Materials and Methods 3.1 Materials - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/30489/12... · Materials and Methods 57 The present work is intended to produce vaccine

Materials and Methods

55

3.1 Materials

All chemicals employed were purchased from Sigma-Aldrich, Hi-

Media, Merck and Qual igens which were of analytical grade unless

stated otherwise.

3.2 Procurement of the HBsAg ‘S’ Gene containing Recombinant

Pichia pastoris strain

This HBsAg ‘S’ gene containing Recombinant Pichia pastoris

strain was developed by India Institute Of Sc ience (IISc), Bangalore.

Professor P N Rangarajan, Department of Bio-Chemistry, IISc is the

principle scientis t in this developmental ac tiv ity, In this technology

recombinant Wild type Pichia pastoris GS115 was used as host,

Fermentat ion and subsequent studies were performed using this

recombinant Pichia pastoris ( IISc , Bangalore) which is able to produce

the HBsAg product. This s train was having a plasmid pPIC3K of 9.0 kb

wherein HbsAg gene was around 680bp.

3.3 Pichia pastoris Expression System

Pichia pastoris, eukaryotic yeast has been developed to be an

outstanding host for the production of foreign proteins . Since its

alcohol oxidase promoter was isolated and c loned; its transformation

was first reported in 1985 (Cregg et al., 1987; Brier ley et a l., 1992).

Compared to other eukaryotic expression systems, Pichia offers many

advantages, because it does not have the endotoxin problem

associated with bacteria nor the viral contamination problem of proteins


56

produced in animal cell culture. Furthermore, P. pastoris can util ize

methanol as a carbon source in the absence of glucose. The P.

pastoris expression sys tem uses the methanol-induced alcohol ox idase

(AOX1) promoter, which controls the gene that codes for the

expression of alcohol oxidase, the enzyme which catalyzes the f irst

step in the metabolism of methanol. This promoter has been

characterized and incorporated into a series of P. pastoris expression

vec tors . As Yeast can be grown rapidly on simple media and to high

cell densi ty and its genetics are more advanced than any other

eukaryote, so that it can be manipulated almost as readily as E. coli.

As a eukaryote, yeast is a suitable host organism for the high-level

production of secreted as wel l as soluble cytosolic proteins . The

fermentation of genetically engineered P. pastoris prov ides an

excellent al ternative to E. coli expression systems. A number of

proteins have been produced using this system, including tetanus toxin

fragment, Bordatel la pertussis pertactin, human serum albumin and

lysozyme. (Chen et a l., 1996; Clare et a l., 1991; Cregg et a l., 1993;

Digan et a l., 1989; Tschopp et al., 1987).

3.4 Medium for growth of P. pastoris

Standard r ich medium for growth of P. pastoris was YP medium

(1% yeast extract, 2 % bacto-peptone) supplemented with carbon

sources to f inal concentrations of 2% dextrose (YPD), 0.5% methanol

(YPM), or 0.2% oleic acid or 0.02% Tween-40 (YPOLT). P. pastoris

cells were routinely cultured at 300C (Dannel et a l., 1993).


57

The present work is intended to produce vaccine agains t Hepatit is

B infection, which inc ludes the production of Hepatit is B Surface

Antigen (HBsAg) (Drug substance / Active Pharma Ingredient) on large

scale with maximum yield and purity, the detail of the work is presented

in the f low sheet.

.


