Title of Invention

PROCESS FOR PRODUCING A SUB UNIT VACCINE AGAINST SALMONELLA FEVERS

Abstract The present invention relates to a process for producing a sub unit vaccine against Salmonella fevers, typhoid and paratyphoid fevers. The process comprises a) fermenting in a semi synthetic liquid medium a highly virulent strain of Salmonella at a temperature between 36°C and 37°C, for a period that is a function of the amount of the inoculum. b) separating the bacterial residue from said fermented strain by centrifuging at moderately high speed. c) precipitating the vaccinating Vi-polysaccharide by in the presence of a precipitating solute of the kind such as herein described.. d) decanting by moderately high centrifuging for purification and dissolving in pyrogen free water and further precipitating with ethanol; e) decanting by moderately high centrifuging for purification and dissolving in pyrogen free water and further precipitating with cold phenol. f) precipitating the vaccinating Vi-antigen by ethanol, and dissolving the precipitation the physiological saline.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)



TITLE OF THE INVENTION
"PROCESS OF PRODUCING A SUB UNIT VACCINE AGAINST SALMONELLA FEVERS AND THE VACCINE SO PRODUCED"
We, CADILA HEALTHCARE LIMITED, a company incorporated under the Companies Act, 1956, of Zydus Tower, Satellite Cross Road, Ahemdabad 380 015, Gujarat, India.
The following specification particularly describes the invention and the manner in which it is to be performed.


PROCESS OF PRODUCING A SUB UNIT VACCINE AGAINST SALMONELLA FEVERS AND THE VACCINE SO PRODUCED
Field of the Invention:
The present invention relates to a process for producing a sub unit vaccine against Salmonella fevers, typhoid and paratyphoid fevers. The present invention also relates a sub unit vaccine against salmonella fever produced by the process of the present invention. Background of Invention/ Prior Art:
Typhoid fever caused by Salmonella typhi, remains a common and serious disease in many parts of the world. The capsular polysaccharide is an essential virulating factor and a protecting antigen ofS.typhi (1). Tacket et al.in J.infect.dis. 154:342-345( 1986) disclose a vaccine made from the Vi-capsular polysaccharide of Salmonella typhi. Field trials in Nepal and Republic of South Africa showed that a single injection of Vi-antigen confers about 70% protection against typhoid fever on old children and in adult (3,4).
The mechanism of protective action, similar to other polysaccharide vaccines, is to elicit a critical level of serum antibody. Bacterial capsular polysaccharide is a T-cell independent Antigen. T-cell independent antigens are characteristically of high molecular weight, with a repeating structure and very slowly degradation. These T-cell independent antigens give rise to predominantly IgM responses, some IgG3 in the mouse, and relatively poor, if any, memory cells.
The Vi-antigen is a capsular polysaccharide of salmonella typhi. It is liner homopolymer of (l-4)-alpha-D-GaIApNAC variably O-actylated at e.sub,3(fig.l) (1,5). Whitiriside and Baker in J.immunol.86:538-542(l96l) and I-andy et al.AM.J.Ilyg.73:55-56(1961) disclose that the)-acetyl groups on Vi-antigen is essential for its antignicity.
The inventors of present invention have identified and characterized a novel strain of Salmonella. They have also found a unique process to use this strain to manufacture vaccines. In this present invention, the use of Cetavlon in cold condition restores the O-acetyl group and this unique process of purification is very useful for purifying Vi-antigen having high value of O-acetyl content.
Objects of the Invention
It is an important object of present invention to provide a bacterial sub unit vaccine for use against Salmonella infections.
It is another important object of present invention to provide a bacterial sub unit vaccine for use against Salmonella infections which overcomes some of the disadvantages of the prior art.
2

It is yet another important object of present invention to provide a process to obtain an active sub-unit, Vi-polysaceharide vaccine with a long life and is easy to administer by injection.
Detailed Description of the invention
In the present invention a novel technique of fermentation, purification and extraction has been developed that makes it possible to obtain an active sub-unit, Vi-polysaccharide vaccine against salmonella fever affecting humans as effective as live vaccine, that is easy to pressurize with a long life and is easy to administer by infection.
Following schemes summarizes the process of the present invention: Scheme: 1 Production of working seed from master seed:




5
Scheme 2) Production of purified Vi-polysaccharide & Control:


Scheme: 3 Purification of the Vi-polysaccharide



ISOLATION AND CHARACTERIZATION OF NEW Salmonella typhi Ty2 STRAIN:
A new strain of Salmonella typhi isolated form a patient suffering from Typhoid
fever in India, is a unique high consistent Vi-producing strain, qualifies the requirement as
per WHO TRS 840 and British Pharmacopoiea for the production of Vi-polysaccharide
typhoid vaccine for commercial production.
SOURCE:
Isolated from patient suffering from typhoid in India.
CHARACTERIZATION:
Isolated culture was identified and characterized In-house and by National and
International agency:
(I) IN-HOUSE CHARACTERIZATION:
The strain is Gram negative showing typical morphology of Salmonella typhi in
Gram staining, motile (hanging drop method) with following biochemical characterization;
utilise glucose without gas production, oxidase negative, salicin negative, maltose positive,
lactose negative, xylose positive, sucrose negative, galactose negative. Agglutinate with
Vi-antiserum and translucent colonies when plate on MacConkey agar plate. Strain was
also characterized by Biolog Microplate using GN2 Microplate which confirm the Strain as
Salmonella typhi by l00(PROB).
7

