Separation Science and Technology

ISSN: 0149-6395 (Print) 1520-5754 (Online) Journal homepage: http://www.tandfonline.com/loi/lsst20

Two-step purification strategy for enhanced

recovery of recombinant hepatitis B surface
antigen from Pichia pastoris

Yew Joon Tam, Nazariah Allaudin Zeenathul, Morvarid Akhavan Rezaei,

Mohd Lila Mohd Azmi, Abdul Rani Bahaman, Sewn Cen Lo, Joo Shun Tan &
Homayoun Hani

To cite this article: Yew Joon Tam, Nazariah Allaudin Zeenathul, Morvarid Akhavan Rezaei,
Mohd Lila Mohd Azmi, Abdul Rani Bahaman, Sewn Cen Lo, Joo Shun Tan & Homayoun
Hani (2016): Two-step purification strategy for enhanced recovery of recombinant
hepatitis B surface antigen from Pichia pastoris, Separation Science and Technology, DOI:
10.1080/01496395.2015.1135949

To link to this article: http://dx.doi.org/10.1080/01496395.2015.1135949

Accepted author version posted online: 15

Jan 2016.

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Download by: [RMIT University] Date: 25 January 2016, At: 11:42

 Two-step purification strategy for enhanced recovery of
recombinant hepatitis B surface antigen from Pichia
pastoris
2-step purification strategy for enhanced recovery of recombinant HBsAg from P. pastoris

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Yew Joon Tam1, 2, Nazariah Allaudin Zeenathul1, 2*, Morvarid Akhavan Rezaei1, 2, Mohd Lila
Mohd Azmi1, Abdul Rani Bahaman1, Sewn Cen Lo 1, 2, Joo Shun Tan3 and Homayoun Hani1

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1
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
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2
Laboratory of Immunotherapeutic and Vaccine Technology (LIVES), Institute of Bioscience,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
3
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School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia

Corresponding Author:
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*Associate Professor Dr. Zeenathul Nazariah Allaudin

Department of Veterinary Pathology and Microbiology,

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Faculty of Veterinary Medicine,

Universiti Putra Malaysia

43400 UPM Serdang,

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Selangor Darul Ehsan, Malaysia.

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Email address: zeenathulnazariah@gmail.com

Telephone number: +60 3 8609 3462

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ABSTRACT

Univariate screening on factors affecting the purification performance of recombinant hepatitis B

surface antigen (HBsAg) on ion exchange chromatography (IEC) and size exclusion

chromatography (SEC) and the establishment of a two-step purification strategy were performed.

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 Amongst four IEC adsorbents examined, the use of Q Sepharose XL IEC adsorbent under

optimised conditions together with optimised SEC purification was able to efficiently purify

HBsAg. An established purification strategy comprised of the two techniques further

demonstrated adaptability for scale-up operations with a final total purification factor (PF) of

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94.82 ± 16.20, HBsAg purity of 95.48% and recovery yield of 78.07%.

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Keywords: Hepatitis B surface antigen, protein purification strategy, ion exchange

chromatography, gel filtration, Pichia pastoris

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INTRODUCTION
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In purification of recombinant hepatitis B surface antigen (HBsAg), recovery of the pure protein
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is often challenging and not a straightforward process, especially where product quality (correct

folding of the recombinant protein with its biological activity retained), product value (purity and
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yield) as well as practicability and robustness of the purification process are considered. For

non-glycosylated HBsAg protein derived from Pichia pastoris [1-3], purification by several means
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of extraction and chromatographic steps has been established, rendering a highly purified,
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biologically-active HBsAg that meets most of the World Health Organization (WHO) safety
[4]
requirements . However, these purification protocols typically faces multiple challenges, such
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as difficulties in production up-scaling, low to moderate yields, incorrect aggregation and

degradation of HBsAg as well as being time consuming [5, 6]. For instance, specific adsorption of

HBsAg to diatomaceous earth was reportedly difficult to perform due to its discrete operation

that included a complicated routine of adsorption, centrifugation, desorption and centrifugation

2
 again, which was susceptible to mechanical stress and resulted in bad performance [7]. In another

instance, affinity chromatography for HBsAg purification was reported to be economically

unfavourable due to multiple factors that included loss and degradation of antibody function,

degradation of support matrix, progressive nonspecific adsorption of contaminants and

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[8, 9]

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incomplete antigen elution . Furthermore, the removal of the affinity tag in the recombinant

protein may prove to be an added burden in certain project applications [10]. More importantly, it

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was also reported that complicated operating procedures involved in multi purification steps
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most often than not resulted in low final product recovery, and not to mention that the extension

of operating time could result in the aggregation and/or dissociation of assembly structure of

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macromolecules [11, 12] leading to increased manufacturing costs [4].
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Currently, chromatographic techniques, such as ion-exchange chromatography (IEC) and size

exclusion chromatography (SEC), were found to be very efficient techniques for recombinant

HBsAg purification due to their high selectivity, feasibility to automate and scaling-up [4, 7].
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This is especially important for intracellularly produced HBsAg protein, known to be associated
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with a large amount of intracellular cytoplasmic proteins together with other contaminants such
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as carbohydrates and lipids , underlining the need for a good separation strategy for HBsAg [4, 13,
14]
. These chromatographic processes have been successfully used in the purification of HBsAg
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[15] [5]
from Chinese hamster ovary (CHO) mammalian cells and Hansenula polymorpha .

Moreover, in certain reports, IEC purification efficiencies were found to be further improved by

the addition of polyethylene glycol (PEG) in the equilibrium and mobile phase of IEC [12], types

3
 [16]
of resin used and the effects of different ligand densities or total ionic capacities of IEC

adsorbents [17].

In view of these previous reports, this present work focuses on examining the parameters

influencing purification performance specific to recombinant HBsAg expressed from P. pastoris

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on both IEC and SEC in a univariate manner for the purpose of establishing a purification

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strategy for HBsAg purification from P. pastoris. Significant parameters for improving the

purification and the recovery level of HBsAg were identified and subsequently, an integrated
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two-step chromatographic approach was adopted to demonstrate the possibility of up-scale

adaptation of the established approach.

EXPERIMENTAL
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Preparation of post-induction cell biomass and cell disruption
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Recombinant HBsAg production was carried out in a two-phase fed-batch protocol using
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laboratory-scale baffled, Erlenmeyer shake flask. Laboratory-scale fed-batch cultivations were

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conducted in 200 mL of buffered glycerol complex medium broth (BMGY) by inoculating the

starter cultures (2.5% v/v) and grown at 30ºC with agitation (250 rpm) until maximum biomass
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concentration was reached (72 h at dry cell weight (DCW) of ~6.64 g/L). Prior to induction

phase, the cultures were pelleted by centrifugation (2,860 x g, for 5 min at 4ºC) and re-suspended

in 200 mL buffered methanol complex medium (BMMY) containing 1% final concentration of

methanol to induce expression. Initial 100% of methanol was added to a final concentration of

4
 1% every 24 h to maintain induction. After the 48 h post-induction, P. pastoris culture was

harvested by centrifugation at 2,860 x g, 10 min, 4ºC. The cell pellet was re-suspended and
[18]
washed twice by centrifugation in ice chilled 20 mM Tris-HCl buffer (pH 7) . Thereafter, re-

suspended cell pellet in the same buffer in different pH according to experiment requirement and

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was further disrupted using high-pressure homogenizer (Avestin Homogenizer Emulsiflex-C50,

Canada) with number of passes at 20 times, 7.70 g/L dry cell weight (DCW) of biomass

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[19]
concentration and pulse pressure at 1,029 bar . Samples collected were added with protease
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inhibitors from a cocktail kit (MP Biomedical, LLC, France) and stored in – 80˚C prior to use.

