103.3 mg protein/g cell, which was 26%, 37% and 49% higher

than that of 15%, 10% and 5% (w/v), respectively. A total of

Fig. 3. The sucrose gradient profile of HBcAg released from E. coli in batch mode bead milling. SDS-PAGE shows the presence of HBcAg, and graphical illustration

indicates the concentration of HBcAg in the sucrose gradient fractions. (a) Samples from batch mode bead milling at 14 m/s. Lane M: molecular mass markers; lanes

1–24: sucrose gradient fractions. The uniform pattern indicates that the HBcAg particles in the 14 m/s tip speed sample comprises evenly of aggregated, ruptured,

partially or fully formed core particles and denatured or unformed monomers. (b) Samples from batch mode bead milling at 10 m/s. Lane M: molecular mass markers;

lanes 1–24: sucrose gradient fractions. The majority of HBcAg was found in the fractions of 9–14 indicating that most of the HBcAg in the disruptate has formed

intact HBcAg particles.

Fig. 4. The antigenicity of the released HBcAg prepared from different tip

speed was tested using ELISA with an anti-HBcAg monoclonal antibody.

Microtiter plate wells were coated with different concentration of HBcAg.

The bound HBcAg was detected by anti-HBcAg monoclonal antibody (1:1000

dilution) and an anti-mouse antibody conjugated to alkaline phosphatase. The

antigenicity of HBcAg was detected by measuring the optical density at 405 nm

after different incubation time with addition of substrate for the enzyme.

Samples tested were HBcAg prepared from 14 m/s tip speed and incubated

for 20 min (*), and 1 h (&); and HBcAg prepared from 10 m/s tip speed and

incubated for 20 min (^). The anti-HBcAg monoclonal antibody was replaced

by milk diluent in the negative control (~). The data represents the

mean � S.D. of triplicate measurements.

C.W. Ho et al. / Process Biochemistry 43 (2008) 206–212210

22.7 mg of HBcAg/g cell was also obtained at the operation at

this biomass concentration, which was 29%, 34% and 62%

higher than that of 15%, 10% and 5% (w/v), respectively.

Besides, the rate constants of HBcAg release recorded at this

operation were 1.6-, 1.9- and three-fold higher than that

recorded at 15%, 10% and 5% (w/v), respectively. This means

that the operation of cell disruption can be operated at a shorter

period of time with higher biomass concentration loading. The

presence of higher biomass in the disruption chamber will

reduce the distances between centres of neighbouring cells and

consequently increases the intensity of mutual interactions of

the micro-organisms [38]. As a result, the cell disruption

efficiency and the rate of protein release are improved. Indeed,

other researchers have observed a similar correlation between

protein release and initial biomass concentration [16,21,39].

Apart from the higher protein release rate, the energy utilization

was also reported to be lower at a higher biomass concentration.

Bunge et al. [40] reported that a significantly lower mechanical

energy input was needed for the disruption of Arthrobacter sp at

biomass concentration of 55% (w/v) compared to that at 10%

(w/v). However, a further increase of biomass concentration

from 20% (w/v) is unfavorable in this study. At higher biomass

concentration, the viscosity of the disruptate was higher, which

poses problems to the subsequent steps of downstream

processing, for instance, chromatographic processes of size

exclusion and expanded bed adsorption. Besides, a viscous

sample not only requires more intense centrifugation force for

solid–liquid separation, but also impedes the separation

performance of centrifuges [41], pumps [41], ultrafiltration

[42], and cross-flow filtration systems [43,44]. High-viscosity

sample also has high-power dissipation under mixing or

agitating mode and thus promote high-heat generation [45].

This explained the moderate temperature increase (to 30 8C)

during the bead milling operation at 20% (w/v) biomass

concentration.