Strain identified and characterizes as Salmonella typhi
(II) INSTITUTE OF MICROBIAL TECHNOLOGY (IMTECH), CHANDIGARH-INDIA
CHARACTERIZATION:
The said strain was characterize by IMTECH, which is one of the Indian leading microbial institution observed as
Colony morphology: Configuration-round, Margin-entire, Elevation-convex, Surface-smooth, Density-transparent, pigment-white.
Gram's Reaction: Gram negative, shape-rods, size-short like coccobacilli, arrangement-single, nonendospore forming, motile and Fluoresence (UV) negative. Growth at temperatures: 4°C - negative, 10°C - negative, 15°C - negative, 22 C - negative, 26°C - positive, 30°C - positive, 37°C - positive, 42°C - positive/negative, 55°C - negative, 65 C - negative.
Growth at pH: pH5.0-positive, pH5.7-positive, pH6.8-positive, pH 8.0-positive, pH9.0-positive, pHl 1.0-positive.
Growth on Nad (%): 2.5-positive, 5.0-positive, 7.0-positive, 8.5-negative, 9.0-negative, 10.0-positive. Growth under Anaerobic condition-positive
Biochemical test: Growth on macConkey agar -lactose nonfemienter, Indole-negative, Methyl red-positive, Voges Proskauer test-negative, Citrate utilization-negative, Casein hydrolysis-positive, Starch hydrolysis-positive, Urea hydrolysis-negative, ONPG hydro lysis-negative, Nitrate reduction-positive, Nitrite reduction-negative, H2S production-positive, Cytochrome oxidase test-negative, Catalase test-positive, Gelatin liquefaction-posilive/negalive, Arginine dihydrolase-positive/negative, Lysine decarboxylase-posilive, Orinithinedecarboxylase-positive/negative.
Acid Production from carbohydrates: Adonito I-negative, Arabinose-negative, Cellobiose-positive, Dextrose-positive, Dulcitol-positive, Fructose-positive, Galactose-positive/negative, Inositol-negative, Insulin-positive, Lactose-positive/negative, Maltose-positive, Mannitol-positive, Mannose-positive, Melibiose-posirtive, Raffinose-positive, Rhamnose-positive, Salic in-negative, Sorbitol-positive, Sucrose-negative, Trehalose-positive, Xylose-positive.
The strain confirmed as Salmonella typhi and preserved as MTCC No.: 3917. (III) STATENS SERUM INSTITUTE, DENMARK (WHO GLOBAL SALMONELLA-SURVEILLANCE CENTRE):
This strain was characterize by Statens Serum Institute, Denmark (WHO Global Salmonella-Surveillance Centre) as Salmonella typhi Ty2
8

Serotyping: -: Vi: D: -. The stain expresses the Vi-antigen so strongly, that the somatic
antigen (O: 9,12) is not expressed neither any R phase antigens expressed in the strain.
Biochemical test: Gram negative, oxidase negative rods and strain utilize glucose without
production of gas. The strain is further maltose-positive, lactose-negative, saccarose-
negative, cellobiose-negative, trehalose-positive, Sorbose-negative, arabinose-negative,
xylose-positive, rhamnose-negative, adonitol-negative, dulcitol-negativc, sorbitol-positive,
mannitol-positive,salicine-negative, inositol negative, Glycerol -positive, melibiose-
positive, raffinose-negative, galactose-positive, fructose-positive, mannose-positive.
Susceptibility test: susceptibility test carried out by use of Rosco tablets on Danish
blodagar.the strain is fully susceptible to streptomycin, Gentamycin, Tetracycline,
Ampicillin,sulphonamide,Mecillinam,Nalidixin,chloramphenicol,spectinomycin,ciprofloxa
cin,apramycin,ceflriaxone,kanamycin,nitroforantoin,Trimethoprim,fosfomycin and
polynyxin.
Riboprint: The strain was cut with PVUI1 and belongs to ribogroup 157-I445-S-2. The pattern was different from the two other Salmonella typhi in their PVD database.
Pulsed Field Gel electrophoresis: Strain molecular typed by PAGE have shown clearly distinguishable pulsed field, when compared to other salmonella typhi in SSI database even though a number of bands in the pattern are seen in all the typhi-in-dictating a common ancestor link-some of the bands in the PAGE patterns in the strain are unique.
Process for production and purification of Vi-polysaccharide
In the first step of the production process of the present invention, the working seed id prepared from master seed lot. The preparation of master seed and working seed is summarised in Scheme 1. One vial of seed is suspended in 0.2ml of S.C.D medium. The master seed lot is obtained by inoculating the suspension on 20ml S.C.D broth and incubating it for 24-48 hours at 37°C.The master seed lot undergoes various tests. Under QC/QA analysis. Following this working seed lot is prepared by inoculating 0.5 ml of the master seed lot on 20 ml of S.C.D broth at 37°C for 24 hours. The working seed lot undergoes further QC/QA analysis before it is approved and is ready for use. Following this the Production of Vi-polysaccharide is carried out
• The fermentation is carried out using the seed lot principle. In this, each production run is initiated from a vial of Salmonella typhi working seed lot.
• The working seed is produced by the method as explained hereinbefore.
• The process of production involves a transfer of matured seed lot to the production
fermentor,
The time of transfer depending upon the OD of the growth.
9