58

3.5 Process flow-chart

Input Process Analytics

Gel Filtration

Chromatography

Resin: Toyopearl HW-65F

Ultracentrifugation For 20-24 Hrs

at 65,000 rpm and 40C

Phosphate buffer

saline, 0.22µm filter

Phosphate buffer

saline

CsCl, Pollyallomer

tubes

Sterile filtration By 0.22µm filter

Refractive index

Protein estimation

Protein, ELISA, DNA,

Lipid, carbohydrate,

purity by SDS-PAGE

& HPLC and sterility

Aerosil adsorption & Desorption

Adsorption for 6 Hrs Desorption at 370C

PEG Precipitation

Incubation for 10 Hrs

Temp.: 4-80 C

Ion-Exchange

Chromatography

Anion-exchange,

Resin: DEAE 650M

Ultra filtration 100 kDa cassette

Aerosil, Wash buffer,

Desorption buffer

Saline

Tris buffer, Saline

pH, conductivity,

test for HBsAg

pH, conductivity,

test for HBsAg

Test for HBsAg

Cell washing & Lysis Cell washing at 2-80C Lysis temp : < 200C

Lysis buffer,

Tween-20

5M NaCl, 50% PEG

OD600, Microscopy,

Test for HBsAg

Fermentation For 96 Hrs

Temp.: 300C, rpm: 500 pH: 5.0, DO: 40%

Fermentation media,

glycerol, methanolantifoam, alkali

and acid

OD600, morphology,

Methanol estimation

Pre-seed & Seed For 48 Hrs

Temp: 300C, rpm : 200

Working Cell Bank

Seed media OD600 ,

morphology


59

3.6 Western blot analysis of S-HBsAg

Culture f i ltrate around 100 ml was ground in 300 µL of Phosphate

Buffer Saline (PBS) supplemented with 0.5% Tween 20. A loading

buffer with Dith iothreitol (DTT), was added to the Culture f i ltrate and

the sample was incubated at 65oC for 15 min. The samples were

centr ifuged at 5000 rpm for 10 minutes. The pellet which was a protein

was col lected. Protein extracts were run on 12% polyacrylamide gel

and then blotted onto a nitrocellulose membrane (Bio-Rad). The blot

was blocked with 5% fat-free milk in PBS and then prote ins were

hybridized with 3000 × diluted polyclonal rabbit serum specif ic to S-

HBsAg. The 10000 × di luted anti-rabbit whole-molecule polyclonal

antibody conjugated with alkaline phosphatase (Sigma) was used as a

secondary antibody. Specif ic S-HBsAg bands can be v isualized after

the reaction with the Bromo-chloro-indolyl phosphate/ni tro blue

tetrazol ium (BCIP/NBT) substrate ((Sigma). Molecular weight of the

bands was estimated by using a pre-stained protein marker (Sigma)

(Laemmli, 1970).

3.7 Upstream Process for HBsAg production

3.7.1 Development of fermentation protocol for HBsAg production

3.7.1.1 Preparation of Pre-Inoculum

Pre-Inoculum was prepared in 100mL baffled flasks hav ing 20 mL

of ster ile glycerol medium. Glycerol medium was prepared in Mill i Q

water and contained: glycerol, 20 g/L; yeast nitrogen base, 13.4 g/L


60

and biotin, 400µg/L. Cells were grown at 300C on an orbi ta l shaker

(Multitron II, Infors A G) at 150 rpm for 48h (Ogata et al., 1969).

3.7.1.2 Preparation of Inoculum

1L of inoculum was prepared in 2L baffled f lasks hav ing 500mL of

ster ile glycerol medium using 10mL of Preinoculum. The inoculum was

prepared using Pichia shake-flask growth medium (Table.3.1). Glycerol

medium was prepared in Mill i Q water and contained: glycerol, 20 gL-1;

10X yeast nitrogen base, 10% by volume; Potassium phosphate

monobasic (anhydrous), 11.5 gL-1 and Potassium phosphate dibasic

(anhydrous), 11.5 gL-1. Cells were grown at 30 0C on an orbita l shaker

(Multitron II, Infors A G) at 150 rpm for 17h (Wegner, 1990).

Table. 3.1: Shake-flask growth medium

Component Weight or

Volume per litre

Potassium phosphate monobasic (anhydrous)

11.5 g

Potassium phosphate dibas ic (anhydrous)

2.7 g

Glycerol 20 g

10X YNB solution 10%

10 X YNB solutions cons ist of 67 g/L YNB without amino acids.

The solution was fi lter-ster il ized and added to other media components

after they were moist heat-ster il ized and cooled. The inoculum was

cult ivated for 40-48 hours at 28°C in a rotary shaker (NBS model G25)

running at 240 rpm. Opt ical Density at 600 nm (OD60 0) should be ≥ 8

and ≤ 12 at inoculation.


61

3.7.1.3 Shake flask Fermentation

The seed stage involved growing a frozen v ial (1 mL) of the cell

bank in 50 mL of media in a 250 mL shake f lask for 24 h in a shaking

incubator at 28 0C with agitation of 250 rpm. 4 mL of th is culture was

subsequently transferred to 396 mL of media in 2 L shake flasks and

grown for a further 24 h under the same condit ions. To seed a 50 L

fermentor, 8 of these 2 L shake flasks would be required.