C.W. Ho et al. / Process Biochemistry 43 (2008) 206–212 211

3.3. Effect of bead loading on batch bead milling

There was no obvious improvement in terms of maximum

total protein and HBcAg released as the bead loading was

increased from 65% to 80% (v/v) in the present study. This is

not surprising since the degree of cell disruption is very similar

at various bead loadings as shown by the measurement of intact

cell particles (OD600 nm; Table 1). This result was somehow not

in accord with that reported by other researchers: higher protein

yield at higher bead loading has been reported by Schutte and

Kula [46] on the disruption of E. coli and baker’s yeast and

Tamer and Moo-Young [20] on the disruption of Alcaligenes

latus. However, a vibrated mixing chamber was used by Schutte

and Kula [46], while a horizontal agitation chamber was used in

the present study. A bead loading in the range of 80–85% (v/v)

is generally accepted as the optimum condition and has been

used in many studies [15,19,21,23]. Bead loading higher than

this range is not preferred for cell disruption due to the high-

heat generation and power consumption, which often out-

weighs the minimal increase in disruption efficiency [47].

Indeed, in the present study, a rise in chamber pressure occurred

when a bead loading of 85% (v/v) was used, which resulted in a

portion of feedstock seeping out from the bead chamber

throughout the process of batch bead milling.

4. Conclusion

In this study, we have developed and optimized a scale-able

cell disruption method for the release of intracellular

recombinant HBcAg from E. coli cells. The highest HBcAg

release rate was achieved at 14 m/s impeller tip speed.

However, the high level of shear stress imparted to the cells

at high-tip speed operation ruptured HBcAg particles and

affected the antigenicity. The yield of protein was increased

with the increasing of biomass concentration due to the higher

mutual interactions, which occur in the grinding chamber. A

slightly increased of temperature to 30 8C was recorded at

biomass concentration of 20% (w/v); therefore a further

increase of biomass concentration is unfavorable. There were

no obvious effects of bead loading on the performance of bead

milling operation for HBcAg release observed. Hence, the

optimal operating conditions for the disruption of E. coli to

release HBcAg is at impeller tip speed of 10 m/s, biomass

concentration of 20% (w/v) and bead loading of 75% (v/v).

Acknowledgement

This study was supported by the IRPA Grant (09-02-04-

0766-EA001) and an E-Science Grant (02-01-04-SF0486) from

the Ministry of Science, Technology and Innovation of

Malaysia.

References

[1] World Health Organization. Introduction of hepatitis b vaccine into

childhood immunization services. Geneva: WHO; 2001. pp. 1–4.

[2] Hoofnagle JH, Gerety RJ, Ni LY, Barker LF. Antibody to hepatitis B core

antigen—a sensitive indicator of hepatitis B virus replication. N Engl J

Med 1974;290:1336–40.

[3] Korec E, Korcove J, Konig J, Hlozanek I. Detection of antibodies against

hepatitis B core antigen using the avidin–biotin system. J Virol Methods

1989;24:321–6.

[4] Tordjeman M, Rabillon V, Abouth D, Trepo C, Hoffenbach A, Somme G.

Specific detection of anti-HBc antibodies with an enzyme immunoassay

using recombinant HBcAg and monoclonal antibodies. J Virol Methods

1992;43:21–30.

[5] Milich DR, McLachlan A. The nucleocapsid of hepatitis B virus is

both a T-cell independent and T-cell dependent antigen. Science

1986;234:1398–401.

[6] Lai KN, Lai FM, Leung NWY, Lo ST, Tam JS. Hepatitis with isolated

serum antibody to hepatitis B core antigen. A variant of non-A, non-B

hepatitis? Am J Clin Pathol 1990;413:79–84.

[7] Tan WS, Dyson MR, Murray K, Hepatitis. B virus core antigen: enhance-

ment of its production in Escherichia coli, and interaction of the

core particles with the viral surface antigen. Biol Chem 2003;384:

363–71.

[8] Tey BT, Chua MI, Chua GS, Ng MYT, Awang Biak DR, Tan WS, Ling TC.

Influence of temperature and level of mixing on the growth and HBcAg

production of Escherichia coli in stirred tank bioreactor.. Biotechnol

Bioprocess Eng 2006;11:164–7.