• Volume to be transferred depends upon the size of the production lot.
• Fermentation is preformed in a fermentor using a semi-synthetic broth medium having composition that is known to the people skilled in the art.
• Fermentation is preformed in a fermentor using a semi-synthetic broth medium having following composition:

S.no Name of component Quantity (in gram)
1 Yeast Extract 5.0
2 Glucose 10
3 Na2HP04 2.5
4 KC1 0.09
5 NaCI 6.0
6 L-glutamine 0.05
7 L-cystein 0.01
8 MgSo4 0.05
9 DL-tryplophan 0.01
10 NR4C1 1.25
• The fermentation is carried out at appropriate temperature in the range of 36 C to 37°C with an aeration.
• The pH during the fermentation is maintained between 7.3 to 7.5 by using sterile NaOH.
• The aeration and agitation is controlled to achieve the dissolved oxygen levels of 50% to 65% of saturation..
• A backpressure of 0.2 is maintained throughout fermentation to facilitate the oxygen transfer and to control the foaming.
• Samples are drawn at regular intervals throughout the exponential growth phase, for checking identity, pH, OD, reducing sugar and microbial purity.
• The total duration of the fermentation is 24 hrs and final OD is around 5.0 at 600nm.
• The fermentation batch is terminated by immediate inactivation using formaldehyde at a concentration of 0.05% for 18 hours in cooling condition of 2°C to 8°C and is checked for inactivation before going ahead in purification. The content of the fermentor is transfer
10

to another sterile tank under sterile condition and is maintained at a temperature of 2 C to
8°C.
The overall summary of Purification of Vi-polysaccharide process is given in scheme: 3
• Formaline inactivated broth is centrifuged in cooling condition at moderately high speed to separate the liquid and bacterial residue, which is discarded.
• The liquid portion is precipitated with a solution of Cetavlon (0.01% to 0.05%), the solute is used in a weak concentration and a pH is adjusted between 7.0 to 7,5 with a strong acid. The process is performed at lowered temperature 0°C to 4°C, to avoid any enzymatic or microbial development.
• Precipitates of the antigen and Cetavlon are collected by centrifugation at a moderately high speed, which is subsequently dissolved in pyrogen free water.
• The dissolved material is subjected to ethanol extraction for 18 hours at 0°C to 4°C.
• Centrifugation at moderately high speed is done to separate, the supernatant liquid and further treated with the ethanol (final concentration of 80°C taking the precipitates dissolved again in water for injection.
• Dissolved material is subjected to cold phenol extraction. Phenol is used in saturated concentration as sodium salt under cooling condition (4°C to 8°C).
• The phenol extracted Vi-polysaccharide is further purified by precipitation using ethanol for 18 hours at 0°C to 8°C.
• Precipitates are separated by centrifugation and are dissolved in physiological saline, the resulting solution is the purified bulk having the Vi-polysaccharide. The vaccinating antigen fraction produced has a molecular weight of nearly 3,50,000 and is used to vaccinate humans.
• Later quality control testing is performed and it has been ascertained in in-house testing using standard, generally accepted methods that the batches manufactured according to the present scheme pass the criteria according to WHO guidelines Ref: TRS.840, page 14-27 & British pharmacopoeia.
Following test are performed to ascertain the quality of the vaccine produced.

