3.0 Materials and Methods - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8542/10/10_chapter...

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3.0 Materials and Methods

3.1 General information:

The experiments were carried out in the R&D laboratory, Bharat

Biotech International Limited, Hyderabad, India.

3.1.1 Materials, Equipments and Reagents:

The strain Salmonella typhi (Ty2) was obtained from Dr. John

Robbins, National institutes of Child Health and Human Development

(NICHD), USA.

Fermentor (Bio Engineering, Switzerland)

Lyophilizer (FTS systems)

Steritest Canisters (Millipore)

GPC column (GE Health Care)

Sepharose CL-4B (GE Health Care)

Purified and Concentrated Tetanus Toxoid obtained from

P.T. Bio Farma, Bandung, Indonesia.

HPLC (Shimadzu systems)

Centrifuge (Sanyo)

pH meter (Polmon)

Colony Counter (Hi media)

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Magnetic stirrer (Tarson)

Orbital shaker

Tangential flow filtration system (Millipore)

Sartocon ultra filtration Cassette PESU (Polyethersulfone)

10kDa(0.1m²), 100kDa (0.6m²), 300 kDa (0.6m²), Sartorius

stedim

Spectrophotometer (Varian, Cary 50)

ELISA 96-well maxisorp (Nunc-immuno plate)

ELISA Reader (Lab systems)

Agape Diagnostics kit

(Anti-mouse–IgG) HRPO conjugate

Alkaline phosphatase labelled goat anti-human-IgG (Conjugate;

Jackson, Sigma)

4-nitrophenyl phosphate disodium salt hexahydrate (Substrate;

Fluka)

Reference Vaccine: Vi polysaccharide Typhoid vaccine

(TYPBARTM) from Bharat Biotech International Limited,

Hyderabad, India

LAL Test Kit (Cape Cod Inc)

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Dehydrated culture media (Hi media)

Analytical grade chemicals (Merck / Sigma)

3.1.2 Media and solution preparation:

All solutions required during the entire study were prepared at

R&D laboratory and the composition of all media and solutions were

prepared as follows:

3.1.2.1 Soyabean Casein Digest Medium (SCDM):

Suspend 30 g of SCDM in 1000 mL of WFI, sterilize by

autoclaving at 121.1º C for 15 minutes.

3.1.2.2 Xylose-Lysine Deoxycholate Agar (XLD):

Suspend 56.68 g in 1000 mL of WFI. Heat with frequent

agitation and boil the medium for complete dissolution. Do not

autoclave. Pour into sterile petriplates.

3.1.2.3 Triple Sugar Iron Agar (TSI):

Suspend 65 g in 1000 Ml of WFI. Sterilize by autoclaving at

121.1º C for 15 minutes. Pour into sterile test tubes.

3.1.2.4 Bismuth Sulphite Agar (BSA):

Suspend 52.23 g in 1000 mL of WFI. Heat with frequent

agitation and boil the medium for complete dissolution. Do not

autoclave. Pour into sterile petriplates.

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3.1.2.5 Tryptone Soya Agar (TSA):

Suspend 30 g of TSA in 1000 mL of WFI, sterilize by

autoclaving at 121.1ºC for 15 minutes. Pour into sterile petriplates.

3.1.2.6 Fluid Thioglycolate Medium (FTM):

Suspend 29.75 g of FTM in 1000 mL of WFI, sterilize by

autoclaving at 121.1ºC for 15 minutes. Distribute into sterile bottles.

3.1.2.7 50% Glycerol:

Take 50 mL of Glycerol, add 50 mL of WFI to it and sterilize by


3.1.2.8 Supplement mix:

Cobaltous chloride 0.260 g

Manganous chloride tetra hydrate 1.560 g

Copper sulphate 0.228 g

Boric acid 0.312 g

Sodium molybdate 0.218 g

Zinc acetate 1.536 g

Ferric citrate 5.20 g

Dissolve all the above chemicals in 500 mL of WFI and sterilize by


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3.1.2.9 Feed medium:

Supplement mix 125 mL

Magnesium sulphate 124.80 g

Calcium chloride 13.52 g

Glucose 4.50 g

Dissolve all the above chemicals in 8 L of WFI and make up to 9 L.

3.1.2.10 50% Ammonia solution:

Take 2.5 L of Ammonia solution, add it to 2.5 L of sterile WFI

and pass it through the 0.22 µ capsule filter.

3.1.2.11 Antifoam solution:

Take 100 mL of silicon antifoam, add it to 900 mL of sterile WFI

and sterilize by autoclaving at 121.1ºC for 15 minutes.