Analytical procedures
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The protein purity of HBsAg was analysed by SDS PAGE and Western blotting . After
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appropriate dilution with 2X SDS sample disruption buffer (62.5 mM Tris-HCl pH 6.8, 25%

(v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue, 5% (v/v) β-mercaptoethanol; 10
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µL), approximately 10 µL of the cell extract prepared as described above was loaded into 12%
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SDS-PAGE gel. Electrophoresis was performed at 32 mA for 90 min using the Mini Protean 3

apparatus (Bio-Rad, USA) immersed in Tris-Glycine buffer (0.025 M Tris-HCl, pH 8.3, 0.192 M
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glycine, 1% (w/v) SDS). After electrophoresis, the SDS PAGE gel was stained with staining

solution (0.1% (w/v) Coomassie brilliant blue R-250, 40% (v/v) methanol, 10% (v/v) acetic acid)
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for 15 min at room temperature with agitation and finally, the gel was de-stained with de-staining

solution (10% (v/v) methanol, 10% (v/v) acetic acid) until the bands became clear (1-3 h).

Western blotting was carried out to determine the presence of HBsAg specific protein. After

electrophoresis, SDS-PAGE gel containing fractionated proteins was equilibrated in transfer

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 buffer (25 mM Tris-HCl, 190 mM glycine, pH 8.0, 20% (v/v) methanol) and transferred to a

nitrocellulose membrane using a semi-dry blotter (Trans-Blot® SD, BioRad, USA) for 1 h at

constant voltage (15V). The presence of HBsAg was detected by using HBsAg primary

monoclonal antibody (Meridian Life Science, USA) and goat anti-mouse secondary antibody

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IgG conjugated with horseradish peroxidase (HRP) (Bio-Rad, California, USA). Finally, the

signals were developed using the Western blotting ABTS substrate. Quantification of HBsAg

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was performed using enzyme linked immunosorbent assay (ELISA) according to the instructions
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provided by the manufacturer (SURASE B-96 ELISA kit specific for HBsAg detection, General

Biologicals Corp, Taiwan). Briefly, cell extracts released from cell disruption were added into 96

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well plates at a volume of 50 µL followed by the addition of another 50 µL of monoclonal anti-

HBsAg antibody conjugated to horseradish peroxidase (HRP) (General Biologicals Corp,

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Taiwan). The mixture was then incubated for 80 min at 37ºC. Thereafter, the well containing the

mixture was washed repeatedly for 3 times with 200 µL washing buffer (phosphate buffered
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saline (PBS), Tween-20) at 30 s intervals. The plate was then dried and 100 µL of 3, 3’, 5, 5’-

tetramethylbenzidine (TMB) (0.6 g/L TMB, citric acid, 0.03% hydrogen peroxide (H 2 O 2 ))
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substrate was added to each well prior to incubation for 30 min at room temperature. 100 µL of
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2N sulphuric acid (H 2 SO 4 ) was added prior to result observation with a reading wavelength of

450 nm and a reference wavelength of 650 nm using an ELISA reader (Tecan, Sunrise,
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Australia). To generate a standard curve, a series of dilutions containing 0 to 250 µg/L of HBsAg

(Biodesign International, USA) was included in each assay. Total protein content was
[22]
determined according to Bradford’s method with bovine serum albumin (BSA) as standard .

Samples and standards were placed in the 96-well micro-plate and the absorbance was measured

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 at 595 nm using the Tecan micro-plate reader controlled by the Magellan 4.0 PC data analysis

software. Both samples and standards were analysed in triplicates. The results were found to be

reliable (the relative coefficient (R2) of the calibration curve of reference protein was above

0.99). Characterization of protein isoelectric point (pI) and molecular weight (MW)

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determination was performed using the Compute pI/MW tool prediction

(http://web.expasy.org/compute_pi/). Observation of HBsAg sample purity was performed with

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reverse phase high performance liquid chromatography (RP-HPLC) using Waters C18 column
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(4.6 x 250 mm internal diameter (I.D.)). The mobile phase was prepared by mixing 100%

methanol: 5 mM potassium dihydrogen phosphate (KH 2 PO 4 ) buffer in the ratio 50: 50 (v/v);

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without pH adjustment. Linear flow rate was kept at 361 cm/h and detection wavelength was set

at 280 nm with a sample injection volume of 10 µL following the protocol as described by

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Shrivastava and Jain [23]. Sample readings were taken in triplicates.
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Purification strategies and chromatographic equipment

All chromatography purification runs were performed at room temperature on an AKTA explorer
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system (GE Healthcare, UK). The columns used for IEC (HiTrapTM DEAE Sepharose FF, ANX
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Sepharose 4 FF (high sub), Q Sepharose FF and Q Sepharose XL, CV= 1 and 5 mL) and SEC

(HiLoad™ 16/600 Superdex™ 75 pg) were also from GE Healthcare, UK. Properties of these
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columns are highlighted in Table 1. Buffers were prepared using chemicals of analytical reagent

grade and filtered through a 0.22 µm cut-off filter before use. For all chromatography runs, the

amount of protein loaded onto the columns was below the maximum binding capacity of the

respective column. Columns were first equilibrated with five column volumes (CV) of binding

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 buffer prior to loading followed by another 5 CV binding buffer post-load washing before the

elution of retained proteins with a linear gradient of 0-100% elution buffer within 10 CV, unless

otherwise stated. For SEC runs, centrifugal filter units (Vivaspin, GE Healthcare, UK) was used

to concentrate partially purified protein solutions from IEC to a concentration of approximately

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1.5 g/L before applying to the equilibrated SEC column. Samples collected subsequently after

each purification step were mixed with 3 M potassium thiocyanate and stored at – 80ºC.

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Cleaning-in-place (CIP) procedures
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Optimal cleaning-in-place (CIP) procedures were established for both IEC and SEC techniques
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by observation of stable baseline during washing of the columns and consistency of binding-

elution for IEC and elution time for SEC was evaluated by using BSA (data not shown). For IEC
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columns, washing was performed with 2 CV of 2 M NaCl, 2 CV of 1 M NaOH, 2 CV of 2 M

NaCl, 2 CV of distilled water and 4 CV of binding buffer at a linear flow rate of 156 cm/h. For
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SEC, washing of the column were done with 1 CV of 0.5 M NaOH followed by equilibration of

the column with 2 CV of start buffer at a linear flow rate of 30 cm/h. CIP procedures were
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performed in reverse flow direction after each purification run.

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Screening for significant factors influencing the purification

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performance of hepatitis B surface antigen (HBsAg)

Four anion exchange chromatography columns with different resin properties (Q Sepharose FF,

DEAE Sepharose FF, ANX Sepharose FF and Q Sepharose XL, CV = 1 mL) were tested. The

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 sample was always loaded onto the IEC columns pre-equilibrated with 20 mM Tris-HCl buffer.

A univariate screening study involving several types of parameters was performed in order to

determine which parameters would significantly influence the performance of HBsAg

purification with IEC. The screening approach set up involves testing of pH of the binding buffer

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ranging from pH 5 to 9 and ionic strength of the elution gradients (0.5 to 2 M NaCl) in

measurement of their binding capabilities and elution efficiency relative to purity as well as their

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correlations on the recovery yield and purification factor (PF) of HBsAg purification with IEC.
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Each screening of parameter was repeated three times with the fraction size measured at 1 mL.

All the columns were eluted with the same linear gradient (1 M NaCl, 10 CV) unless otherwise

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specified with a flow rate of 156 cm/h at room temperature. Similarly, for SEC, a univariate

screening approach was adopted, exploring the influence of linear flow rate (9 to 45 cm/h) and
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sample volume (0.5 to 5 mL) on recovery yield and PF of HBsAg purification. The screening of

parameter was repeated three times and fraction size measured at 5 mL. All SEC runs were
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performed using 20 mM Tris-HCl, pH 8 buffer with a linear flow rate of 30 cm/h at room

temperature unless otherwise specified.

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Strategy for up-scaling of hepatitis B surface antigen (HBsAg)

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purification
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In order to improve the strategy for further up-scaling, another series of univariate screening

approach was performed to determine parameters that significantly influence the performance of

IEC column in HBsAg purification. Sample loading volume (2 to 32 mL, 2.5 CV) and length of

elution gradient in CVs (5 to 30 CV) on an IEC column with increased CV size (CV = 5 mL)

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 were evaluated and their effects on column performance were analysed including peak height,

peak area and peak area/total area and as well as their correlations on recovery yield and PF of

HBsAg purification. Determination of the peak height (mAU), peak area (mAU*mL) and peak

area/total area (%) were automatically calculated by the Unicorn software version 5.0 (GE

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Healthcare). Results obtained was further verified by increasing the loading volume of cell

disrupted samples accordingly to the size of column from 5 mL to 15 mL (serial attachment of Q

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Sepharose XL columns, CV = 5 mL x 3) while SEC column remained the same because the
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loaded volume was still less than the capacities of the SEC maximum loading volume.

Data analysis
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Adsorption efficiency (AE) (%) of the IEC columns performed under different pH was
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calculated based on the initial amount of HBsAg concentration in crude (mg/L) loaded (I) versus

the amount of HBsAg concentration (mg/L) present in the flow-through (F), according to
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Equation 1 [24]:
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AE (%) = Amount of HBsAg (mg/L)

(loaded – flow-through) x 100 (1)

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Amount of HBsAg (mg/L) loaded

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To check for possible losses and potential degradation of the HBsAg protein during the process,

the recovery yield (%) of HBsAg concentration (YH) and total protein yield concentration (YP)

were calculated by summing the total HBsAg concentration from flow-through and all eluted

fractions, according to Equation 2 and 3 [25]:

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 YH (%) = HBsAg concentration

(mg/L) (fractions + flow-through) x 100 (2)

HBsAg concentration (mg/L) (loaded)

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YP (%) = Total protein (g/L) (fractions +

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flow-through) x 100 (3)

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Total protein concentration (g/L) (loaded)
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Elution efficiency (EE) (%) of HBsAg protein in IEC columns was performed with different salt

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(NaCl) concentrations in buffers that have the same pH with the binding buffer. The calculation

of EE present during the purification step was based on Equation 4 [24]:

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EE (%) = Amount of HBsAg (mg/L) recovered

x 100 (4)
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Amount of HBsAg protein bound (mg/L)

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To evaluate the success of purification steps, the total HBsAg protein concentration (mg/L) and
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total protein concentration (g/L) in the crude extract, the flow-through fractions and the eluted

fractions were measured and their respective specific activities (mg/g) and recovery yield (%)
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were determined. Specific activities of the crude extract and of the fractions as well as the

purification factor (PF) was calculated according to Equation 5 and 6 while the recovery yield of

HBsAg was calculated by the ratio of initial amount of HBsAg in the loading sample to HBsAg

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 in fractions according to Equation 7. The purity of HBsAg recovered during purification step

was calculated using Equation 8 [25]:

Specific activity (mg/g) = HBsAg activity (mg/L) (5)

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Total protein (g/L)

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PF = Specific activity HBsAg fraction (before purification) (mg/g) (6)
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Specific activity HBsAg fraction (after purification) (mg/g)

Recovery yield (%) = Amount of HBsAg recovered (mg/L) x 100 (7)

Amount of HBsAg loaded (mg/L)

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Purity (%) = Amount of HBsAg (mg/L) recovered x 100 (8)
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Amount of total protein (mg/L)

RESULTS
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Characterization of crude extract from cell disruption and pre-

purification
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Production of intracellular HBsAg recombinant protein was from P. pastoris (GS115 strain,

Mut+) cultivated using fed-batch fermentation and the cell disrupted to obtain crude extract.

Total protein concentration of the crude extract was about 13.15 ± 2.7 g/L. Based on ELISA, the

12
 total HBsAg concentration was about 147.07 ± 6.0 mg/L and a specific activity of 11.19 mg/g

with an initial PF at 1. The pI and MW of monomeric HBsAg protein product was calculated

theoretically in un-glycosylated form and found to be 8.2 and 25.4 kDa, respectively.

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Influence of binding buffer pH on protein adsorption of ion exchange

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chromatography (IEC)

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The study of HBsAg protein adsorption was performed on the IEC columns pre-equilibrated with
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different pH buffer conditions (pH 5 to 9) and eluted with the same linear gradient (20 mM Tris-

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HCl, 0 to 1 M NaCl, 10 CV). The quantified amount of HBsAg protein in the flow-through used

to calculate the adsorption efficiency showed that purification runs performed for the screening
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of AE were between 93% and 100% for both YP and YH. Thus, it was assumed that there were

no HBsAg lost or denatured during the process. The AE values of HBsAg are depicted in Table
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2. Based on the result obtained, HBsAg protein AE was observed to increase as pH increases in

all the IEC columns screened, with an optimum AE achieved in pH 8. Further increase in the pH
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of the buffer from pH 8 to 9 showed little significant increase in AE of HBsAg protein.

Generally, a better AE was seen to be achieved by using Q Sepharose XL while ANX Sepharose
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FF attained poorer AE. The highest AE (84.5%) of HBsAg was achieved with Q Sepharose XL
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in pH 8. Both DEAE Sepharose FF and Q Sepharose FF showed similar success in producing

good AE of HBsAg, demonstrating an optimum AE at pH 8 with 78.3% and 81%, respectively.

Influence of salt ionic strength on elution gradient behaviour of ion

exchange chromatography (IEC)

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 The influence of NaCl concentration on the elution gradient behaviour of the IEC columns at

their optimum binding pH was examined. Elution procedure of HBsAg protein from IEC

columns performed under different NaCl concentrations (0.5 to 2 M) was evaluated and the

results obtained based on EE and PF calculations described in Table 3. An improvement of EE of

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HBsAg was observed for all the IEC columns with an increment of NaCl concentration.

However, for PF obtained, improvement can only be seen to a certain extent, beyond which a

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further increase in NaCl concentration demonstrated either non obvious increment or decreased
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effect on purification of HBsAg. For ANX Sepharose FF and DEAE Sepharose FF, it was

observed that optimum EE were achieved at 1 M NaCl concentration.

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For Q Sepharose FF and Q Sepharose XL columns, saturation of EE was observed when 1.5 M
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NaCl concentration was used in the elution of HBsAg. The highest EE (95.6%) and PF (3.1 ±

0.1) of HBsAg was achieved with Q Sepharose XL in the presence of 1.5 M NaCl concentration.
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Generally, the lowest EE and PF of HBsAg protein were achieved with 0.5 M NaCl

concentration for all IEC columns. The lowest EE was observed by using Q Sepharose XL
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(47.7%) while the lowest PF was obtained with ANX Sepharose FF (1.06 ± 0.1) with the same

NaCl concentration. Based on the results obtained, the Q Sepharose XL column has the highest
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in AE (84.5%, pH 8) and EE (95.6%) while attaining high PF (3.1 ± 0.1) for HBsAg,

demonstrating good potential as an adsorbent for subsequent experiments in the purification of

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HBsAg.

Influence of polyethylene glycol (PEG) on ion exchange

chromatography (IEC) purification performance

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 In order to further improve the performance of IEC purification, PEG with molecular weight of

10,000 at a concentration of 1% as described by Bi et al. [12] was added to the binding and elution

buffers to study its influence on the separation of HBsAg by IEC. It was observed that upon the

addition of PEG into the mobile phase of both binding and elution buffer, separation of proteins

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from crude samples decreased, as proteins accumulated to form a single peak as opposed to

multiple peaks demonstrated in the absence of PEG. The addition of PEG in just the elution

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buffer produced minute significance on the changes on protein separation with IEC (as compared
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to protein profile generated in the absence of PEG). Thus, for subsequent IEC experiments, PEG

was omitted from use in the mobile phase.

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Influence of sample loading volume and elution gradient length on the
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purification behaviour of ion exchange chromatography
ed

The sample loading volume (mL) of the Q Sepharose XL in an up-scaled (CV = 5 mL) column
[26]
for HBsAg protein was determined and calculated using linear regression analysis , based on
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the curves generated from peak height, peak area as well as the PF and recovery yield of HBsAg

from the elution profiles shown in Figure 1. Linear regression analysis of sample loading volume
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against all variables produces good linearity (R2 ≥ 0.99) during initial increase in loading
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volumes (2 to 8 mL). The sample loading volume to achieve maximum HBsAg binding capacity

for Q Sepharose XL column was determined at 8 mL of sample containing an average of 147.07

± 6.0 mg/L of HBsAg. Further increase in loading of samples (16 to 32 mL) produced a

significant breakthrough of HBsAg binding capacity where unbound protein losses was seen to

occur as indicated by the curving of linear graphs based on peak height and peak area (Figure

15
 1A) as well as reduction of PF and yield of HBsAg recovered (Figure 1B). Therefore, to avoid

the possibility of HBsAg losses, 4 mL sample volume of disrupted samples was loaded to

column for subsequent experiments.

The influences of elution gradient length in various CVs (5 to 30) on behaviour of HBsAg

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purification in Q Sepharose XL column are shown in Figure 2. As observed, the occupancy of

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the peak area for HBsAg over total area declines as the elution length of purification runs

increases up to 20 CV (26.56%). This is attributed to the appearance of additional protein peaks

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as the elution length increases where greater separation of proteins was observed. The PF of

HBsAg recovered was increased with the highest to be observed from elution length of 20 CV
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(13.1 ± 0.2) and thus, was chosen to be the optimum elution length.
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Influence of sample volume and flow rate on chromatographic behaviour

of size exclusion chromatography

ed

After purification by IEC column, SEC was employed as a second step to further purify HBsAg.
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Prior to loading, collected samples from IEC were concentrated with centrifugal filtration to a
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concentration of approximately 1.5 g/L. Based on obtained results as shown in Figure 3, it was

possible to inject up to 5 mL per sample volume, which corresponds to 4% of 1 CV, at an

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applied linear flow rate of 30 cm/h. Correlation of sample volumes to both peak height and peak

area demonstrated good linearity values of (R2 ≥ 0.99) indicating that the purification

performance of HBsAg by SEC was not limited by the proposed sample volumes. In addition,

16
 the increase in sample volume demonstrated an increase to both recovery yield and PF of

HBsAg. Thus, for subsequent experiments, 5 mL of sample volume was used.

In order to obtain more information on this system and to improve operation conditions for the

purification process, further analysis for the possible influences of flow rate on the responses of

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recovery yield and PF of HBsAg purification by SEC was carried out. Five mL of the said

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concentrated protein was loaded into Superdex 75 at different flow rates. It was observed that the

influences of flow rate have little significance within the limit tested on purification of HBsAg
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with SEC, demonstrating similarity in separation behaviour and retention volume independent

from the flow rate applied. On the average, HBsAg was found to be eluted at a retention volume
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at a range from 45 to 60 mL after injection of the IEC samples. Overall, an average PF of 5.0 ±
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0.68 was obtained from the purification runs with SEC and an average of 98% recovery yield of

HBsAg. However, due to the consideration for pressure limit of the column, 1mL/min flow rate
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was chosen for subsequent experiments.

Up-scaling of purification strategy of hepatitis B surface antigen and

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analysis
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Concentration and purification of the HBsAg protein in a two-step integrated purification

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strategy using IEC (Q Sepharose XL) and SEC (Superdex 75) were demonstrated under definite

process conditions. In order to facilitate the need for practical requirements of a low cost process

in the ease of up-scaling, purification of HBsAg with IEC was up-scaled to a CV of 15 mL by

serial attachment of Q Sepharose XL, CV = 5 mL columns. Here, the sample volumes were

17
 increased simultaneously according to the capacity of the columns used and purification runs

were repeated three times. As observed, the separation profiles of the purification processes

relevant to the increase in scalability bore minimal changes, with the exception in peak retention

volume and peak volume size. Thus, it was deduced that the up-scaling of the IEC purification

t
ip
process could be performed by serial attachment of the columns to accommodate larger sample

volumes. Hence, further purification of clarified disrupted samples of recombinant HBsAg

cr
protein was performed with the optimized operation conditions using the up-scaled IEC with
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us
initial loading of 12 mL sample volume and existing SEC column size. The analytical results of

HBsAg purification strategy were summarized in Table 4. The purity of the protein recovered

an
was further identified using RP-HPLC.
M
The elution profiles obtained from the purification was detected with the SDS-PAGE (Figure 4)

and Western blotting (Figure 5) against monoclonal anti-HBs (Meridian Life Science, USA),
ed

confirming the presence of HBsAg protein bands. It was observed that a large amount of

unwanted proteins from the host cells after disruption were unbound and flow through the IEC
pt

column while the rest of the proteins including HBsAg bound were divided into 5 peak fractions

by linear gradient elution. Samples were collected from the peak fraction 2 which contained the
ce

presence of HBsAg as determined by ELISA with an average HBsAg recovery yield at 81% and

a PF of 16.86 ± 1.87. Overall, the elution of HBsAg protein was observed to occur in a range of
Ac

0.09 to 0.20 M of the linear elution gradient by IEC. The remainder of the unwanted proteins was

further removed by applying SEC as a second purification step further dividing the existing IEC

samples into 3 peak fractions. Samples from SEC were collected from peak fraction 1 with a

final purity of more than 95% with a recovery yield of 78% and a PF of 5.04 ± 0.38. The purity

18
 of HBsAg was increased throughout the purification strategy according to the assay of SDS

PAGE and RP-HPLC.

Characteristics of hepatitis B surface antigen aliquots during purification

t
ip
Aliquots of the HBsAg were placed at room temperature, 4°C, – 20ºC and – 80ºC during the

duration of the purification study. It was noticed that samples containing recombinant HBsAg

cr
released from P. pastoris demonstrated an eventual built-up of insoluble particles in the solution
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us
after prolonged storage (Figure 6). These insoluble particles can be seen as early as 3 days in

room temperature, followed by 14 days at 4°C and subsequently 12 months at – 20°C. So far, no
an
appearance of this phenomenon occurred in samples stored at – 80°C. In this event, further

purification of disrupted samples under such condition by IEC after removal of the particles by
M
centrifugation produced chromatograms that depicted the absence of prior mentioned peak

fraction containing HBsAg from its protein separation profile followed by a shift in HBsAg
ed

distribution to other peak fractions. In addition, a loss in HBsAg activity was also observed in the

aforementioned samples compared to when analysed immediately with the use of ELISA as
pt

detection assay.
ce

DISCUSSION
Ac

During preliminary investigations, it was observed that HBsAg protein obtained from IEC

exhibited poor binding onto the cationic adsorbents of SP Sepharose FF and CM Sepharose FF

columns (data not shown) in a pH range either above or below the calculated pI value of HBsAg

19
 [5, 16]
protein, indicating unsuitability of the cationic exchange columns for HBsAg purification .

Hence, using anionic exchange adsorbents, our results revealed that Q Sepharose XL produced

the highest AE (pH 8) at 84.5% with the highest EE (1.5 M NaCl) at 95.6% as compared to the

other IEC columns tested. The presence of long quaternary dextran chains in the matrix of IEC

t
ip
column, used as surface extenders in forming a lattice that distributes the ligands throughout the

porous space of the resin interior would allow protein molecules to fully utilize the available

cr
space within the resin, creating a better retention capability within equivalent resin ligand
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[27]
density . As a result, a higher AE can be achieved in HBsAg adsorption while a higher NaCl

concentration was needed for efficient elution. However, obtained results in this study were

different to reports by Huang et al. an[17]

and Pessela et al. [28]
. In the purification of Chinese

hamster ovary mammalian cell line (CHO) based HBsAg by IEC, chromatographic efficiency
M
was improved by using lower ligand density adsorbents, demonstrating a higher disassociation of
[17]
HBsAg protein with increasing ligand density of DEAE adsorbents . Dissociation of HBsAg
ed

protein (which is capable of spontaneous assembly into larger structures) was explained due to

the occurrence of intense multipoint adsorption of the protein to absorbent, making it

pt

[28, 29]
increasingly difficult to desorb the protein . On the other hand, other similar studies have
ce

also observed that multipoint attachment was not attained during adsorption of large protein

structures to IEC near to pI value [30, 31]. In this study, the Q Sepharose XL column used was is a
Ac

high ligand density adsorbent with 0.18-0.26 mmol Cl–/mL medium yet disassociation of HBsAg

protein was not observed, with a good demonstration of high YH. Moreover, HBsAg protein was

eluted by low ionic strength (0.09-0.20 M) from Q Sepharose XL column, implicating that pH

20
 chosen for binding and elution of HBsAg protein too may play a role in determining the outcome

on the structure of HBsAg recovered from IEC [6, 16, 27, 29].

It was previously reported that the addition of PEG increases partition efficiency by altering
[12,
adsorption and retention behaviour of proteins in IEC and thus, was able increase selectivity

t
ip
13, 32, 33]
. In this study, a different behaviour was observed in the separation profile of HBsAg

cr
protein when 1% PEG 10,000 was included in the purification step by IEC. The reverse effect on

the impact of PEG could be due to the differences in composition and structures of P. pastoris
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host proteins being more susceptible to PEG as a purification chaperone [34] altering the retention

and elution behaviour of the proteins during IEC.

an
Results from the optimization of elution length of linear gradient showed improved purification
M
performance of HBsAg with a better separation of the protein, leading to achievement of a higher

PF (13.1 ± 0.2) when elution length was determined to be at 20 CV in length. Tennikova and
ed

[35]
Svec pointed out that the increase in elution length could improve resolution of the protein

purified whereby a single compound band can be further separated into several additional peaks
pt

due to the lack of homogeneity of the solid surface of the separation medium. In this study, the
ce

extension of elution length was also found to improve resolution of HBsAg purification up to a

limit. A further increase of elution gradient length produced an adverse effect on the HBsAg
Ac

purification where peak area/total area was higher than of the elution length of 20 CV (28.58%)

and of a lower PF (8.1 ± 0.2). This result suggests a peak broadening effect on elution profile,

possibly due to the occurrence of mass transport resistances between the protein mixtures and the

21
 adsorbent, giving rise to dilution of the solute with reduced resolution and less purity in HBsAg

protein recovered [36, 37].

In purification of HBsAg with SEC, appearance of HBsAg was found only in the first peak

fraction recovered from the separation of concentrated IEC sample. Prior to the addition of

t
ip
potassium thiocyanate in IEC samples, HBsAg activity was initially detected in all 3 peak

cr
fractions generated from SEC purification (data not shown), consistent with previous suggestions

where yeast derived HBsAg travels in a mixture of heterogeneous protein structures during in
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[6, 38, 39]
vitro purification . The difference of elution seen could be attributed to the addition of

potassium thiocyanate, which was reported to promote correct assembly of HBsAg protein [39-41].
an
By SDS PAGE detection under reducing conditions, HBsAg purified was found to appear in
M
multiple bands rather than a single band representing molecular weight of 25.4 kDa. These

additional bands were proven their correspondence to the 25.4 kDa monomer by Western
ed

Blotting, indicating a mixed-form of fully assembled large protein structures and incomplete

assembly of HBsAg [38, 42, 43].

pt

After prolonged storage, both clarified disrupted samples and samples collected from IEC as well
ce

as SEC experienced an eventual built-up of insoluble particles in the solution. The differing rate

of built-up observed depended on the storage temperature. Even after centrifugation, further use
Ac

of the prolonged stored clarified disrupted samples led to shifting of HBsAg distribution in IEC

and a loss in HBsAg antigenicity. This could be one of the reasons for low recovery yield in the
[6]
purification of HBsAg. Previously, Bardiya reported a loss in HBsAg activity in disrupted

samples after 4 weeks of storage at 4°C and –20°C. The cause was suggested to be due to the

22
 effects of the pressure employed during cell disruption in the presence of detergent in the sample.

However, the issue of insoluble particles built-up during storage and its effect on HBsAg

distribution during IEC were not raised. Since clarified disrupted sample used in our study was

prepared in the absence of detergent, there could be other influencing factors contributing to the

t
ip
shift in distribution and loss of HBsAg activity during storage. It is speculated that it could be
[14, 44]
due to the formation and progression of protein aggregation during storage . There is a

cr
possibility of further aggregation into large particles with irreversible structure change whilst
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retaining only about 20% of the original HBsAg activity [45]. This tendency to aggregate is most

probably due to the hydrophobic domains of HBsAg protein, which became uncovered after cell

45]
an
disruption and subsequently caused by stress conditions that occurs in process environments

. In this matter, Tleugabulova et al. [44]

[42,

demonstrated that addition of potassium thiocyanate

M
into HBsAg samples were able to be stored for 2 days at 60°C and 7 days at 37°C without

observation of changes on the protein and further suggested that aggregation of the HBsAg
ed

protein can be prevented in the presence of potassium thiocyanate during storage. In our study,

addition of potassium thiocyanate into the HBsAg samples improved recovery of HBsAg from
pt

centrifugal filtration (relatively high recovery yield) as compared to the previously reported low
ce

[13, 45]
recoveries of HBsAg due to aggregation . Analysis with RP-HPLC on the SEC purified

HBsAg showed the formation of a single peak suggesting charge uniformity and thus
Ac

representing the absence of HBsAg aggregation in the final product recovered.

In chromatography up-scaling, there is a general practice of keeping linear flow rates, gradient

slope and column length constant while increasing the column diameter. However, these constant

kept parameters are often affected by limitations such as column compression, process time

23
 [46, 47]
involved and changes in buffer consumption, thus making up-scaling process difficult .

Therefore, in this study, up-scaling of HBsAg purification with IEC was instead performed with

the application of iso-resolution curve concept in which the column length was adjusted [47, 48]. It

was shown that the up-scaling of IEC process with serial attachment of the IEC columns is

t
ip
possible, demonstrating a close similarity of separation profiles in the purification of HBsAg on

either small-scaled (CV = 5 mL) or up-scaled level (CV = 15 mL) by IEC. Although there were

cr
noticeable differences in the peak retention volume and peak volume size amongst the separation
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us
profiles due to a longer column length in the up-scaled process, several studies in the literature

have indeed supported the fact that the influence of column length has only a small effect on the

an
resolution of large molecules such as proteins in IEC operated under retention mode with linear

gradient elution [35, 46, 49].

M
In the present study, clarified disrupted samples from P. pastoris containing HBsAg was
ed

successfully purified using the up-scaled two-step purification strategy resulting in an average

recovery yield of 78.07%, purity of more than 95% and an average total PF of 94.82 ± 16.20.
pt

Compared to other studies, our results are within the acceptable range for purification of HBsAg,
[14]
with a higher recovery yield. For instance, in a study performed by , purification of HBsAg
ce

derived from P. pastoris incorporating the use of ultrafiltration and immuno-purification

produced results with more than 95% purity and an average recovery yield of 71%. In another
Ac

[5]
instance, Huang et al. was able to achieved results with an average PF of more than 80 and

purity of more than 99% in the purification of HBsAg derived from Hansenula polymorpha

incorporating the use of chromatography techniques of IEC, Hydrophobic interaction (HIC) and

SEC. However, the recovery yield obtained was about 21%. Taken together, analysis based on

24
 the ELISA results and confirmation by Western blotting revealed that the purified HBsAg

protein with the established purification strategy is antigenic and therefore could further be used

as a diagnostic agent as part of detection assay development.

t
CONCLUSION

ip
cr
In the present study, a variety of common factors known to influence protein purification

performance of IEC and SEC were screened in a univariate manner to establish an optimal
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purification strategy for the purification of recombinant HBsAg produced from P. pastoris.

• an
For IEC, clarified disrupted samples can be applied directly without prior diafiltration or

buffer exchange preparation onto Q Sepharose XL column with conditions of 4 mL sample

M
volume; equilibration and elution with Tris-HCl binding buffer and elution buffer containing 1.5

M NaCl at pH 8 with an elution gradient length of 20 CV.

ed

• Addition of 1% PEG 10,000 into the mobile phase of IEC was found to have an adverse
pt

effect on the performance of purification while it was observed that the addition of potassium

thiocyanate or at a temperature of – 80°C was able to improve stability of HBsAg during storage;
ce

• For SEC, sample volume of 5 mL which corresponds to 0.04 CV and a linear flow rate of
Ac

30 cm/h can be applied.

• Established purification strategy in an up-scaled level provided an overall PF of more

than 94 with an improved recovery yield of HBsAg with an average 78% from the initial loading

of clarified disrupted sample.

25
 • The application and integration of IEC and SEC techniques were preferable for their high

selectivity and feasibility for automation and scaling-up. Thus, the two-step purification strategy

in HBsAg purification is undeniably a convenient alternative for an efficient process-scale.

t
ABBREVIATIONS

ip
cr
HBsAg Hepatitis B surface antigen
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IEC ion exchange chromatography

SEC size exclusion chromatography

Mut+ Methanol utilization plus

an
M
DCW Dry cell weight
ed

CV column volume

PF purification factor
pt
ce

ACKNOWLEDGEMENTS
Ac

The authors would like to thank and acknowledge the Ministry of Science and Technology of

Malaysia (MOSTI) for providing financial support under the research grant number 03-02-04-

0563-SR0008/05-02. In addition, we thank Universiti Putra Malaysia for providing the research

environment and necessary facilities.

26
 REFERENCES

(1) Lünsdorf, H.; Gurramkonda, C.; Adnan, A.; Khanna, N.; Rinas, U. (2011) Virus-like

t
particle production with yeast: ultrastructural and immunocytochemical insights into Pichia

ip
pastoris producing high levels of the Hepatitis B surface antigen. Microb Cell Fact, 10: 48.

cr
(2) Cregg, J.; Tschopp, J.; Stillman, C.; Siegel, R.; Akong, M.; Craig, W.; Buckholz, R.;
Downloaded by [RMIT University] at 11:42 25 January 2016

us
Madden, K.; Kellaris, P.; Davis, G. (1987) High-level expression and efficient assembly of

hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Nat. Biotechnol., 5(5):

479-485.

(3)
an
Bo, H.; Minjian, L.; Guoqiang, H.; Zhaoxia, L.; Zhenyu, Z.; Lin, L. (2005) Expression of
M
hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-

HBs antibody. J. Biochem. Mol. Biol., 38(6): 683.

ed

(4) Hardy, E.; Martínez, E.; Diago, D.; Diaz, R.; González, D.; Herrera, L. (2000) Large-
pt

scale production of recombinant hepatitis B surface antigen from Pichia pastoris. J. Biotechnol.,

77(2-3): 157-167.
ce

(5) Huang, Y.; Bi, J.; Zhang, Y.; Zhou, W.; Li, Y.; Zhao, L.; Su, Z. (2007) A highly efficient
Ac

integrated chromatographic procedure for the purification of recombinant hepatitis B surface

antigen from Hansenula polymorpha. Protein Expression Purif., 56(2): 301-310.

(6) Bardiya, N. (2006) Expression in and purification of Hepatitis B surface antigen (S-

protein) from methylotrophic yeast Pichia pastoris. Anaerobe, 12(4): 194-203.

27
 (7) Schaefer, S.; Piontek, M.; Ahn, S.-J.; Papendieck, A.; Janowicz, Z.A.; Gellissen, G.

(2001) Recombinant Hepatitis B Vaccines Characterization of the Viral Disease and Vaccine

Production in the Methylotrophic Yeast, Hansenula polymorpha in K. Dembrowsky, P.S. (Ed)

Novel Therapeutic Proteins: Selected Case Studies;VCH:Weinheim

t
ip
(8) Sánchez-Romeu, J.; País-Chanfrau, J.M.; Pestana-Vila, Y.; López-Larraburo, I.; Masso-

Rodríguez, Y.; Linares-Domínguez, M.; Márquez-Perera, G. (2008) Statistical optimization of

cr
immunoaffinity purification of hepatitis B surface antigen using response surface methodology.
Downloaded by [RMIT University] at 11:42 25 January 2016

us
Biochem. Eng. J., 38(1): 1-8.

(9) Antonsen, K.P.; Colton, C.K.; Yarmush, M.L. (1991) Elution conditions and degradation
an
mechanisms in long-term immunoadsorbent use. Biotechnol. Prog., 7(2): 159-172.
M
(10) Arnau, J.; Lauritzen, C.; Petersen, G.E.; Pedersen, J. (2006) Current strategies for the use

of affinity tags and tag removal for the purification of recombinant proteins. Protein Expression
ed

Purif., 48(1): 1-13.

(11) Ye, H. (2006) Simultaneous determination of protein aggregation, degradation, and

pt

absolute molecular weight by size exclusion chromatography–multiangle laser light scattering.

ce

Anal. Biochem., 356(1): 76-85.

(12) Bi, J.-X.; Zhou, W.-B.; Li, Y.; Huang, Y.-D.; Zhang, Y.; Dong, A.-H.; Su, Z.-G. (2005)
Ac

Polyethylene glycol accompanied ion-exchange chromatography to purify recombinant hepatitis

B virus surface antigen. Chin. J. Biotechnol., 21(6).

28
 (13) Zhou, W.; Bi, J.; Janson, J.-C.; Dong, A.; Li, Y.; Zhang, Y.; Huang, Y.; Su, Z. (2005)

Ion-exchange chromatography of hepatitis B virus surface antigen from a recombinant Chinese

hamster ovary cell line. J. Chromatogr. A, 1095(1): 119-125.

(14) Ottone, S.; Nguyen, X.; Bazin, J.; Bérard, C.; Jimenez, S.; Letourneur, O. (2007)

t
ip
Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for

in vitro diagnosis. Protein Expression Purif., 56(2): 177-188.

cr
(15) Belew, M.; Mei, Y.; Li, B.; Berglöf, J.; Janson, J.-C. (1991) Purification of recombinant
Downloaded by [RMIT University] at 11:42 25 January 2016

us
hepatitis B surface antigen produced by transformed Chinese hamster ovary(CHO) cell line

growth in culture. Bioseparation, 1(5): 397-408.

(16)
an
Yamamoto, S.; Miyagawa, E. (1999) Retention behavior of very large biomolecules in
M
ion-exchange chromatography. J. Chromatogr. A, 852(1): 25-30.

(17) Huang, Y.; Bi, J.; Zhou, W.; Li, Y.; Wang, Y.; Ma, G.; Su, Z. (2006) Improving recovery
ed

of recombinant hepatitis B virus surface antigen by ion-exchange chromatographic supports with

low ligand density. Process Biochem., 41(11): 2320-2326.

pt

(18) Tam, Y.J.; Zeenathul, N.A.; Morvarid, A.R.; Azmi, M.L.M.; Bahaman, A.R.; Lo, S.C.;
ce

Tan, J.S. (2014) Two-phase fed-batch modification for 48 hour peak expression of hepatitis B

surface antigen in Pichia pastoris shake flask system. Cent. Eur. J. Biol., 9(8): 749-760.
Ac

(19) Tam, Y.J.; Allaudin, Z.N.; Lila, M.A.M.; Bahaman, A.R.; Tan, J.S.; Rezaei, M.A. (2012)

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from

Pichia pastoris using response surface methodology. BMC Biotechnol., 12(1): 70.

29
 (20) Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature, 227(5259): 680-685.

(21) Morvarid, A.; Zeenathul, N.; Tam, Y.; Zuridah, H.; Mohd-azmi, M.; Azizon, B. (2012)

Effect of glycerol feed in methanol induction phase for hepatitis B surface antigen expression in

t
ip
Pichia pastoris strain KM71. Pertanika J. Sci. & Technol., 20(1): 31-42.

cr
(22) Kruger, N.J. (2002) The Bradford method for protein quantitation in Walker, J.M. (Ed)

The Protein Protocols Handbook;Humana Press

Downloaded by [RMIT University] at 11:42 25 January 2016

us
(23) Shrivastava, V.; Jain, U. (2009) A validated RP-HPLC method for estimation of hepatitis

(24)
an
B surface antigen (HBsAg) in bulk preparation. Orient. J. Chem., 25(3): 763.

Raksha, S.; Tan, W.S.; Hamid, M.; Ramanan, R.N.; Tey, B.T. (2014) A Single-Step
M
Purification of the Glycoprotein of Nipah Virus Produced in Insect Cells using an Anion

Exchange Chromatography Method. Sep. Sci. Technol., 49(2): 249-257.

ed

(25) Spadiut, O.; Rossetti, L.; Dietzsch, C.; Herwig, C. (2012) Purification of a recombinant

plant peroxidase produced in Pichia pastoris by a simple 2-step strategy. Protein Expression
pt

Purif., 86(2): 89-97.

ce

(26) Kutner, M.H.; Nachtsheim, C.; Neter, J.; Li, W. (2004) Applied linear statistical models,

5th Ed.;McGraw-Hill/Irwin:New York

Ac

(27) Hart, D.S.; Harinarayan, C.; Malmquist, G.; Axén, A.; Sharma, M.; van Reis, R. (2009)

Surface extenders and an optimal pore size promote high dynamic binding capacities of

antibodies on cation exchange resins. J. Chromatogr. A, 1216(20): 4372-4376.

30
 (28) Pessela, B.C.; Munilla, R.; Betancor, L.; Fuentes, M.; Carrascosa, A.V.; Vian, A.;

Fernandez-Lafuente, R.; Guisán, J.M. (2004) Ion exchange using poorly activated supports, an

easy way for purification of large proteins. J. Chromatogr. A, 1034(1): 155-159.

(29) Huang, Y.; Bi, J.; Zhao, L.; Ma, G.; Su, Z. (2010) Regulation of protein multipoint

t
ip
adsorption on ion-exchange adsorbent and its application to the purification of macromolecules.

Protein Expression Purif., 74(2): 257-263.

cr
(30) Yamamoto, S.; Ishihara, T. (1999) Ion-exchange chromatography of proteins near the
Downloaded by [RMIT University] at 11:42 25 January 2016

us
isoelectric points. J. Chromatogr. A, 852(1): 31-36.

(31)
an
Yamamoto, S.; Ishihara, T. (2000) Resolution and retention of proteins near isoelectric

points in ion-exchange chromatography. Molecular recognition in electrostatic interaction

M
chromatography. Sep. Sci. Technol., 35(11): 1707-1717.

(32) Gagnon, P.; Godfrey, B.; Ladd, D. (1996) Method for obtaining unique selectivities in
ed

ion-exchange chromatography by addition of organic polymers to the mobile phase. J.

Chromatogr. A, 743(1): 51-55.

pt

(33) Lu, X.; Zhao, D.; Ma, G.; Su, Z. (2004) Polyethylene glycol increases purification and
ce

recovery, alters retention behavior in flow-through chromatography of hemoglobin. J.

Chromatogr. A, 1059(1): 233-237.

Ac

(34) Mattanovich, D.; Graf, A.; Stadlmann, J.; Dragosits, M.; Redl, A.; Maurer, M.;

Kleinheinz, M.; Sauer, M.; Altmann, F.; Gasser, B. (2009) Genome, secretome and glucose

transport highlight unique features of the protein production host Pichia pastoris. Microb. Cell

Fact., 8(1): 29.

31
 (35) Tennikova, T.B.; Svec, F. (1993) High-performance membrane chromatography: highly

efficient separation method for proteins in ion-exchange, hydrophobic interaction and reversed-

phase modes. J. Chromatogr. A, 646(2): 279-288.

(36) Gibbs, S.J.; Lightfoot, E.N. (1986) Scaling up gradient elution chromatography. Ind. Eng.

t
ip
Chem. Fundam., 25(4): 490-498.

cr
(37) Frey, D.D. (1990) Asymptotic relations for preparative gradient elution chromatography

of biomolecules. Biotechnol. Bioeng., 35(10): 1055-1061.

Downloaded by [RMIT University] at 11:42 25 January 2016

us
(38) Tleugabulova, D. (1998) Sodium dodecylsulfate polyacrylamide gel electrophoresis of

an
recombinant hepatitis B surface antigen particles. J. Chromatogr. B Biomed. Sci. Appl., 707(1):

267-273.
M
(39) Wampler, D.; Lehman, E.; Boger, J.; McAleer, W.; Scolnick, E. (1985) Multiple

chemical forms of hepatitis B surface antigen produced in yeast. Proc. Natl. Acad. Sci. U.S.A.,
ed

82(20): 6830-6834.

(40) Zhao, Q.; Wanga, Y.; Freedb, D.; Fub, T.-M.; Gimeneza, J.A.; Sitrina, R.D.;
pt

Washabaugh, M.W. (2006) Maturation of recombinant hepatitis B virus surface antigen particles.
ce

Hum Vaccines, 2(4): 174-180.

(41) Gurramkonda, C.; Zahid, M.; Nemani, S.K.; Adnan, A.; Gudi, S.K.; Khanna, N.;
Ac

Ebensen, T.; Lünsdorf, H.; Guzmán, C.A.; Rinas, U. (2013) Purification of hepatitis B surface

antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their

immunogenic properties. J. Chromatogr. B, 940: 104-111.

32
 (42) Tleugabulova, D. (1998) Size-exclusion chromatographic study of the reduction of

recombinant hepatitis B surface antigen. J. Chromatogr. B Biomed. Sci. Appl., 713(2): 401-407.

(43) Lau, F.W.; Bowie, J.U. (1997) A method for assessing the stability of a membrane

protein. Biochemistry, 36(19): 5884-5892.

t
ip
(44) Tleugabulova, D.; Falcón, V.; Sewer, M.; Pentón, E. (1998) Aggregation of recombinant

cr
hepatitis B surface antigen in Pichia pastoris. J. Chromatogr. B Biomed. Sci. Appl., 716(1): 209-

219.
Downloaded by [RMIT University] at 11:42 25 January 2016

us
(45) Li, Y.; Bi, J.; Zhou, W.; Huang, Y.; Sun, L.; Zeng, A.-P.; Ma, G.; Su, Z. (2007)

an
Characterization of the large size aggregation of hepatitis B virus surface antigen (HBsAg)

formed in ultrafiltration process. Process Biochem., 42(3): 315-319.

M
(46) Ishihara, T.; Kadoya, T.; Yamamoto, S. (2007) Application of a chromatography model

with linear gradient elution experimental data to the rapid scale-up in ion-exchange process
ed

chromatography of proteins. J. Chromatogr. A, 1162(1): 34-40.

(47) Yamamoto, S.; Kita, A. (2005) Theoretical background of short chromatographic layers:
pt

optimization of gradient elution in short columns. J. Chromatogr. A, 1065(1): 45-50.

ce

(48) Ishihara, T.; Yamamoto, S. (2005) Optimization of monoclonal antibody purification by

ion-exchange chromatography: application of simple methods with linear gradient elution

Ac

experimental data. J. Chromatogr. A, 1069(1): 99-106.

(49) Carta, G.; Jungbauer, A. (2010) Effects of dispersion and adsorption kinetics on column

performance in Carta, G., Jungbauer, A. (Eds) Protein Chromatography: Process Development

and Scale-up;Wiley-VCH Verlag GmbH:Weinheim

33
 Tables

Table 1

IEC

t
ip
Properti Matrixe Mean Type of Charged Total Dynam Type of ligand

cr
es s cross particl medium group ionic ic
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linkage e size (anion) capacit binding

us
(%) (µm) y capacit

an (mmol

Cl-
y
M
/mL)
ed

ANX 4 45-165 Weak - 0.13- 43 g Diethylaminopro

Sepharo N+(C 2 H 5 ) 2 0.17 BSA/L pyl

pt

se 4 FF H

(high
ce

sub)
Ac

DEAE 6 45-165 Weak - 0.11- 110 g Diethylaminoeth

Sepharo N+(C 2 H 5 ) 2 0.16 HSA/L yl

se FF H

34
 Q 6 45-165 Strong - 0.18- 120 g Quaternary

Sepharo N+(CH 3 ) 3 - 0.25 HSA/L amine

se FF

t
ip
Q 6 45-165 Strong - 0.18- > 130 g Long quaternary

N+(CH 3 ) 3 -

cr
Sepharo 0.26 BSA/L amine

se XL
Downloaded by [RMIT University] at 11:42 25 January 2016

us
SEC

Properti Matrix Mean

an
Separati Column
M
es particl on range volume

e size (M r ) (mL)
ed

(µm)
pt

Superde Dextran 34 3 × 103-7 120

ce

x 75 covalent × 104

prep ly bound
Ac

grade to highly

cross-

linked

35
 Downloaded by [RMIT University] at 11:42 25 January 2016

Tables
Ac agarose

ce
pt
ed

36
M
an
us
cr
ip
t
 Table 2

IEC Columns pH

5 6 7 8 9

t
ip
AE (%)

cr
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us
ANX 27.53 50.18 51.78 57.78 57.71

Sepharose FF

(high sub) an
M
DEAE 35.60 53.35 54.04 78.25 73.45

Sepharose FF
ed

Q Sepharose 34.07 51.96 57.53 81.02 79.71

pt

FF
ce

Q Sepharose 37.31 52.04 60.44 84.47 82.04

Ac

XL

Tables

37
 Table 3

IEC NaCl (M)

Columns

t
ip
0.5 1 1.5 2

cr
EE (%) PF ± EE (%) PF ± EE (%) PF ± EE (%) PF ±
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us
SD SD SD SD

ANX

Sepharose
58.01

0.13
an
1.06 ± 88.24 1.27 ± 87.97

0.14
1.23 ± 87.65

0.05
1.15 ±

0.03
M
FF (high

sub)
ed

DEAE 57.26 1.29 ± 90.80 1.68 ± 91.70 2.36 ± 90.87 1.88 ±

pt

Sepharose 0.01 0.02 0.01 0.13

ce

FF
Ac

Q 51.49 1.11 ± 88.84 1.79 ± 91.01 1.85 ± 90.08 1.58 ±

Sepharose 0.05 0.03 0.11 0.02

FF

38
 Q 47.66 1.58 ± 86.69 2.53 ± 95.64 3.11 ± 95.04 2.86 ±

Sepharose 0.00 0.14 0.06 0.05

XL

t
ip
cr
Tables
Downloaded by [RMIT University] at 11:42 25 January 2016

us
an
M
ed
pt
ce
Ac

39
 Table 4

Purification Volume Total Total HBsAg Recovery PF

steps (mL) protein (g) HBsAg purity yield (%)

protein (%)

t
ip
(mg)

cr
Clarified
Downloaded by [RMIT University] at 11:42 25 January 2016

us
disrupted 157.75 ± 1588.41 ±

sample 12 32.89 72.24 1.01 100 1

an 1292.61 ± 16.86 ±
M
IEC 25 7.61 ± 0.05 51.02 16.98 81.38 1.87
ed

Centrifugal 1283.22 ± 1.12 ±

filtration 5 6.77 ± 0.01 41.92 18.95 99.27 0.18

pt
ce

1240.05 ± 5.04 ±

SEC 10 1.30 ± 0.07 50.43 95.48 96.64 0.38

Ac

94.82 ±

Total 78.07 16.20

40
 Downloaded by [RMIT University] at 11:42 25 January 2016

Ac
ce
pt
ed

41
M
an
us
cr
ip
t
 Downloaded by [RMIT University] at 11:42 25 January 2016

Ac
ce
pt
ed

42
M
an
us
cr
ip
t
 Downloaded by [RMIT University] at 11:42 25 January 2016

Ac
ce
pt
ed

43
M
an
us
cr
ip
t
 Downloaded by [RMIT University] at 11:42 25 January 2016

Ac
ce
pt
ed

44
M
an
us
cr
ip
t
 Downloaded by [RMIT University] at 11:42 25 January 2016

Ac
ce
pt
ed

45
M
an
us
cr
ip
t
 Downloaded by [RMIT University] at 11:42 25 January 2016

Ac
ce
pt
ed

46
M
an
us
cr
ip
t