S.n0 Name of test Reference:
1 Protein content (see annuxer-1)
2 Vi-polysaccharide content (see annexuer-2) ,
3 Nucleic acid content (see annexure-3)
II

4 Phenol content (see annexure-4)
5 Serological identity (see annexure-5)
6 O-acetyl content (see annexure-6)
7 Molecular size determination (see annexure-7)
8 pH (see annexure-8)
9 Clarity (see annexure-9)
10 Pyrogen testing (see annexure-10)
11 Abnormal toxicity (see annexure-11)
12 Endotoxin content (see annexure-12)
The overall results of five consecutive batches of production are indicated in the following
Tables:
TABLE-1
Determination of protein content of the purified Vi-polysaccharide.
Determination by Lowry's method. Each lot contain less than 10 mg of protein content per gram of polysaccharide.
The protein reacts with Folin-Ciocalteau reagents to give colors complex. The color so formed is due to the reaction to protein material. The standard reagents are used in the process of protein determination.
I) Reagents

S.No Name of reagent Concentration /description
A Alkaline sodium carbonate solution 20g/l Na2C03 in 0.1 M/I NaOH
B CuS04-sodium potassium tartarate solution 5g/l CuS04.5H2O in l0g Na-K tartarate
C Alkaline solution Mixture of 50ml of (a) and l ml of(b)
D Foline- Ciocalteau reagents 50% diluted solution
E Standard protein BSA solution of 1mg/ml
Add 5ml of the alkaline solution to I ml of the solution. Mix thoroughly and allow to stand for l0 min or longer. Add 0.5ml of the diluted Foline- Ciocalteau reagent rapidly with immediate mixing. After 30 mins. Read the extinction against the appropriate blank at 600nm.
12

Estimate the protein content of the unknown solution after preparing a standard curve from the standard Bovine serum albumin solution. Results

BatchNo. Mean OD of sample at 600nm Mean OD of blank with phenol at 600nm Protein content (mg/ml)
1A 0.310 0.324 Nil
2A 0.275 0.333 Nil
3A 0.334 0.375 Nil
4A
5A
TABLE-2
Determination of the Vi-polysaccharide content of purified Vi-polysaccharide
Determination by Rocket immuno electrophoresis (RIE).
The Vi-polysaccharide (antigen) reacts with the antibody and form precipitin rockets.
The height of the precipitin rocket is proportional to the antigen concentration and thus Vi-content can be calculated by interpolation from height of the precipitin rocket of reference material. Reagents: The standard reagents are used in the process of RIE are:

S.No Name of reagent Concentration /description
A Barbitone buffer 0.15MpH8.6
B Agarose solution l%w/v
C Agarose solution 4% w/v
D Vi antiserum Polyclonal whole serum
E Standard Vi-polysaccharide Of known concentration
F Phosphate buffer saline pH7.2
Procedure:
1 On a glass slide, over lay 1% agarose with 4% agarose solution, which has 12.5% Vi antiserum, mixed with Barbitone buffer.
2 Bore 2mm wells in the agarose and fill them with 3μL of the test solution and that of reference standard.
13

3 Mount the glass plant on the horizontal electrophoresis unit and run the apparatus for three hours at 10-15 mA.
4 Measure the height of the precipitin rocket after 4 hours of storage of the plate in phosphate buffer saline at 4-8°C.
Results:

Batch No. Vi- content (mg/ml)
1A 1.13
2A 1.29
3A 1.29
4A 1.13
5A 1.00
TABLE-3
Determination of the Nucleic acid content of purified Vi-polysaccharide:
Each lot of purified Vi-polysaccharide shall contain less then 20mg of nucleic acid per gram of polysaccharide.
The final diluted sample is analyzed for nucleic acid content by measuring absorbance at 260nm where 1 O.D is equivalent to 50 μg/ml nucleic acid. Results:

Batch No. Nucleic acid - content (mg/gram of Vi polysaccharide)
1A Nil
2A 5.5
3A Nil
4A Nil
5A 18.5
TABLE-4
Determination of the phenol content of purified Vi-polysaccharide
The standard reagents are used are:

S.No Name of reagent Concentration /description
A Phenol standard solution 1 mg/ml
14

B Borate buffer pH:9.0
C Potassium ferricyanide lgm/l00 ml
D Aminoantipyrine solution 0.lgm/ml in buffer(pH:9.0) Borate
Procedure:
1: Mix 1.5ml of test solution with 1.5 ml of borate buffer.
2: Add 1.5 ml of Aminoantipyrine solution and 1.5 ml of potassium ferricyanide to the
above mixture.
3: Allow the mixture to stand at room temperature for ten minutes.
4: Take the OD of the resulting solution at 546 nm.
5:Calcultae the phenol content from a calibration curve prepared from 1.5 ml quantities of
series of the solutions of phenol containing 5 μg, 10μ.g, I5μ,g, 20μg, 30μg of phenol per
ml., treated in the same manner as mention in above mentioned steps.