3.1.2.12 0.4M Cetrimide:

Cetrimide 134.0 g

WFI 1 L

Sterilize by autoclaving at 121.1ºC for 15 minutes.

3.1.2.13 1M NaCl:

Sodium chloride 58.60 g

WFI 1 L

Sterilize by autoclaving at 121.1ºC for 15 minutes.

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3.1.2.14 0.5M NaOH solution:

Dissolve 20g Sodium hydroxide in 1000 mL of sterile WFI and

sterilize by autoclaving at 121.1ºC for 15 minutes.

3.1.2.15 0.05M NaOH solution:

Dissolve 2 g sodium hydroxide in 1000 mL of sterile WFI and

sterilize by autoclaving at 121.1ºC for 15 minutes.

3.1.2.16 Ethanol:

Sterilize the ethanol using a 0.22 µ sterile capsule filter.

3.1.2.17 2% Sodium acetate solution:

Take and dissolve 20 g of sodium acetate in 1000 mL of sterile

WFI and sterilize by autoclaving at 121.1º C for 15 minutes.

3.1.2.18 0.4M Sodium carbonate/Sodium bicarbonate buffer

(pH 10.5):

Sodium bicarbonate 36.04 g

Sodium carbonate 5.04 g

WFI 1 L

Dissolve in WFI and sterilized by passing through 0.22 µ sterile

capsule filter.

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3.1.2.19 0.05M Cyanogen bromide:

Cyanogen bromide 5.2 g

Dissolve 5.2 g of Cyanogen bromide in 1L acetonitrile solution.

3.1.2.20 Adipic acid dihydrazide (ADH) in 1M Sodium bicarbonate

(pH 8.5):

Adipic acid dihydrazide 30.0 g

Sodium bicarbonate (1M) 1 L

Dissolve 30 g of ADH in 1 L of 1M Sodium bicarbonate and sterilize by

passing through 0.22 µ sterile capsule filter.

3.1.2.21 1M Sodium bicarbonate:

Sodium bicarbonate 84.01. g

WFI 1 L

Dissolve 105.99 g Sodium bicarbonate in 1L of WFI and sterilize by


3.1.2.22 Phosphate buffer (pH 7.2):


Di sodium hydrogen ortho phosphate dihydrate 13.7 g

Sodium dihydrogen ortho phosphate monohydrate 3.20 g

Dissolve in 1 L of WFI and Sterilize by autoclaving at 121.1ºC for

15 minutes.

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3.1.2.23 MES buffer (pH 6.1):

MES (2-morpholinoethanesulfonic acid) 21.33 g


Dissolve in 1 L of WFI and sterilize by passing through 0.22 µ filter.

3.1.2.24 EDC solution:

Dissolve 75 g N-(3-Dimethylaminopropyl)-N'-ethyl carbodiimide

hydrochloride (EDC) in 1 L of WFI.

3.1.2.25 0.1M Na2HPO4/NaH2PO4, 0.05M EDTA buffer (pH 8.0):

Disodium hydrogen ortho phosphate dihydrate 16.86 g

Sodium hydrogen ortho phosphate monohydrate 0.730 g

EDTA 1.86 g

Dissolve in 1 L WFI and Sterilize by autoclaving at 121.1ºC for 15

minutes.

3.1.2.26 0.1M Na2HPO4/NaH2PO4, 1.5M NaCl PBS buffer (pH 7.2):

Disodium hydrogen orthophosphate dihydrate 16.86 g

Sodium hydrogen ortho phosphate monohydrate 0.730 g

NaCl 88.0 g

Dissolve in 1 L WFI and Sterilize by autoclaving at 121.1ºC for 15

minutes.

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3.1.2.27 0.02M TRIS buffer (pH 7.0):

Dissolve 2.42 g of Tris buffer in 1 L of WFI and sterilize by


3.1.2.28 Alkaline hydroxylamine solution:

Mix equal volumes of 2 M Hydroxylamine Hydrochloride and

3.5 M sodium hydroxide.

3.1.2.29 10mM Potassium phosphate buffer (pH 7.5):

K2HPO4 1000 mL

KH2PO4 1000 mL

80 mL of 1M K2HPO4 and 19.8 mL of 1M KH2PO4 to be mixed and

made up to 100 mL with WFI, sterilize by autoclaving at 121.1ºC for

15 minutes.

3.1.2.30 Coomassie blue staining solution:

Coomassie blue 100 mg

Methanol 50 mL

Glacial acetic acid 10 mL

Mix the components and make up to 100 mL with WFI.