[9] Fish NM, Lilly MD. The interactions between fermentation and protein

recovery. Biotechnology 1984;2:623–7.

[10] Datar R, Rosen CG. Studies on the removal of Escherichia coli cell debris

by aqueous two-phase polymer extraction. J Biotechnol 1986;3:207–19.

[11] Foster D. Optimizing recombinant product recovery through improve-

ments in cell-disruption technologies. Curr Opin Biotechnol 1995;6:

523–6.

[12] Currie JA, Dunnili P, Lilly MD. Release of protein from bakers’ yeast

(Saccharomyces cerevisiae) by disruption in an industrial agitator mill.

Biotechnol Bioeng 1972;14:725–36.

[13] Marffy F, Kula MR. Enzyme yields from cells of brewer’s yeast disrupted

by treatment in a horizontal disintegrator. Biotechnol Bioeng

1974;16:623–34.

[14] Limon-Lason J, Hoare M, Orsborn CB, Doyle DJ, Dunnill P. Reactor

properties of a high-speed bead mill for microbial rupture. Biotechnol

Bioeng 1979;21:745–74.

[15] Canales M, Buxado JA, Heynngnezz L, Eriquez A. Mechanical disruption

of Pichia pastoris yeast to recover the recombinant glycoprotein Bm86.

Enzyme Microb Technol 1998;23:58–63.

[16] Ricci-Silva ME, Vitolo M, Abhahao-Neto J. Protein and glucose 6-

phosphate dehydrogenase releasing from baker’s yeast cells disrupted

by a vertical bead mill. Process Biochem 2000;35:831–5.

[17] Chow YM, Tey BT, Ibrahim MN, Ariff A, Ling TC. The disruption of

Saccharomyces cerevisiae cells and release of glucose 6-phosphate dehy-

drogenase (G6PDH) in a horizontal Dyno Bead Mill operated in con-

tinuous recycling mode.. Biotechnol Bioprocess Eng 2005;10:184–288.

[18] Rehacek J. Continuous disintegration of micro-organisms in a new

laboratory apparatus. Experientia 1971;27:1103–4.

[19] Woodrow JR, Quirk AV. Evaluation of the potential of a bead mill for the

release of intracellular bacterial enzymes. Enzyme Microb Technol

1982;4:385–9.

[20] Tamer IM, Moo-Young M. Disruption of Alcaligenes latus for recovery of

poly (b-hydroxybutyric acid): comparison of high-pressure homogeniza-

tion, bead milling and chemically induced lysis. Ind Eng Chem Res

1998;37:1807–14.

[21] Bury D, Jelen P, Kalab M. Disruption of Lactobacillus delbrueckii ssp.

Bulgaricus 11,842 cells for lactose hydrolysis ion dairy products: a

comparison of sonication, high-pressure homogenization and bead

milling.. Innov Food Sci Emerg Technol 2001;2:23–9.

[22] Hopkins T. Laboratory cell disruptors: a review of apparatus & techniques.

Int Lab News 2000;16–7. October.

[23] Garcia FAP. Cell wall disruption. In: Kennedy JF, editor. Cabral JMS

recovery process for biological materials. New York: John Wiley & Sons;

1993. p. 47–69.

C.W. Ho et al. / Process Biochemistry 43 (2008) 206–212212

[24] Melendres AV, Unno H, Shiragami N, Honda H. A concept of critical

velocity for cell disruption by bead mill. J Chem Eng Jpn 1992;25:354–6.

[25] Middelberg APG. Process cell disruption of micro-organisms. Biotechnol

Adv 1995;13:491–551.

[26] Harisson STL. Bacterial cell disruption: a key unit operation in the

recovery of intracellular products. Biotechnol Adv 1991;9:217–40.

[27] Tan WS, McNae IW, Ho KL, Walkinshaw MD, Crystallization. X-ray

analysis of the T=4 particle of hepatitis B capsid protein with an

N-terminal extension. Acta Cryst 2007;F63:642–7.

[28] Tey BT, Yong KH, Ong HP, Ling TC, Ong ST, Tan YP, Ariff A, Tan WS.

Optimal conditions for hepatitis B core antigen production in shaked flask

fermentation. Biotechnol Bioprocess Eng 2004;9:374–8.

[29] Laemmli UK. Cleavage of structural proteins during the assembly of the

head of bacteriophage T4. Nature 1970;227:680–5.

[30] Bradford MM. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein–dye

binding. Anal Biochem 1976;72:248–54.

[31] Ho CW, Tan WS, Kamaruddin S, Ling TC, Tey BT. The direct recovery of

recombinant hepatitis B core antigen (HBcAg) from disruptate derived

from continuous flow bead milling. Biotechnol Appl Biochem 2007. doi:

10.1042/BA20070088.

[32] Ng MYT, Tan WS, Abdullah N, Ling TC, Tey BT. Heat treatment of

unclarified Escherichia coli homogenate improved the recovery effi-

ciency of recombinant hepatitis B core antigen. J Virol Methods

2006;137:134–9.

[33] Ho CW, Chew TK, Ling TC, Kamaruddin S, Tan WS, Tey BT. Efficient

mechanical cell disruption of Escherichia coli by an ultraconicator and

recovery of intracellular hepatitis B core antigen. Process Biochem

2006;41:1829–34.

[34] Kula MR, Schutte H. Purification of proteins and the disruption of

microbial cells. Biotechnol Prog 1987;3:31–42.

[35] Dyson MR, Murray K. Selection of peptide inhibitors of interactions

involved in complex protein assemblies: association of the core and

surface antigens of hepatitis B virus. Proc Natl Acad Sci USA

1995;92:2194–8.

[36] Ohori H, Shimizu N, Yamada E, Onodera S, Ishida N. Immunological and

morphological properties of HBeAg subtypes (HBeAg/1 and HBeAg/2) in

hepatitis B virus core particles. J Gen Virol 1984;65:405–14.

[37] Naito M, Ishii K, Nakamura Y, Kobayashi M, Takada S, Koike K. Simple

method for efficient production of hepatitis B virus core antigen in

Escherichia coli. Res Virol 1997;148:299–305.

[38] Heim A, Kamionowska U, Solecki M. The effect of micro-organism

concentration on yeast cell disruption in a bead mill. J Food Eng

2007;83:121–8.

[39] Morohashi S, Okada S, Hataya T, Sasaki T, Hoshino K, Sasakura T.

Release process of protein from Baker’s yeast by disruption in agitating

bead mill. J Chem Eng Jpn 1997;30:182–6.

[40] Bunge F, Pietzsch M, Mfiller R, Slydatk C. Mechanical disruption of

Arthrobacter sp. DSM 3747 in stirred ball mills for the release of

hydrantoin-cleaving enzymes. Chem Eng Sci 1992;47:225–32.

[41] Li WG. Effects of viscosity of fluids on centrifugal pump performance and

flow pattern in the impeller. Int J Heat Fluid Flow 2000;21:207–12.

[42] Wang SS. Effect of solution viscosity on ultrafiltration flux. J Membr Sci

1988;39:187–94.

[43] Brocklebank MP. Downstream processing plant and equipment. In:

Asenjo JA, editor. Separation processes in biotechnology. New York:

Marcel Dekker; 1990. p. 617–740.

[44] Fane AG, Radovich JM. Membrane systems. In: Asenjo JA, editor.

Separation processes in biotechnology. New York: Marcel Dekker; 1990.

p. 209–62.

[45] Khalaf WG, Sastry SK. Effect of fluid viscosity on the ohmic heating rate

of solid–liquid mixtures. J Food Eng 1996;27:145–58.

[46] Schutte H, Kula MR. Analytical disruption of micro-organisms in a mixer

mill. Enzyme Microb Technol 1988;10:552–8.

[47] Geciova J, Bury D, Jelen P. Methods for disruption of microbial cells for

potential use in the dairy industry—a review. Int Dairy J 2002;12:541–53.