Results:
Batch No. Phenol - content (ug/ml)
1A 625.00
2A 547.60
3A 651.33
4A5A 693.66
702.33
TABLE-5
Determination of the Serological identity of purified Vi-polysaccharide
The serological identity of the Vi-polysaccharide is determined by Ouchterlony immuno-double diffusion assay.
Reagents:
The standard reagents used in the process of Ouchterlony immuno-double diffusion assay are:

S.No Name of reagent Concentration /description
A Agarose solution l%w/v
15

B Vi antiserum Polyclonal whole serum
C Standard Vi-polysaccharide Of known concentration
D Phosphate buffer saline pH7.2
Procedure:
1: On a glass slide, lay 1 % agarose solution.
2: Bore 2mm wells in the agarose at the corners of the imaginary hexagon and 5mm bore at
the center of the hexagon.
3: Fill 200μL of Vi-antiserum in the central bore and 40 μLof test solution in the bores at
the corners of the hexagon.
4: Incubate the plate at 2-8°C for 24 hours in a wet chamber having phosphate buffer saline.
5: Appearance of the full regular hexagon of the pacipitin around the central bore is
indicative of the serological identity of the Vi-polysaccharide.
Results:

Batch No. Serological identity
IA Identical
2A Identical
3A Identical
4A Identical
5A Identical
TABLE-6
Determination of the O-acetvl content of purified Vi-nolvsaccharide
O-acetyl group react with hydroxlyamine in alkali pH to form hydroxamic acid. The Hydroxamic acid formed is measured by the formation of a purple brown complex with Fe3+ in acidic pH. Standard reagents are used in O-acetyl group determinations are:

S.No Name of reagent Concentration /description
A Hydroxylaminehydrochloride(cold) 2mol/L
B Sodium hydroxide 3.5 mol/L
C Hydrochloric acid Concentrated
16

D Ferric chloride hexahydrate in 0.l mol/L HCI 0.37mol/L
E Standard Solution of acetylcholine chloride Relative molecular 181.7 wt:
F Sodium acetate 0.001mol/L,pH:4.5
Procedure:
1 : Preparation of Standard series of Standard Solution:
Prepare a standard series of standard solution of acetylcholine chloride appropriately diluted with 0.001 sodium acetate to make final concentration of O-acetyl of 0.83, 1.66, 2.48, 3.32, 4.15 micromol/ml.
2 : The test solution having concentration of 1.0 G/L of Vi-polysaccharide in water is to be
used.
3: Mix equal parts of reagent A & B and add 2ml of it to 1ml of test solution and standard
series.
4: Add 1ml HCI .and regent D to all test tubes if step 3.
5: Blank solution is prepared for both test solution and standard solution by adding HCI
before adding NaOH to hydroxylamine hydrochloride solution.
6: Take absorbance of die resulting solutions of test, standard and blank solutions at a
appropriate wave length at which net absorbence between 520nm and 540nm is highest.
7: Subtract the sample blank reading from that of the test sample and subtract the standard
blank reading from those of the standard dilution readings.
8: From the absorbance obtained from the sample, the corresponding number of
micromoles of O-acetyl per Milligram of Vi-antigen can be read out from the reference
curve.
Results:

Batch No. O-acetyl content (millmole/gram Vi-antigen)
1A 5.95
2A 6.25
3A 5.96
4A 6.38
5A 6.48
TABLE-7
Determination of the molecular size of purified Vi-polysaccharide
17

Sepharose gel column chromatography is used to determine the relative molecular size of the Vi-polysaacride. Standard reagents are used are:

S.No Name of reagent Concentration /description
A Sepharose CI-4B gel
B Sodium chloride solution 0.2 mol/L
C BlueDextran2000 -
D Sodium azide -
Procedure:
1: Preparation of gel filtration column:
Prepare one in three part mixture (v/v) of Sepharose (C1-4B gel) in Sodium chloride solution, allow the sediments to settle down, remove the supernatant, and repeat the same procedure 3-4 times.
2: Packing of the column:
Pack the column form the washed slurry of Sepharose (C1-4B gel) and allow the gel to get settle down under recommended operating pressure for 16 hrs.
3: Calibration of the Column:
Dissolve 200mg of blue Dextran in 100 ml 0.2mol/l sodium chloride solution containing 500mg of sodium azide. Apply I ml of this solution to the packed column and collect fraction of about 2.0ml each.
The void volume (V0) is determined with Blue Dextran and the total volume (Vt) with sodium azide by detecting absorbence at 206nm or 260nm.
4: Assay of the purified Vi-polysaccharide:
One ml of the Vi-polysaccharide is loaded on the top of the column. Collect 2ml fractions.
Make two pools of the fractions: Pool I: Fraction eluted before a distribution constant (KD) of 0.25 is reached. Pool 2: Fraction eluted after a distribution constant (KD) of 0.25 is reached. Quantify the Vi-polysaccharide in each pool by determining the O-acetyl content.
Results:

Batch No. Vi-polysaccharide in pooled fraction-1 (%)
IA 73
18

2A 74
3A 63
4A 54
5A 68
TABLE-8
Determination of the pH of purified Vi-polysaccharide
A three point calibrated pH electrode is used for determining the pH of the purified Vi-polysaccharide. The pH electrode calibrated at 4.0, 7.0 and 8.5 pH is used.
Results:

Batch No. pH of Vi-polysaccharide
1A 7.0-7.2
2A 7.2-7.3
3A 7.2-7.3
4A 7.2-7.3
5A 7.2-7.3
ANNUXER-9
Determination of Clarity of the purified Vi-polysaccharide
The final solution of the purified Vi-polysaccharide should be free from any type of particulate matter.
Result:

Batch No. Clarity of Vi-polysaccharide
1A Clear
2A Clear
3A Clear
4A Clear
5A Clear
TABLE-10
Test of pyrogenicity for the purified Vi-polysaccharide
Each lot of the purified Vi-polysaccharide prepared is tested for pyrogenicity by intevenious injection into rabbits.
19

Procedure:
I: Rabbits which are healthy and passing in sham test are used.
2: Test for pyrogen conducted as per the guidelines mentioned in Indian
pharmacopoeia are followed.
3: Dilute 1.0ml of the purified Vi-polyaccride with pyrogen free saline to final dilution of25ng/ml.
4: Inject diluted vaccine in the dose of 1 ml/kg intravenously in the ear marginal vein slowly, within a period of maximum 10 minutes.
5: Temperature of the rabbit is determined by using a electronic thermometer probe inserted in the rectum of the rabbit at least 7.5cm deep.
6: Record temperature of each animal every 30 minutes for 3 hours after injection. The average of 2 readings taken 30minuts apart before injection is taken as average body temperature.
Results:

Batch No. Sum of difference in temperature of three rabbits (in °C)
1A 0.2
2A 0.6
3A 1.0
4A 0.8
5A 0.4
TABLE-11
Test of Abnormal toxicity for the purified Vi-polysaccharide
Each lot of the purified Vi-polysaccharide prepared is tested for abnormal toxicity .by intraperitoneal injection of one human dose into each of the five mice (weighing 17-22 grams) injection into rabbits.
Procedure:
1: Test for pyrogen conducted as per the guidelines mentioned in Indian pharmacopoeia are followed.
2: intraperitoneal injection of one human dose into each of the five mice
(Weighing 17-22 grams) and intraperitoneal injection of one human dose
into each of two guinea-pigs(( Weighing :250-350grams).
3: The weight of the subjected animals should be monitored for seven days.
20

Results:

Results
Batch No. Survival for seven days Weight loss seven days during
1A Yes Nil
2A Yes Nil
3A Yes Nil
4A Yes Nil
5A Yes Nil
TABLE-12
Test of endotoxin content of the purified Vi-polysaccharide
The determination of endotoxin with limulus amoebocyte lysate (LAL) is based on the gel formation of a mixture containing of a solution of the gram-negative bacteria with a solution of the lysate.
Procedure:
1:AI1 the procedure followed for reconstituting all reagents and performing LAL test are as per the recommendation of the manufacturer of the LAL kit.
2: The end point dilution of the appropriately diluted purified Vi-polysaccharide is determined.
3: The End point dilution is multiplied with the lysate sensitivity to get the endotoxin content in EU/ml
Results:

Batch No. Endotoxin content(EU/ml)
1A 1.25
2A 1.25
3A 1.25
4A 1.25
5A 1.25

TABLE-13
Results of five consecutive batches of the claimed vaccine
S Parameter Defined range as per Batch No. Re
21

WHO guidelines IA 2A 3A 4A 5A mar
N0 ks
1 Protein content 10mg/g of Vi-polysaccharide Nil Nil Nil Nil Nil Pass
2 Nucleic 20 mg/g of Vi- Nil 5.5 Nil Nil 18.5 Pas
acid content polysaccharide mg/g mg/g s
3 O-acetyl content NLT2mmol/ g of Vi-polysaccharide 5.95 6.25 5.96 6.38 6.48 Pas s
4 Molecular sizedeterminati on At least 50% eluted before KD0.25 73% 74% 63% 54% 68% Pass
5 Serological Identical Identi Identi Identi Identi Identi Pas
identity cal cal cal cal cal s
6 PH 6.5-7.5 7.0- 7.2- 7.2- 7.2- 7.2- Pas
7.2 7.3 7.3 7.3 7.3 s
7 Pyrogen testing Sum of difference in temperature of three rabbits should not exceed 1.5°C 0.2 0.6 1.0 0.8 0.4 Pass
8 Abnormal Survive for at least No No No No No Pas
toxicity seven days with out Wt. Wt. Wt. Wt. Wt. s
weight loss loss loss loss loss loss
9 Clarity Clear Clear Clear Clear Clear Clear Pas s
Thus it is shown that all consecutive five batches had passed the Criteria according to WHO guidelines Ref: TRS.840, page 14-27 & British pharmacopoeia
Further, clinical trials [open label, comparative type] were conducted to assess the purity & the comparative immunogenicity and safety of Vi capsular polysaccharide Typhoid vaccine manufactured by the present process with already marketed purified Vi-polysaccharide Typhoid vaccine in India i.e. Typherix of M/s GlaxoSmithKine. The study