3.1.2.31 Destaining solution:

Methanol (10%) 10 mL

Glacial acetic acid (7%) 7 mL

Made up to 100 mL using WFI

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3.1.2.32 Reagent-A:

8.4 mL of 1 M Hydrochloric acid made up to 100 mL with WFI.

3.1.2.33 Reagent-B (pH 6.8):

Deoxycholate (1%) 100 mg

WFI 10 mL

pH to be adjusted using reagent- A.

3.1.2.34 Reagent-C:

0.1M Sodium hydroxide 0.40 g

WFI 1000 mL

3.1.2.35 Eluent-A (pH 7.2):

Disodium hydrogen orthophosphate dihydrate 1.37 g

Sodium dihydrogen orthophosphate monohydrate 0.32 g


Dissolve Disodium hydrogen orthophosphate dihydrate, Sodium

dihydrogen orthophosphate monohydrate and 8.8 g of sodium chloride

in 800 mL of WFI.

3.1.2.36 Physiological saline:

Dissolve 9 g of Sodium chloride in 1 L of WFI and sterilize by

autoclaving at 121.1ºC for 15 minutes.

3.1.2.37 Coating buffer:

1x PBS (pH 7.4) Filter the solution using 0.45 µ capsule filter.

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3.1.2.38 Washing buffer:

Sodium chloride 0.85%

Brij 35 0.1%

Sodium nitrite 0.02%

Filter the above solution using 0.45µ capsule filter.

3.1.2.39 Dilution buffer:

1x PBS (pH 7.4)

Brij 35 0.1%

BSA%


3.1.2.40 Substrate buffer (pH 9.8):

1M Tris 1000 mL

1M MgCl2 3 mL


3.1.2.41 Blocking buffer:

1 X PBS (pH 7.4)

1% BSA

Filter the above solution using 0.45 µ capsule filter.

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3.1.2.42 10mM Phosphate buffered saline (PBS) (pH 7.4):


Potassium dihydrogen phosphate 0.210 g

Disodium hydrogen orthophosphate 1.150 g

Potassium chloride 0.20 g

WFI 1000 mL

Sterilize by autoclaving at 121.1ºC for 15 mins.

3.1.2.43 Blocking solution:

Skimmed milk powder 1.0 g

10mM Phosphate buffered saline (pH 7.4) 100 mL

3.1.2.44 Wash buffer (Phosphate buffered saline-tween PBST):

Tween 20 0.5 mL

10 mM Phosphate buffered saline (pH 7.4) 1000 mL

3.1.2.45 Substrate buffer (0.05M Citrate-phosphate buffer):

0.2 M Disodium hydrogen phosphate solution 25.30 mL

0.1 M Citric acid solution 24.70 mL

WFI 50 mL

Orthophenylene diamine dihydrochloride 0.10 g

Hydrogen peroxide solution 100 µL (30%)

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3.1.2.46 Stopping solution:

2N Sulphuric acid

Take 805 ml of WFI in a 1 L glass bottle, to this add 196 mL of

sulphuric acid slowly through the walls of the glass bottle.

3.2 Methodology of Bioprocess:

3.2.1.1 Bacterial growth and characterization:

The cultre received from NICHD, USA was confirmed and

identified in our lab as Salmonella serovar typhi by identification of

the following characteristics:

Gram staining, glucose positive without gas formation, and H2S

positive on a XLD Agar, and positive serology with Vi polysaccharide.

The purity of the strain was confirmed on different selective

media such as Xylose Lysine Deoxycholate agar (XLD agar), Bismuth

Sulfite Agar (BSA), Triple Sugar Iron (TSI) agar.

3.2.1.2 Preparation of master seed lot:

Salmonella typhi Ty2 was grown on Soyabean Casein Digest

(SCDM) medium at 37±1ºC, for 12 hours. The bacterial culture was

centrifuged and the pellet was resuspended in sterile glycerol (50%).

0.5mL aliquots of the glycerol suspension in 1mL cryovials were

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prepared and stored at -70ºC.Viable cell count of the master seed was

also carried out.

3.2.1.2 Preparation of working seed lot:

The contents of cryovial of the Master seed lot was inoculated

into SCDM broth and incubated at 37±1ºC for 12 hours. The bacterial

culture was centrifuged and the pellet was resuspended in sterile

glycerol (50%). Viable cell count was carried out. Aliquots of the

glycerol suspension in cryovials were prepared and stored at -70ºC.

The master and working seed lots were characterized by grams

staining, utilization of glucose (Durham's method), oxidase test,

agglutination test and viable cell count. (1mL of the sample was

serially diluted up to 1010. This was plated on Tryptone Soya Agar

(TSA) and incubated at 37ºC for 48 to 72 hours. Colony count was

performed using colony counter).