The shake flask experiments were carried out in 500 mL baffled

f lasks (50 mL working volume). The minimum glycerol medium (1.34%

yeast ni trogen base, 2% glycerol, 0.0004% biotin) was used. The

culture was incubated at 30o C with shaking (300 rpm). At an

absorbance of 20 (OD), cells were harvested and re-suspended in

50mL minimum methanol medium (1.34% yeast nitrogen base, 0.0004%

biotin). Methanol (f ina l concentration was respectively at 0.5%, 1.0%,

1.5%, 2%, 2.5%, 3%) was added every 24 h during the methanol

induction phase. After 96 h induction, the supernatant was collected

and the quantitative analysis of recombinant Fab fragment was carr ied

out (Deng et a l. , 2005).

3.7.1.4 Pilot-scale (50L) production of HBsAg

Fermentat ion studies were performed at 50 L scale using Bio-

engineering Fermentor. Fermentation working volumes were set at 60-

80% and inoculation was performed with 5-10 % working volume of the


62

fermentor. For t imed-based induction, f i lter ster il ized methanol was

added for induction when the glycerol level had depleted. For auto-

induction, methanol was added together with glycerol at the start of

induction fermentation. Fermentation was carried out for a total of 48 to

90 h with pH maintained at 5.5 ± 0.1 with automated addit ions of acid

and alkal i (Ortho Phosphoric acid and Ammonia) Fermentation level

was monitored using a foam probe with automated addit ions of

antifoam. Measurements of temperature, pH, agitation speed, and

dissolved oxygen tension (DOT) and airf low rate were recorded

automatically using the Bio-command software (SCADA – System

Contro l and Data Acquisit ion) integrated to the fermenter. Regular

samplings were performed to allow of f l ine analysis of optical density

(OD) measurements at 600nm, wet cel l mass and dry cell weight

analysis (Joyce et al., 1995).

Following fermentation, cells were harvested using a High speed

centr ifuges (Beckman, USA) which has 6 X 1 L volume centri fuge

bottles and is operated in a batch mode. For cell harvesting, the

centr ifuge rotational speed was set at 8,000 rpm. Cel ls recovered in the

form of a solid paste were stored in polyethylene carboys at -80 0C.

3.7.1.5 Large scale (300L) Fermentation of Pichia pastoris culture

to produce HBSAg surface antigen

A standard 300L Fermentation equipment (Sartor ius) was used to

grow Pichia pastoris in a fed batch mode of fermentat ion. A Bio


63

Command Plus ® superv isory software was used to control the feed

schedule, achiev ing 100 g/L dry cell weight (DCW). Then, a Gas-Mix

Contro ller was added, and the fermentation was repeated with oxygen

supplementation of the sparge gas. This second run achieved a very

high dry cell weight of 160 g/L. Neither run was ful ly optimized, but the

descriptions of procedures and materials, as well as the data

discussion will be useful to operators of s imilar fermentors.

3.7.1.5a The Fermentor Vessel

Sartorius Fermentor was equipped with a heat-blanketed

( insulated) 300 L fermentation vessel with nominal 250 L working

volume. Al l fermentation vessels are configured with a 4-baffle

stainless-steel insert, dual Rushton agitation impellers, and a h igh-

speed, direct-dr ive agitation sys tem with mechanical face-seal.

Dissolved oxygen and pH probes (Mettler Toledo) are also included, as

are a variety of items such as liquid addit ion bottle kits (3), cables,

tubing and c lamps.

3.7.1.5b Control System

The four control modules included with the Advanced Fermentation

Kit were used for the Fermentat ion runs.


64

3.7.1.5c Control Set-points

Set-points were keyed into the controller pr ior to inoculation and,

except for DO which remained high until culture was introduced; the

vessel was allowed to equil ibrate prior to inoculation.

Temperature 30°C

pH 5.0

Dissolved Oxygen 40%

Agitation 100 - 500 rpm

(Responding automatically to oxygen demand)

3.7.1.5d Dissolved Oxygen (DO) Control

The DO probe was calibrated at 0%, (obtained by brief ly

disconnecting the cable), and at 100% (obtained using 500 rpm

agitation and 5 L/m (1 vvm) airflow. After calibration, DO remain

approximately at 100% until inoculation.

An agitation cascade was selected in the controller to maintain DO

at set-point through automatic adjustment of agitation speed. The

agitation cascade increases agitation speed with increasing oxygen

demand. To set up the cascade, the DO control display was used and

keypad on the PCU to select:

Cascade Agitation

Minimum RPM 100

Max imum RPM 500


65

3.7.1.5e Nutrient Feed

Init ial feed was 50% glycerol solution with 12 mL/L of trace metals.

Bio-Command began this feed automat ically when the dissolved oxygen

showed a sudden r ise above the set-point, a wel l-known carbon

exhaustion indicator. After all the glycerol was consumed, a brief

starvation phase was allowed, then changed the feed to 80% methanol

+ 20% Glycerol solution with 12 mL/L of trace metals solution.

Glycerol Pump 2 of the 4-Pump Module

Methanol Pump 3 of the 4-Pump Module

Transfer tubing: Sil icone tubing as supplied (1.4mm inner diameter

and 4.8mm outer diameter)

Vessel in let : Tr i-port adapter in the vessel head plate

Contro l Setup:

1) Pump 2 plugged into "Pump A" power-outlet of the Power Contro ller.

2) Pump 3 plugged into "Pump B" power-outlet of the Power Contro ller.

3) Pumps A & B: Manual mode, controlled by Bio-Command

3.7.1.5f pH Control

Liquid base was used to maintain pH at set-point, relying on the

acid-produc ing culture to lower pH if needed. The pH control

parameters were:

Base: Ammonium hydroxide, 30% solution

Pump: Pump 1 of the 4-Pump Module

Transfer tubing: Sil icone tubing,


66

Vessel in let: Tri -port adapter in the vessel head plate.

Contro l Setup:

1) Pump 1 plugged into "BASE" power-outlet of the Power Control ler

2) pH Control Selections : Mult iplier = 25%

Dead-band = 0

PID values: factory defaults

A feed control program was created using software, It turned on

the glycerol feed-pump when the dissolved oxygen (DO) level rose

above 60%. Each time DO exceeded 60%, the glycerol pump turned on;

each time it fell below 60%, the pump turned off. General ly 40% is a

high DO level, indicative of reduced metabolism due to carbon

exhaustion. The rationale for this s trategy is that the DO increases due

to reduced growth of the cells, which is a result of nutr ient depletion.

Approximately one hour after a r ise in DO which indicated depletion of

the supplementary glycerol, the program automatically turned on the

methanol feed-pump.

Table. 3.2: pH control parameters

Control Module Function

Primary Control Unit (PCU)

User interface for up to four vessels

Power Control ler Agitation and temperature contol ; power outlets

for f ive peristalt ic pumps

Four-Pump Module

Adding and remov ing liquids

pH/DO Controller Maintain ing dissolved oxygen and pH at

setpoints


67

3.7.1.5g Fermentation media evaluation

The fermentation media compos it ion and preparation methods were

given here for the production of HBsAg from recombinant Pichia

pastoris . In v iew of the poor cell growth observed using the Yau (2005)

media (Table.3.3), a second media originally developed for the large

scale production of the HPV VLP vaccine (Joyce et al., 1998), was

investigated for HBsAg production.

Table.3.3: Fermentation Medium components

Component Weight or Volume per litre

H3PO4 27 ml

CaSO 4. 2 H2O 0.9 g

K2SO4 18 g

MgSO4. H2O 15 g

KOH 4.13g

Trace Metals Solution 4.4 ml

Glycerol 40 g

Trace Metals Solution

Component Weight or Volume per litre

Cupric sulfate . 5 H2O 6.0 g

Sodium iodide 0.08 g

Manganese sulfate . H2O

3.0 g

Sodium mo lybdate 0.2 g

Boric acid 0.02 g

Cobalt chlor ide 0.5 g

Zinc chloride 20 g

Ferrous sulfate . 7 H2O 65.0 g

Biotin 0.2 g

Sulfur ic acid 5.0 ml


68

3.7.1.5h Feed Solutions

Glycerol Feed Solution: 50% glycerol with 12 ml/L trace metals solution

Methanol Feed Solut ion: 100% glycerol with 12 ml/L trace metals

solution

3.7.1.5i Fermentation Protocol

A frozen v ial of 1 ml P. pastoris sample was inoculated into a 1 L

shake flask with 150 mL Yeast Nitrogen Base (YNB)-glycerol medium. A

variety of genetically engineered P. pastoris strains were used, many of

which are slow growing on methanol (muts) and engineered to produce

proteins of interest. The culture was incubated at 30°C, 240 rpm, for 14

hours in an env ironmental incubator shaker (Remi Scientif ic). The entire

150 mL volume of inoculum was transferred to a 3.3 L fermentor vessel

( total volume) containing 1.5 L of basal salts medium and 4.4 mL/L trace

metal solution. The temperature was controlled at 30°C. The dissolved

oxygen was set at 30% and pH is at 5.0. Ammonium hydroxide solution

(30%) was used as the base solution to adjust the pH. After 20 hours of

batch culture, the optical density (OD) reaches 42. The glycerol fed-batch

process was then init iated. The feeding medium consisted of 50% glycerol

and 12 mL/L of trace metal solution. The feed rate was 24 mL/L/h, which

was adjusted automatically based on the DO reading. DO control was

maintained by the proportional integral derivative (PID) cascade controller,

which changes the speed of agitation. Pure oxygen was automatically

suppl ied to the fermentor to keep the DO level at the set-point after the

agitation speed reached the maximum allowable set-point. After the growth


69

phase, a half-hour carbon-source starvation period was established before

the culture was switched to the production phase.

The production phase (methanol feeding) was started after 43 hours of

cell growth. The production feed medium cons ists of 80% methanol and 12

mL/L trace metal solution. Feeding rates were div ided into three stages: 6

hr induction, 40 hr in a high-feed-rate stage and 10 hr in a low-feed-rate

stage. The feeding rate of the induction stage was ramped from 1 to 10.9

mL/L/hr, which was controlled by the computer program. Feeding rates in

the high and low rate stages were 15 and 2 mL/L/hr respectively. The total

volume of feed was 2 L. During the fermentation, oxygen demand can be

quite high and oxygen was added to the air stream automatically. pH is

usual ly adjusted to inhib it the ac tiv it ies of proteinases existing in the

culture broth during the production phase. Furthermore, since a host strain

that is protease defic ient was used, it was not necessary to change the pH

level when culture was shifted from cel l growth phase to production phase

(Table.3.4) (James et a l., 2000; Cereghino and Cregg, 2000).

Table.3.4: Fermentation Conditions Time (Hrs)

Stage Mode Feed Substance Feed Rate (ml/L/hr)

0-20 Growth Batch None NA

20-42.5 Growth Fed-Batch 50% Glycerol 24**

42.5-43 Starvation Batch None NA

43-49 Induction Fed-Batch 80% Methanol+20%

Glycerol

1-10.9***

49-90 Production Fed-Batch 15

90-100 Production Fed-Batch 2

* Al l feed solutions contain 12ml/L of a trace metal solution.

** Feed rate adjusted based on dissolved oxygen levels.

*** L inear ramp programmed v ia the Time Profi le.


70

3.8 Downstream Process for HBsAg production

3.8.1 Cell Harvest and Washing

At the end of fermentation, a sample was taken for testing

(Microscopic contamination check, A6 00nm, culture purity, and

homogeneity). If no contamination was observed, the fermentor broth

was cooled to 4 to 10oC. The cells are separated from the spent broth,

which were collected in a S. S. Sti r vessel in the form of a paste. The

cell concentrate was re-suspended in Wash Buffer (phosphate buffer

contain ing EDTA, pH 7.7 0.3) to give a final Cell Density, A60 0 = 400

20, maintained at 4 to 8ºC. A sample was taken for measurement of

pH, conductiv ity and A60 0 (Jose et al., 2004).

3.8.2 Homogenisation for Cell Disruption

Before homogenizat ion, the cell paste was resuspended in 0.1M

sodium phosphate, pH 7.2, with 0.5 M NaCl containing

phenylmethylsulfonyl f luoride (PMSF), a protease inhibitor, prepared in

to a concentration of 0.2M in isopropanol and

added to the phosphate buffer to achieve a f inal concentration of 2 mM

of PMSF. Cell paste to buffer ratio for resuspens ion was 1:3 (w/v). The

temperature of the machine was maintained at below 6oC by f lowing

glycol coolant through the external coi ls throughout the process.

Homogenisation was performed for 2-3 passes at 1200 bar unless

stated otherwise. To produce crude cell homogenate, the cells were

disrupted in a roller glass ball mil l. The ball mill chamber was

maintained at 2 to 4°C by pumping a refr igerant into the chamber jacket


71

throughout the cell breakage process. For cel l breakage, the f inal cell

suspension was treated with Tween 20 prior to pumping it in to the

roller mil l chamber. The temperature of the c rude cell homogenate

( lysate) coming out of the roller ball mil l chamber was monitored

continuously. Homogenate was stored in a s ti rred tank at 2 to 8°C

(Roster, 1992; Cross, 1999).

3.8.3 Detergent treatment for HBsAg liberation

In experiments for comparing the eff ic iency of different detergents

and chemicals for HBsAg l iberation, solutions of Triton X-100, Triton X-

101, Polysorbate 20 and 80 (PS20 and PS80) and CHAPS were

prepared in their typical operating concentrations of 0 - 1% v /v and

ethyl butyl ether (EB) in the range of 0 - 30% w/v in 0.01 M phosphate

buffer (pH 7.5). Yeast cell homogenate was added to 2 volumes of the

solutions prepared and these were allowed to mix for 2 h in a vessel.

The removal of cellular material was performed subsequently by

centr ifugation (Westfalia continuous centr ifuge) at 12,000 rpm with an

ejection t ime of 200 Seconds. The supernatant samples were recovered

for analys is (Kay et a l., 1988).

For all other studies, Triton X-100 detergent, which was found to

give the highest degree of HBsAg liberation, was employed. For

screening studies to determine the optimal working concentration of

Triton X-100, solutions of Triton X-100 in 0.01 M phosphate buffer (pH

7.5) were prepared in the range of 0 - 1% v /v.


72

3.8.4 Polyethylene Glycol (PEG) Precipitation and clarification

Crude cell homogenate was prec ipitated overnight using NaCl and

Polyethylene Glycol (PEG-6000). Cold 5 M NaCl solution was slowly

added under stirred condit ion to the crude cell homogenate followed by

the addit ion of cold 3% , 5%, 8% and 10% (w/v) PEG solution. Cell

debris and precipi tated proteins were removed from the PEG-crude cell

homogenate suspension by centr ifugation. PEG supernatant was

collected in a S.S. stir vessel. Approximately 2 to 8oC temperature were

maintained throughout the precipitation and centr ifugation steps. The

total volume of the PEG Supernatant was determined basing on the

Precipitation Time points at 3, 6, 10 and 12 Hours (Polson, 1968;

Giese, 2009).

3.8.5 Aerosil Adsorption and Desorption

To the PEG supernatant, aerosil suspension ( in wash buffer at 2 to

8oC) was added to give a f inal concentration of 1.5 % w/v and s ti rred

and then allowed to sett le. The aerosil sediment was then resuspended

in 0.9 % saline by agitat ion. The saline-washed aerosil was s ti rred

briefly and allowed to re-sediment. The aerosil sediment was

transferred to another vessel and Desorption buffer was added and

stirred for 4 Hrs at 37oC and fi ltered through Depth f i l ter to make it free

from any recombinant culture and aerosol (Yigzaw et a l., 2006;

Schirmer et a l., 2010).


73

3.8.6 Ion Exchange Chromatography (IEC)

The column (BPG 450–Pharmac ia) contain ing anion exchange

resin (Toyopearl DEAE 650M from TosoHaas) was used.

3.8.6.1 Cleaning and Sanitization of the column

The column was washed with 0.5 N NaOH solutions prior to

neutralis ing with Elution Buf fer-II (0.5M NaCl). Neutralisation was

carr ied out with loading buffer (50 mM tr is buffer) at a f low rate of 1.2

cm/min. The pH of the eluant should be identical to the pH of Elution

Buffer-II. The column was washed at a f low rate of 1.2 cm/min with ~2

column volumes of loading buffer until stable baselines for Absorbance

A28 0nm and conductiv ity were obtained.

3.8.6.2 Column loading

The fi ltered Desorbate (adjusted using 5 M NaCl to conductiv ity in

the range of 13-16 mS/cm) was loaded into the column at a f low rate of

1.2 cm/min. The flow through for the main IEC run was collected. The

column was washed with loading buffer until stable baselines for A28 0nm

and conductiv ity are obtained.

3.8.6.3 Elution step I

The prote in from the column was eluted with Elution Buffer- I at a

f low rate of ~1.2 cm/min in the form of a single A2 80nm peak. Elution

was continued until A280 nm reaches the baseline. About 2 column

volumes of Elut ion Buffer- I are necessary in th is elution step.


74

3.8.6.4 Elution step II

Further protein elution was performed with Elution buffer-II at a

f low rate of 1.2 cm/min until A28 0nm absorbance achieves a stable

basel ine (about 2 column volumes).

According to the difference of the DEAE l igand density, di fferent

elution gradients were optimized. The absorbent with a l igand density

of 0.130 mmol DEAE/ml absorbent was pre-equilibrated with buffer B

(20 mM sodium phosphate, pH 7.0) and eluted s tepwise with 11, 12 or

13% buffer C (20 mM sodium phosphate, pH 7.0, added 1.0 M NaCl)

and 100% buffer C in sequence. The other absorbents with lower ligand

density were pre-equilibrated with buffer B and then eluted stepwise

with lower gradients and 100% buffer C in sequence. The eluted

fractions were collec ted for the further analys is (Belew et a l., 1991).

3.8.7 Concentration by Ultra filtration

The ac tive fractions eluted from Ion-Exchange column under the

optimum condit ions were pooled and concentrated by ultrafiltration

using a 100 kDa molecular weight cross-flow filtration system hav ing

Biomax Pellicon ultra-f i ltrat ion cassette [media – polyethersulphone

(PES)],. The concentrated sample was stored at 4-80C for further

purification by Density gradient separation (Gonçalves et al., 2003;

Pinto et a l., 2006).


75

3.8.8 Density Gradient Ultra-Centrifugation

A 6 M ice-cold solution of Caesium chloride was slowly mixed with

concentrated IEC pool to give a f inal concentration of 1.5 M, and

loaded in Ultracentr ifuge tubes. The HBsAg partic les were separated

from contaminat ing proteins and lipids using an ultracentr ifuge

(Beckman) by centrifuging at 65,000 rpm for < 24 h, the tubes were

harvested by puncturing with a 18 gauge injection needle f i tted to a

syringe at the location of the brownish yellow band, corresponding to a

refractive index = 1.35 to 1.36 and a sample was withdrawn for analysis

(Wingfield et al., 1995; Zlotn ick et a l. , 1996).

3.8.9 Gel Filtration Chromatography (GFC)

The HBsAg from the CsCl pool was desalted using a Pharmacia

column with s ize exc lusion gel matrix (Toyopearl HW 65F - TosoHass)

Column sanitization

The column was sanit ized with 2 column volumes of 0.5 N NaOH

solutions. The column was neutralized with ~2 column volumes of

Desalt ing Buffer until the pH and the conductiv ity of the eluant show a

constant baseline value to those of desalting buffer.

Desalting

CsCl pool was loaded onto the column at a f low rate of <0.4

cm/min, followed by elution with desalt ing buffer. Separation of the

HBsAg partic les from the salt and other impurit ies was monitored by a

UV monitor and a conductiv ity meter. Eluted frac tions were collected


76

and HBsAg containing fractions were pooled. Exhaustive washing of the

column with Desalt ing buffer was done to remove traces of CsCl. The

column was cleaned with 2 column volumes of 0.5 N NaOH. A sample

from the GFC pool (containing the protein peak) was analysed (Chen et

al., 2007).

3.8.10 SDS-PAGE/Densitometry

SDS-PAGE is the most commonly used method for separation of

proteins based on size v iz. the higher the size, the lesser the migrat ion

in the gel. 20µg HBsAg (ultra or bulk) samples were loaded in each wel l

and the samples were for 1h. The gels were stained with coomassie

blue and were then scanned using densitometer. The intensity of

contaminating protein band(s) was calculated by measuring the optical

densi ty of each band using Biorad Quantity 1 software. BSA (1%, 2%,

and 3%) was used as a standard to determine the % contamination.

The intensi ty of contaminating bands was measured and compared with

intensi ty of BSA bands and relative % of contaminant protein was

calculated (Laemml i, 1970).

The concentration of non-HBsAg in the samples was determined by the

following equation:

The average OD of BSA (1%, 2% and 3%) X Average conc. of BSA

OD of Indiv idual non-HBsAg bands

The % purity of the HBsAg was determined by the following equation:


77

The quant i ty of HBsAg loaded in the wel l (20 µg) – the conc. of contaminant] X 100 Conc. of HBsAg (20 µg)

For discriminat ing HBsAg from non-HBsAg in various bulk samples,

western blott ing was performed (Sambrook et a l., 1989).

3.8.11 RP-HPLC

Reverse Phase (RP)-HPLC is a powerful tool for determining the

concentration and purity of macromolecules. The principle is based on

the hydrophobic interaction between the s tationary phase (column

material) and the analyte; the higher the hydrophobicity of the analyte,

the higher the retention t ime. Unlike SDS-PAGE and SE-HPLC where

separation depends on size (physical property), the separation is based

on the chemistry of the analyte (protein). Since no two proteins wil l

have s imilar chemistry, one can easily get good resolution and eff ic ient

separation of proteins .

In brief, the sample containing the analyte is loaded onto reverse

phase column in the presence of aqueous (water) mobile phase

contain ing small amount (5%) of organic (acetonitr i le) phase. With

t ime, there will be a gradual increase in acetonitr i le phase. As

mentioned earlier, different proteins (any macromolecules) wil l be

eluted with different RTs from the column depending on the

hydrophobicity (O'Keefe and Paiva, 1995).


78

Methodology

Column: Waters Symmetry C4, 150 mm Length X 4.6 mm Internal

Diameter, 300 Aº Pore Size.

Mobile Phase: Gradient, Mobile Phase-A: 0.1% TFA in degassed WFI

water & Mobi le Phase-B: 0.1% TFA in (100%) Acetonitri le.

Sample concentration : 0.5 – 1.0 mg/mL, Inj Vol: 1 - 200 µL. Flow

Rate: 1.0 mL/min. Wavelength: 220 nm / 280 nm

Samples Preparation: 320 µl of bulk sample (1mg/ml) sample pipetted

out into 1.5 ml of microfuge tube and mixed with 80 µl of 400 mM DTT

the samples were mixed thoroughly, subjected to heat treatment for 10

min by placing the microfuge tubes in water bath kept at 95oC. The

tubes were then centr ifuged for 5 min at 10000 rpm and 100 µg of

supernatant was used for analysis.

The following Gradient Run Program was optimized for analyzing

various HBsAg bulk samples.

3.8.12 Sterile Filtration of Bulk and Storage

Each lot of GFC Pool was steril ized by f i ltration in a Class A

laminar f low hood using dual membrane (0.8 / 0.2 m) absolute f i l ter

cartr idge (Type-Polyethersulfone) hav ing low protein b inding property.

The final HBsAg bulk after f i lter ster il ization was stored in 20 L

borosilicate glass bottles at 2 to 8 0C. The sterile f i ltered HBsAg was

tested for protein concentration, l ipid content, carbohydrate content,

DNA content, antigen content by ELISA and s teril ity.


79

3.8.13 Processing Philosophy

The movement of materia l throughout the entire process was done

via closed pre-sanit ized tubings into precleaned and sanit ized vessels.

All buffers and solution used in the process were either autoclaved or

f i lter ster il ized. All glasswares used were depyrogenized in the Dry

Heat Steri l izer. Any open handling procedure and aseptic operations

such as seeding of the cul ture, harvesting of ultracentr ifuge tubes and

sterile f i ltration through 0.2 m absolute f i lter were carr ied out in Class

A Laminar Flow Hood. The sterile f i lters used were tested for integrity.

Documentation of experimental data

The observations in each experiment were periodically recorded

and documented.

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