had a total of 79 adult healthy volunteers of either sex between age of 18-44 year who fulfilled the inclusion and exclusion criteria of the vaccine were enrolled in the study. 40 of the 79 were administered vaccine as per the present invention, while 39 reed. Typherix.
The volunteers were randomly administered 1 intermuscular (I.M.) dose of either the claimed vaccine or the already marketed vaccine on the day 0 (visit 1) in the deltoid muscle of the arm.
Each full I.M dose (0.5ml) of the both vaccines contained 25ug of Vi capsular polysaccharide antigen against S.typhi.
ASSESSMENT OF EFFICACY:
A positive seroconversion for the study was defined as follows:
a) Development of Vi-antibodies in the volunteers who had no antibody titers at baseline.
b) >(or equal to) 4 fold rise in Vi-antibody titers in volunteers who had some antibody titers at base line.
Overall, all 37 of the 40 volunteers (92.5%) receiving Vi-purified capsular Polysaccharide Typhoid Vaccine manufactured by the claimed process, seroconverted after immunization.
Of the 40 volunteers (100%) who had no Vi-antibody titers at baseline seroconverted after 3 weeks of vaccination. Of the 5 volunteers, who had some Vi-antibody titers at baseline, two volunteers seroconverted after 3 weeks, with one of the volunteer showing, 32 fold increase in the antibody titers post-vaccination, and the other volunteer showing a 8 fold increase in the Vi antibody titers. Of the remaining 3 volunteers in the group who did not seroconvert, two volunteers showed a 2-fold increase in their antibody titers one volunteer had no change in the antibody titers at the end of the week 3 as compared to baseline.
Overall, 36 of the 39 volunteers (92.3%) receiving Typerix of M/s GlaxoSmithKline in our stud, seroconverted after immunization.
Of these 39 volunteers, 30 volunteers (76.9%) had no Vi antibody titers at baseline while 9 volunteers (23.1%) had some Vi antibody titers at base line.
All the 30 volunteers (100%) who had no Vi antibody titers at baseline seroconverted after 3 weeks of vaccination. Of the 9 volunteers, who had some Vi-antibody at baseline, a total of
6 volunteers seroconverted after a 16-fold increase and three volunteers having an 8-fold increase in the Vi-antibody titers post-vaccination
The results of the above open comparative multicentric phase III clinical trial
clearly demonstrate that the Purified Vi-Capsular Polysaccharide Typhoid Vaccine
23

manufactured by the claimed process is as immunogenic as Typherix of M/s GlaxoSmithKline with an almost equal number of volunteers seroconverting in both the study groups.
To conclude, Purified Vi-Capsular Polysaccharide Typhoid Vaccine manufactured by the claimed process is as immunogenic & as well as well tolerated as another already marketed Purified Vi-Capsular Polysaccharide Typhoid Vaccine i.e.Typherix of M/s GlaxoSmithKline.
24

Claims:
1. A process for producing and purification of a bacterial sub unit vaccine for use
against Salmonella infections comprising :
a) fermenting in a semi synthetic liquid medium a highly virulent strain of Salmonella at a temperature between 36°C and 37°C, for a period that is a function of the amount of the inoculum.
b) separating the bacterial residue from said fermented strain by centrifuging at moderately high speed.
c) precipitating the vaccinating Vi-polysaccharide by in the presence of a precipitating solute of the kind such as herein described..
d) decanting by moderately high centrifuging for purification and dissolving in pyrogen free water and further precipitating with ethanol;
e) decanting by moderately high centrifuging for purification and dissolving in pyrogen free water and further precipitating with cold phenol.
f) precipitating the vaccinating Vi-antigen by ethanol, and dissolving the precipitation the physiological saline.

2. A process as claimed in claim 1 wherein said precipitating solute is Cetavlon.
3. A process as claimed in claim I or 2 wherein said precipitation of Vi-polysaccharide is carried out a temperature in the range of 0°C to 4°C, at a pH of 7.0 to 7.5 with a residence time of about 18 hrs.
4. A process as claimed in any preceding claim wherein said precipitation of Vi-antigen by ethanol is carried out a residence time of about 18 hrs at temperature between 0°C to 4°C.
5. A process as claimed in any preceding claim wherein said precipitated Vi-antigen is extracted in the presence of phenol at about 0°C to 4°C.
6. A process as claimed in any preceding claim wherein said fermenting is carried out at neutral pH or slightly above neutrality, in average aerobiosis.
7. A bacterial sub unit vaccine for use against Salmonella infections prepared by the process as claimed in any preceding claim.