3.2.2 Fermentation process:

3.2.2.1 Inoculum development:

One cryovial of the working seed lot was taken from the freezer

and thawed at room temperature using a water bath. The culture was

inoculated into 10mL sterile SCDM containing culture tube and

incubated in an orbital shaker (RPM 150±10) at 37±1ºC for 12 hours.

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Incubated culture was further equally transferred to 2 x 50 mL

medium containing flasks and incubated in an orbital shaker (RPM

200±10) at 37±1ºC for 12 hours and then transferred to 4 x 500 mL

medium containing flasks and incubated in an orbital shaker (RPM

250±10) at 37±1ºC for 12 hours. At every stage of the culture the

purity and morphological characteristics were checked by gram

staining and by microscopy.

3.2.2.2 Batch mode fermentation:

Table 3.1 Fermentation parameters and specification limits

Parameters Specification limits

pH 6.9±0.2

DO 70-90 %

Stirrer speed 250±10 RPM

Temperature 37±2ºC

Air flow 0.5±0.1 VVM

Eighty liters of SCDM was prepared in 100L S.S vessel and

transferred to the fermentor. This was sterilized in situ at 121ºC for 15

minutes. The medium was cooled to 37ºC by circulating chilled water

through fermentor jacket. At this stage sterile supplement mix was

pumped into the fermentor through the addition port. To maintain pH

and as a nitrogen source, 50% ammonia solution in a bottle was

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connected to the addition port of fermenter and set in auto mode. The

seed inoculum was transferred into the fermentor and the

fermentation process was continued accordingly with the above-

mentioned parameters up to 22 - 24 hours. The growth was checked

spectrophotometrically by taking the OD values at 600nm

periodically.

3.2.2.3 Fed batch mode fermentation:

To the early stationary phase culture the feed medium

containing carbon source along with inorganic salts and minerals was

pumped incrementally into the fermenter throughout the fermentation

process. The pH was maintained at 6.90 and dissolved oxygen level

was maintained between 40-60%. Ammonia solution (50%) was

supplied as a nitrogen source along with the feed medium. Foaming

was controlled by pumping antifoam solution through the addition

port aseptically. The growth was checked spectrophotometrically by

taking the OD values at 600 nm periodically. Process was continued

up to 22-24 hours and then the bacterial culture was inactivated with

0.5% formaldehyde and kept under mild stirring in chilled condition

(below 15°C) until it was processed.

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3.2.3 Downstream process:

3.2.3.1 Cell separation:

The harvested culture was centrifuged in a bowl centrifuge at

4200 rpm (8000g) for 30 minutes at 4ºC. The supernatant was

collected in sterile vessels. A sample was taken from the supernatant

and assayed for O-acetyl content.

3.2.3.2 Concentration and diafiltration:

The supernatant was diafiltered by using tangential flow

filtration (TFF) system using 100 kDa membrane. The supernatant

was concentrated to 1/10th of the original volume and further

diafiltered with water for injection (WFI) till the required concentrate

was obtained. O-acetyl content of the concentrate was assayed. The

cassettes were washed with 10 L of 0.5 M sodium hydroxide solution

and stored in 0.05 M Sodium hydroxide solution for further use.

3.2.3.3 Cetrimide precipitation:

To the concentrate 0.4 M cetrimide was added and incubated at

(5º±1ºC) for 3±1 hours. The contents were centrifuged at 4200 RPM

(8000 g) for 30 minutes at 4ºC. The pellet collected was suspended in

the required volume of 1 M NaCl. The O-acetyl content of the pellet

suspension was determined.

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3.2.3.4 Ethanol precipitation:

One volume of ethanol and 2% of sodium acetate were added to

the resuspended cetrimide precipitate; the contents were stirred for

20±5 minutes using a magnetic stirrer. Contents were centrifuged at

4200 rpm (8000 g) for 30 minutes at 4ºC. The supernatant was

collected into a sterile bottle and the pellet was discarded. To the

supernatant, two volumes of ethanol were added (100%) under

continuous stirring for a period of 60±10 minutes. 2% of sodium

acetate was added to the above content under continuous stirring.

After 1 hour of incubation, the contents were centrifuged at 4200 RPM

(8000 g) for 30 minutes at 4ºC. The supernatant was discarded; pellet

was suspended in sterile cool WFI and transferred to sterile bottle.

Sample was checked for O-acetyl content.

The ViPs bulk thus obtained was re-extracted with cetrimide

and precipitated with ethanol. Finally, the bulk was concentrated and

diafiltered using a 300 kDa cassette (known as concentrated bulk) as

mentioned above. The O-acetyl content was assayed after each

process.

3.2.3.5 Filtration:

The concentrated ViPs bulk was passed through 0.22 µ capsule

filter (Sartopore, Sartorius). This sterile filtered purified bulk of ViPs

was assayed for O-acetyl content.

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3.2.3.6 Lyophilization:

The purified ViPs bulk was dried under vacuum by subjecting it

to low temperature vaccum dryer. The ViPs powder was aseptically

transferred to sterile bottle for further analysis. The lyophilized powder

was tested for O-acetyl content, endotoxin content, protein content,

serological identification, moisture content, nucleic acid content and

molecular size distribution.

3.2.4 Conjugation process of Vi polysaccharide with Tetanus

toxoid:

3.2.4.1 Modification of Vi polysaccharide (ViPs):

The functional groups of Vi Polysaccharides were activated by

cynalating reagents with cyanogen bromide. 6.0 mg/mL of

Vi Polysaccharides was treated with alkaline buffer (0.4M Sodium

carbonate/bicarbonate buffer, pH 10.5). After 2 hours of mild stirring

5M cyanogen bromide was added and incubated at 2-8°C for

15 minutes. To this mixture, 0.8M adipic acid dihydrazide (ADH) in

1M sodium carbonate buffer was added and reaction was continued

upto 16 hours at 2-8°C. This reaction mixture was diafiltered against

phosphate buffer (pH 7.2) and MES buffer using a 10 kDa membrane.

The reaction volume was concentrated and assayed for ViPs content.

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3.2.4.2 Concentration and Diafiltration of Tetanus toxoid (TT):

Tetanus toxoid (8.0 mg/mL) was concentrated and buffer

exchanged against MES buffer using 10 kDa membrane cassettes.

The protein content was measured.

3.2.4.3 Coupling of modified Vi polysaccharide (Vi Ps) - protein

(TT) molecules:

The modified polysaccharides and protein molecules were

coupled under controlled conditions in the presence of EDC. The

reaction continued upto 4 hours under mild stirring at 2-4°C. The

reaction was terminated by addition of 0.1M Na2HPO4/NaH2PO4,

0.005M EDTA (pH 8.0) buffer. The reaction mixture was concentrated

and buffer exchanged through 0.1 M Na2HPO4/NaH2PO4 , 1.5M NaCl

PBS buffer, pH 7.2 followed by MES buffer.

3.2.4.4 Purification of coupled molecules:

The modified ViPs-protein conjugates were loaded in a gel

permeation column (GPC) to separate the conjugated molecules from

the unconjugated ones. The fractions were collected and analyzed for

Vi content, protein content and Vi/TT ratio. Fractions complying

within the Vi/TT ratio of 0.5-1.5 were pooled. The pooled fractions of

the conjugate were concentrated and buffer exchanged, using 10 kDa

membrane slice cassette, with 20 mM Tris buffer, pH 7.0. The final

conjugate bulk was labeled as ViPs-TT conjugate bulk. This was sterile

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filtered by passing through 0.22 µ filter membrane and stored at 2-

8°C. O-acetyl content, protein content, free protein, free Vi poly-

saccharide, and sterility were estimated in this sample. The molecular

size distribution of the ViPs-TT conjugate bulk was also determined.

3.3 Formulation and Filling:

The ViPs-TT conjugate bulk was formulated in physiological

saline and two batches were prepared. These were labeled as

Vi-TT/BB/01 and Vi-TT/BB/02. Each batch was further filtered

through0.22 µ filter and filled into the sterile 2 mL vials each

containing 0.5 mL of vaccine. The filled lots Vi-TT/BB/01 and

Vi-TT/BB/02 were analyzed for pH, O-acetyl content, Vi content, free

ViPs content and sterility.

Vaccine composition per dose/Single human dose (0.5 mL):

Purified capsular Vi polysaccharide of typhoid

(covalently Bound to Tetanus toxoid (ViPs - TT ) : 25 µg

Sodium chloride I.P. : 4.5 mg

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3.4 Quality control testing of Vi polysaccharide bulk, ViPs-TT

conjugate, Formulated and Filled final lots including mice

immunogenicity and challenge test in mice models:

3.4.1 Assay for O-acetyl content:

Determination of O-acetyl content was performed by the

method of Hestrin. (Hestrin, 1949). The amount O-acetyl in the

sample was proportional to the amount of Vi-content expressed in

mg/mL.

0.5 mL of 3.6 N HCl and 1 mL of alkaline hydroxylamine

solution were added to the test samples 3 mL and mixed thoroughly.

The mixture was kept at room temperature for 2 minutes and 0.5 mL

of ferric chloride solution added and mixed well. The absorbance was

measured at 540 nm. The O-acetyl content was calculated as follows:

O-acetyl (µmoles/mL) =

Test OD x Standard concentration

_____________________________________ x Dilution factor

Standard OD

Factor for O-acetyl to ViPs content conversion =

O-acetyl µmoles/mL x 0.294 (25/0.085/1000) = ViPs content (mg/mL)

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3.4.2 Endotoxin test:

The level of bacterial endotoxin of the vaccum dried samples of

ViPs was measured by LAL test kit. The quantities of endotoxins are

expressed in Endotoxin units (EU).

3.4.3 Serological test:

A precipitin reaction in agarose gel was performed by

Ouchterlony technique (Bailey, 1996). The gel was stained with 0.1%

solution of Coomassie brilliant blue in acetic acid, methanol and water

for 10 minutes at room temperature. The excess stain was removed by

washing the gel with the same solution without the dye.

3.4.4 Protein content:

The protein content in the samples was estimated by Lowry

method (Lowry et al., 1951).

3.4.5 Moisture content:

The moisture content in ViPs was checked by Karl fischer

method (Daniele et al., 2010).

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3.4.6 Nucleic acid content (European Pharmacopeia, 2007):

Nucleic acid was determined in ViPs by measuring absorption

at 260 nm assuming an extinction co-efficient of 20 for a solution of

1.0 mg of DNA/mL in a 1 cm cuvette.

3.4.7 Molecular size determination:

The molecular size of the samples was determined by using Gel

permeation column with Sepharose CL-4B as stationary phase. GPC

column with a length of 90 cm and 1.6 cm internal diameter was

equilibrated with Phosphate buffered saline (pH 7.2). 5 mg of

polysaccharide (ViPs) or polysaccharide-protein conjugate (ViPs-TT)

was loaded to the column and eluted at about 1mL/min. using a

peristaltic pump. 3-5 mL fractions were collected. The point

corresponding to the kDa 0.25 was determined as Vi polysaccharide

and the point corresponding to the 0.3 kDa determined as

polysaccharide-protein conjugate. The kDa Value of the fractions was

determined as follows:

kDa = Ve – Vo

Vt - Vo

Where:

kDa - Kilo Dalton

Ve - Bed Volume

Vo - Void Volume

Vt - Total Volume

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3.4.8 Quantification of free ViPs (DOC method):

To 75-100 µg of the ViPs-TT sample was added 80 µL of 1% w/v

deoxycholate (DOC) and incubated at 2°-8°C for 30 minutes. To this

was added 50 µL of reagent A and vortexed. Samples were centrifuged

at 5000 rpm (x1000g) for 45 minutes at 5°C. The supernatant and

pellet were separated. The supernatant was subjected to analysis of

O-acetyl content by Hestrin method and the pellet was dissolved in

0.1M NaOH.

The free ViPs content was calculated as follows:

ViPs content (mg) = O-acetyl content x 0.294

3.4.9 Detection of free protein by HPLC:

Free Protein was detected by using the HP-GPC column Shodex

columns Ohpak 805-804 HQ columns mounted in series. The HP-GPC

column was first equilibrated with Eluent A to a final isocratic flow

rate of 1 mL/minute. The blank was injected (Eluent A) to check for

the base line values. Samples were run in duplicates on the HPLC

column and the resulting peaks were integrated.

3.4.10 HPLC analysis:

ViPs-Protein conjugates were loaded into HPLC system

(Shimadzu) equipped with RI and UV detectors. ViPs, ViPs-TT

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conjugate and free protein were detected by analysing the

corresponding peaks.

3.4.11 ViPs/protein ratio:

The ViPs/Protein ratios were calculated.

3.4.12 Sterility test:

The sterility of the conjugate bulk and vaccine was determined

according to the British pharmacopeia - 2000, 2009 and WHO

Technical Report Series-530. The samples were filter-sterilized using

0.45 µ membrane. The steritest canister device was aseptically

inoculated into 100 mL of Fluid Thioglycollate Media (FTM) (for aerobic

and anaerobic bacteria), and Soyabean Casein Digest Medium (SCDM)

(for fungi and aerobic bacteria). FTM was incubated at 30°C - 35°C

and SCDM at 20°C- 25° C for 14 days.

3.4.13 pH of final vaccine:

The pH value of the vaccine was determined by a digital pH

meter.

3.4.14 Mice immunogenicity test:

Immunogenicity test was performed as per the method

described in British Pharmacopoeia-2000. In this study, female

BALB/c mice weighing 17-22 g were used. 32 groups of each

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containing 3 mice each were set up for the test plan. Eight groups of

mice each was allocated to the control group (Saline), Reference

vaccine and the two test vaccines (i.e., 4 x 8 groups each). The set if

eight groups received the same dose of vaccines/saline.

Vaccine containing 25 µg/0.5 mL (single human dose) was

diluted to 6.5 µg/0.5 mL i.e.,¼ dilution in case of reference vaccine

(TYPBARTM) and test vaccines (Vi-TT conjugate vaccines -Vi-TT/BB/01

& Vi-TT/BB/02) 0.5mL of these vaccines and 0.5 mL of saline were

injected subcutaneously into predetermined mice groups (4 x 8 groups

each) respectively; All the vaccine group mice were injected with

2 doses of reference and test vaccines on 0th day and 14th day and

saline group with saline on 0th day and 14th day. The animals were

bleed and sera obtained on 21st day for antibody evaluation by ELISA

method.

3.4.14.1 Enzyme linked immunosorbent assay (ELISA):

Microtitre plates (96-wells plates) were pre-coated with

tyraminated Vi polysaccharide typhoid antigen (10 µg/mL) diluted in

10 mM PBS. The ELISA plates were incubated at 2°C to 8°C overnight

for coating The contents of the microtitre plate were discarded and the

wells were blocked each with 200 µL of the blocking solution and

incubated at 25°C±1°C for 1 hour. The contents were discarded and

the wells were washed with wash buffer (Phosphate buffered saline

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tween, pH 7.4) to remove residual buffer in the wells. 50 µL of samples

(test, positive and negative control samples) were added in the

identified wells as per the test plan. 50 µL of 0.5% blocking solution

was added in all the sample wells and incubated under rocking at

room temperature for 1 hour. Contents of the wells were discarded

and 50 µL of detector antibody, (Anti-mouse–IgG) HRPO conjugate,

was added to each well. The plates were incubated at 25ºC±1ºC for

1 hour. The contents of the wells were discarded and the residual

buffer was also removed. To these wells, 100 µL of substrate buffer

(0.05 M Citrate-phosphate buffer) was added and incubated at

25ºC±1°C in dark for 10 minutes. The reaction development was

stopped by the addition of the stopping solution and the microtitre

plates were read at the absorbance of 492 nm.

3.4.15 Challenge test in mice model:

The 50% lethal dose (LD50) values were calculated according to

Reed and Muench (1938), using four different dilutions of S. typhi

culture to four groups of mice, each containing 10 per group.

The calculation was based on the number of survivors on day 15. The

LD50 was determined as 1 x 105.3 organisms of Salmonella typhi Ty2.

Healthy adult mice receiving varying doses of test vaccine

(Vi-TT/BB/01) and reference vaccine sample were challenged with

S. typhi cultures based on the LD50 detemined. Three dilutions

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(1/4, 1/8, 1/16) of test vaccine (Vi-TT) and reference vaccine

(TYPBARTM) were prepared in PBS and injected intraperitoneal to the

pre-labelled mice groups. Two doses of vaccine were given on 0 day

and 7th day. 10 mice were set aside for control test. All immunized

mice were challenged with S. typhi Ty2 strain at 20 LD50

(i.e., 20 x 105.3 LD50 organisms) intraperitoneally 14 days after the

second dose of vaccine. All the mice were observed for another 15

days. Deaths occurring after the 5th day were included for the

calculation. The calculation of the potency was done according to the

method developed by Reed and Muench (1938).

3.5 Pre-Clinical toxicity study:

3.5.1 Abnormal toxicity test:

Test for innocuity was performed as per the method described

in Indian Pharmacopoeia (2007). This test is also called the abnormal

toxicity test to detect other toxic substances in vaccine.

The Test vaccine Vi-TT/BB/01 and Vi-TT/BB/02 (ViPs-TT

conjugate), 25µg/0.5mL was injected intraperitoneally into 5 mice

weighing 17-22 g and 2 guniea pigs weighing 250 - 350 g. The animals

were observed for the abnormalities in health and activity for 7 days.

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3.5.2 Acute toxicity study:

This test evaluated the toxic characteristics of the final test

vaccine. Two groups of Swiss albino mice weighing about 17-22 g were

set up with 12 mice in each group. Group-I was set up as control

animals and injected subcutaneously with 0.1 mL of sterile saline.

Group II contained experimental animals, and injected with 0.05 µg in

0.1mL of test vaccine, (ViPs-TT conjugate) Vi-TT/BB/01.

Two groups of rabbits (1500 - 2000 g) were set up with 12 in

each group. Control group-I was injected intramuscularly with 0.5 mL

of sterile saline and group-II with 2.5 µg in 0.5mL of test vaccine

(ViPs-TT conjugate) Vi-TT/BB/01. All the animals (mice and rabbits)

were observed for 14 days for signs of bleeding, change in physical

appearance, body weight, food intake and mortality.

3.5.3 Systemic toxicity study:

Two groups of Swiss albino mice weighing about 17–22 g were

used with 20 mice in each group. Group-I was set up as control

animals and injected subcutaneously with 0.1 mL of sterile saline.

Group II was the test group and each animal was injected with

0.05 µg in 0.1 mL of test vaccine (ViPs-TT Conjugate) Vi-TT/BB/01.

Two groups of rabbits each containing 12 per group

(1500 - 2000 g) were also used. Group-I was control rabbits and

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injected intramuscularly with 0.5 mL of sterile saline; Group II was

injected with 2.5 µg in 0.5 mL of test vaccine (ViPs-TT Conjugate)

Vi-TT/BB/01.

All the animals (mice and rabbits) were given four doses of the

final test vaccine on 0th, 7th, 14th and 21st day and observed for 28

days. Body weight and food consumption of mice and rabbits were

determined. Blood samples were collected from the animal on 0th day

and 28th day to check the total WBC count and differential leucocyte

count. Serum was separated and the amount of aspartate

aminotransferease, alanine aminotransferease, alkaline phosphatase,

total protein, albumin, sodium and potassium chloride were checked.

The assay kit was procured from Agappe Diagnostics and the assays

were carried out as per the instruction manual. All the animals were

euthanised on 28th day and gross necropsy was carried out. This

systemic toxicity study was performed under the supervision of

qualified veternarian following Good Laboratory practice regulations

currently in effect and the Schedule-Y of the Indian Drugs and

Cosmetics Act, 1940 and these rules under. In addition the study

complies with the ‘OECD (Organisation for Economic Co-operation

and Development) Principles of good laboratory practices’.

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3.6 Human clinical trials:

A total of 95 subjects (age group 2-17 years) were enrolled for

human trials from various hospitals in Hyderabad, India; to assess

safety and the humoral immune response (Vi-IgG antibody level)

against the typhoid conjugate vaccine [Test vaccine (Vi-TT/BB/01)].

Typhoid Vi polysaccharide (TYPBARTM) vaccine was used as a reference

vaccine. Serum anti Vi-antibodies were measured by ELISA (Szu et.al.,

1994). The subjects were grouped as follows on the basis of their age

and administered varying doses of the vaccine.

Table 3.2 Study groups

Age Group

(Years)

No. of

Subjects Product Dose

13 - 17

15 Test vaccine 25 µg / 0.5 mL

10 Reference 25 µg / 0.5 mL

6 - 12

20 Test vaccine 25 µg / 0.5 mL

10 Reference 25µg / 0.5 mL

2 - 5

15 Test vaccine 25 µg / 0.5 mL as double

dose with 6 weeks interval

5 Reference

(TYPBARTM) 25 µg/0.5 mL

2 - 5

15 Test vaccine 15 µg / 0.5 mL as double

dose with 6 weeks interval

5 Reference

(TYPBARTM) 25 µg/ 0.5 mL

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Blood samples were collected from the candidates after the stipulated

period and serum was separated. The antibody titre in the serum was

determined.

Vi antigen was diluted so as to contain 2 µg/mL with coating

buffer. Microtitre wells were coated with 100 µL of the antigen and

incubated at 25°C overnight. The plate was washed with washing

buffer and shake dried. Pipetted 100 µL per well of blocking solution.

Covered tightly and incubated at 25°C for 2 hours. Washed the plates

twice with washing buffer, shaken to dry. Diluted serum samples

(1/10 to 1/2000) with dilution buffer. Pipetted 100 µL of dilution

buffer in all wells except for the first row (Row A). 200 µL of the diluted

serum sample was transferred to row A of the microtitre plate. This

was serially diluted from row A by transferring 100 µL to other rows

(B to H). Covered the plate tightly and incubated at 25°C overnight.

Washed plates twice and dried. Add 100 µL of 1:5000 diluted

conjugate IgG in dilution buffer to all wells and incubated at 37°C for

4 hours. Washed the plates twice and dried. Added 100 µL of freshly

prepared substrate (1 mg/mL in substrate buffer) to all rows and read

the plate at 405 nm within two minutes of time duration.

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