25

Abstract
The present invention relates to a process for producing a sub unit vaccine against Salmonella fevers, typhoid and paratyphoid fevers. The process comprises
a) fermenting in a semi synthetic liquid medium a highly virulent strain of Salmonella at a temperature between 36°C and 37°C, for a period that is a function of the amount of the inoculum.
b) separating the bacterial residue from said fermented strain by centrifuging at moderately high speed.
c) precipitating the vaccinating Vi-polysaccharide by in the presence of a precipitating solute of the kind such as herein described..
d) decanting by moderately high centrifuging for purification and dissolving in pyrogen free water and further precipitating with ethanol;
e) decanting by moderately high centrifuging for purification and dissolving in pyrogen free water and further precipitating with cold phenol.
f) precipitating the vaccinating Vi-antigen by ethanol, and dissolving the precipitation the physiological saline.
11 AUG2006
26

Documents:

942-MUM-2005-ABSTRACT(10-12-2010).pdf

942-mum-2005-abstract(11-8-2006).pdf

942-MUM-2005-ABSTRACT(17-12-2009).pdf

942-mum-2005-abstract(amended)-(17-12-2009).pdf

942-mum-2005-abstract(granted)-(23-12-2010).pdf

942-mum-2005-abstract(provisional)-(12-8-2005).pdf

942-mum-2005-abstract.doc

942-mum-2005-abstract.pdf

942-MUM-2005-CANCELLED PAGE(17-12-2009).pdf

942-mum-2005-cancelled pages(10-12-2010).pdf

942-MUM-2005-CLAIMS(AMENDED)-(10-12-2010).pdf

942-MUM-2005-CLAIMS(AMENDED)-(16-9-2010).pdf

942-MUM-2005-CLAIMS(AMENDED)-(17-12-2009).pdf

942-mum-2005-claims(granted)-(23-12-2010).pdf

942-MUM-2005-CLAIMS(MARKED COPY)-(16-9-2010).pdf

942-mum-2005-claims.doc

942-mum-2005-claims.pdf

942-MUM-2005-CORRESPONDENCE(15-9-2010).pdf

942-MUM-2005-CORRESPONDENCE(17-9-2010).pdf

942-MUM-2005-CORRESPONDENCE(19-02-2010).pdf

942-MUM-2005-CORRESPONDENCE(2-8-2010).pdf

942-MUM-2005-CORRESPONDENCE(22-6-2010).pdf

942-MUM-2005-CORRESPONDENCE(26-5-2010).pdf

942-MUM-2005-CORRESPONDENCE(27-02-2009).pdf

942-MUM-2005-CORRESPONDENCE(3-11-2010).pdf

942-MUM-2005-CORRESPONDENCE(3-12-2010).pdf

942-mum-2005-correspondence(9-1-2008).pdf

942-mum-2005-correspondence(ipo)-(24-12-2010).pdf

942-mum-2005-correspondence-received-ver-010806.pdf

942-mum-2005-correspondence-received-ver-080805.pdf

942-mum-2005-correspondence-received-ver-101105.pdf

942-mum-2005-description (complete).pdf

942-mum-2005-description(granted)-(23-12-2010).pdf

942-mum-2005-description(provisional)-(12-8-2005).pdf

942-MUM-2005-FORM 1(10-12-2010).pdf

942-mum-2005-form 1(12-8-2005).pdf

942-mum-2005-form 18(9-1-2008).pdf

942-mum-2005-form 2(granted)-(23-12-2010).pdf

942-mum-2005-form 2(provisional)-(12-8-2005).pdf

942-MUM-2005-FORM 2(TITLE PAGE)-(10-12-2010).pdf

942-mum-2005-form 2(title page)-(11-8-2006).pdf

942-mum-2005-form 2(title page)-(granted)-(23-12-2010).pdf

942-mum-2005-form 2(title page)-(provisional)-(12-8-2005).pdf

942-mum-2005-form-1.pdf

942-mum-2005-form-2.doc

942-mum-2005-form-2.pdf

942-mum-2005-form-3.pdf

942-mum-2005-form-5.pdf

942-MUM-2005-OTHER DOCUMENT(26-5-2010).pdf

942-MUM-2005-POWER OF AUTHORITY(17-12-2009).pdf

942-MUM-2005-REPLY TO EXAMINATION REPORT(17-12-2009).pdf

942-MUM-2005-REPLY TO HEARING(10-12-2010).pdf

942-MUM-2005-REPLY TO HEARING(16-9-2010).pdf

942-MUM-2005-SPECIFICATION(AMENDED)-(17-12-2009).pdf


Patent Number 244897
Indian Patent Application Number 942/MUM/2005
PG Journal Number 53/2010
Publication Date 31-Dec-2010
Grant Date 23-Dec-2010
Date of Filing 12-Aug-2005
Name of Patentee CADILA HEALTHCARE LIMITED
Applicant Address Zydus Tower, Satelite Cross Road, Ahmedabad-380015,
Inventors:
# Inventor's Name Inventor's Address
1 SINHA AMARES Zydus Tower, Satelited Cross Road, Ahmedabad-380015,
2 PATEL, P.R. Zydus Tower, Satelited Cross Road, Ahmedabad-380015,
PCT International Classification Number A61P31/04 C07K14/255
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA