FORM 2
THE PATENT ACT, 1970,
(39 OF 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
(SEE SECTION 10; RULE 13)
Applicant(s): BIOBRIDGE HEALTHCARE SOLUTIONS PVT LTD,
a company incorporated under the Companies Act, 1956 of 13, Rachna Blossom, Jagdish Nagar, Aundh, Pune 411 007 (M.S)
TITLE: Multivalent vaccine composition having increased stability and activity.
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates in particular to a multivalent vaccine composition. More particularly to a multivalent adjuvant based vaccine composition comprising recombinant Hepatitis B, Diphtheria toxoid, Tetanus toxoid, whole cell pertussis vaccine and Haemophilus type b conjugate vaccine and is stable for one month at 37°C.
BACKGROUND OF THE INVENTION
Throughout this application, various references are referred to. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Infections with Hepatitis B virus and bacteria such as Corynebacterium diphtheriae, Clostridium tetani, Hemophilus influenza and Bordetella pertussis are a widespread problem in infants and vaccines for which have been used for mass immunization are now available individually and preparations are very well know in art and applied successfully.
However it is often necessary or desirable to administer separate vaccines such as Hepatitis B vaccine, Hib conjugate vaccine, DPT vaccine and this can involve multiple injections as part of immunization schedule.
Mere combining immunogenic antigens does not ensure a stable and immunogenic preparation as ail the antigens are of different nature for e.g. Diphtheria and Tetanus are detoxified toxins, Pertussis is a inactivated whole cell preparation, Haemophilus influenza b is a conjugate vaccine (polysaccharide conjugated to a carrier protein such as Tetanus toxoid) and Hepatitis B is a subunit vaccine viz. a surface protein expressed in a host e.g. yeast Pichia pastoris.

There is published literature for combining various antigens to prepare multivalent vaccines. However none reveal methods to prepare combinations stable at 37°C for 1 month.
Therefore there exists a need for a consistent process to prepare a stable combined immunogenic vaccine comprising all the antigens in a vaccine formulation capable of preventing diseases caused by Hepatitis B virus, Haemophilus influenzae, Bordetelia pertussis, Clostridium tetani and Corynebacterium diphtheriae.
OBJECT OF THE INVENTION
Therefore the object of the present invention is to overcome the drawbacks of the prior methods for attempting preparation of multivalent vaccine which has less stability.
Yet another object of the present invention is to provide a process for the preparation of multivalent adjuvant based vaccine having multivalent antigenic composition comprising recombinant Hepatitis B, Diphtheria toxoid, Tetanus toxoid, whole cell pertussis vaccine and Haemophilus type b conjugate vaccine.
Yet another object of the present invention is provide a multivalent adjuvant based vaccine having antigenic composition comprising recombinant Hepatitis B, Diphtheria toxoid, Tetanus toxoid, whole cell pertussis vaccine and Haemophilus type b conjugate vaccine stable for one month at 37°C.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, become better understood with reference to the following description, appended claims, and accompanying drawings in which like characters represent like parts throughout the drawings

Figure 1 represents the integrated process flow diagram for production of Figure 1 represents the integrated process flow diagram for production of multivalent adjuvant based vaccine having multivalent antigenic composition comprising recombinant Hepatitis B, Diphtheria toxoid, Tetanus toxoid, whole cell pertussis vaccine and Haemophilus type b conjugate vaccine.
DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description and explanation of the present invention with some examples thereof. In order to better appreciate the invention, it is described with reference to the figures which illustrate various embodiments of the invention.
The present invention relates in particular to a multivalent vaccine composition. More particularly to a multivalent adjuvant based vaccine composition comprising recombinant Hepatitis B, Diphtheria toxoid, Tetanus toxoid, whole cell pertussis vaccine and Haemophilus type b conjugate vaccine and is stable for one month at 37°C.
As shown in figure 1, a process for the preparation of multivalent vaccine is provided (10). Separate antigenic composition is prepared with adjuvant Aluminium Phosphate and preservative wherein Item 1 is DTwP (12). Formulation of antigenic composition is prepared with adjuvant Aluminium Phosphate and preservative wherein Item 2 is HBsAg (14) followed by independent adsorption of Item -1 (16) and Item 2 (18) further mixing Items 1 and 2 in step (20) and adsorption of mixed Items 1 and 2 in step (22). Item 3 having Hib-TT is mixed with adsorbed DTwP-HepB (24) for formulation of final pentavalent vaccine composition. The bulk prepared vaccine composition is further stored (26) at about 5°C.
The present invention provides Diphtheria, Tetanus, w-Pertussis, Hepatitis B (rDNA) & Haemophilus influenzae type b Conjugate Vaccine (DTwP-Hep-B-Hib) which is a sterile and uniform suspension of Diphtheria toxoid, Tetanus toxoid,

whole cell Pertussis vaccine, Hepatitis B surface antigen and conjugated Haemophilus influenzae type b (PRP-TT) vaccine adsorbed on Aluminium phosphate and suspended in isotonic sodium chloride solution. 2-Phenoxyethanol or Thiomersal or Benzethonium chloride can be used as a preservative preferably thiomersal is added as a preservative.
The preparation of the antigens and adsorption procedure with the adjuvants are well known in the art. Diphtheria and tetanus toxoids are obtained by detoxification of respective toxins by formalin. Pertussis vaccine is a suspension of heat-killed Bordetella pertussis of all the three major agglutinogens viz. 1, 2 and 3.
Surface antigen of Hepatitis B virus is obtained from cultures of transformed yeast by insertion in its genome of the gene coding for the surface antigen (HBsAg) using recombinant DNA procedures. Haemophilus influenzae type b (PRP-TT) vaccine is derived from highly purified capsular polysaccharide isolated from Haemophilus influenzae type b and coupled with Tetanus toxoid.
The final multivalent vaccine contains preferably the following Items in 0.5 ml which is pediatric dose -
Diphtheria Toxoid 20 Lf
Tetanus Toxoid 7.5 Lf
Inactivated w-B pertussis 12 OU
rec-HBsAg 10 mcg
H. influenzae type b polysaccharide 10 mcg
Conjugated to Tetanus toxoid (PRP-TT)
Aluminium (AI3+) 0.25 mg
(As Aluminium Phosphate gel)
Thiomersal 0.025 mg
Physiological saline qs
The potency of the vaccine per single human dose is at least 30 lU for diphtheria, 40 IU for tetanus (determined in guinea pig) or 60 IU for tetanus (determined in mice), 4 IU for whole cell pertussis, the upper confidence limit of the relative

potency for Hepatitis B surface antigen (in vivo test) should be > 1.0 and 10 mcg for Hib (PRP-TT). Suitable antigens for use in vaccines according to the invention are already commercially available and details may be obtained from the World Health Organization.
The present invention provides process for the preparation of multivalent vaccine by addition of specified concentration of various antigens, Aluminium phosphate, normal saline and Thiomersal required for the identified batch size based on the bulk API concentrations.
Specified concentration of Aluminium phosphate is added to the known concentration of aluminium phosphate adjuvant to a reactor 1, further addition of specific concentration of diphtheria toxoid takes place to aluminium phosphate adjuvant with stirrinp for about 15 min at about 150 rpm.
Specific concentration of Tetanus toxoid is added to the reactor 1 containing aluminium phosphate and diphtheria toxoid with stirring for about 15 min at about 150 rpm, further specific concentration of normal saline (0.85%) is added to make up the volume. To reactor 2, specific concentration of whole cell pertussis is added followed by addition of thiomersal solution (of about 1% stock solution).
Contents of reactor 1 and reactor 2 are transferred to mixing vessel with stirring at about 150 rpm for about 1 hour followed by addition of water for injection equivalent to the water loss taking place during sterilization by adjusting pH using 1N NaOH/ 1N HCI to adjust in between 6-7 further incubating the mixture at about 35°C for about 48 hours.
In reactor 3, specified concentration of Aluminium phosphate is added to the know concentration of adjuvant further specific concentration of Hepatitis B surface antigen is added to said aluminium phosphate adjuvant with stirring for about 15 min at about 150 rpm followed by addition of pre defined concentration of thiomersal, saline solution and water for injection equivalent to the water loss taking place during sterilization with stirring at about. 150 rpm for about 1 hour and adjusting the pH using 1N NaOH/ 1N HCI to adjust in between 6-7. Contents

of reactor 1, 2 and 3 are mixed followed by stirring at about 150 rpm for 1 hour and incubating entire mixture at about 350C for about 48 hours.
In reactor 4, specific concentration of Hib-TT vaccine is added to the pre-defined concentration of saline with a mixing and stirring at about 150rom for about 15 minutes. Mixing all the Items of reactor 1 to 4 with stirring at about 150rpm for about 1 hour followed by adjusting pH between 6-7 using 1 N NaOH or 1N HCI.
The final vaccine is now ready for dispensing in appropriate containers, stoppered and sealed. The sealed vaccines should be stored at about 2°C to about 8°C for optimum shelf life.
The invention is described hereinafter, with reference to the following examples,
which are illustrative only and should not be construed to the limit of the scope of
present invention.
Example 1
Method for the preparation of 5 Liter of stable vaccine preparation
Begin calculations for the ingredients for preparation of effective multivalent adjuvanted 5 L vaccine comprising of 5 antigens viz. recombinant Hepatitis B, Diphtheria toxoid, Tetanus toxoid, whole cell pertussis vaccine and hemophilus type b conjugate vaccine as below:

CALCULATION for vaccine ingredients
Constituents of Final batch (viz a, b and c)
a Solution 1 containing Hib-TT 150 ml
b Solution 2 containing DT, TT, wP, Aluminium phosphate and thiomersal 2425 ml
c Solution 3 containing Hep B, aluminium phosphate and thiomersal 2425 ml

Final batch volume (a+b+c) contains details as per Part A and Part B 5000 ml


Par tA Calculations for antigens
Diphtheria toxoid bulk @ 2400 Lf/ml required at final conc of 42 Lf/ml 87.5 ml
Tetanus toxoid bulk @ 2000 Lf/ml required at final conc of 15.75 Lf/ml 39.37 ml
Whole cell Pertussis bulk @ 250 OU/ml required at final conc of 25.2 OU/ml 504 ml
Hep B antigen bulk @ 800 mcg/ml required at final conc of 21 mcg/ml 131.25 ml
Hib-TT antigen bulk @ 1500 mcg/ml required at final conc of 21 mcg/ml 70 ml
Total Part A 832.12

A

Par tB Calculations for excipients
Aluminium phosphate gel bulk @ 5 mg/ml required at a final cone of 0.5 mg/ml AI3+ 500 ml
Thiomersal solution bulk @ 10 mg/ml required at a final conc of 0.05 mg/ml 165 ml
Physiological saline @ 0.85% solution 3502.88 ml
Total Part B 4167.88 B

Final batch volume (A+B) 5000 ml
Although the preferred embodiments of the present invention and their respective variations have been described, people having ordinary skills in the art would envision various modifications of those embodiments. Accordingly, the present invention should not be limited to precise forms and manners in the above disclosure and description but should simply be taken by way of examples. Thus,

the present invention can be varied and modified without departing the true scope and spirit thereof as defined in the appended claims.

WE CLAIM:
1. A combined multivalent stable vaccine composition comprising: Hepatitis B surface antigen, Hib-TT conjugate vaccine, diphtheria toxoid, tetanus toxoid and whole cell pertussis vaccine in combination with an adjuvant such as aluminium phosphate with a shelf life of one month at about 37° C without any substantial loss in immunogenicity of any of the antigenic component.
2. A combined multivalent vaccine composition as claimed in claim 1, is prepared by mixing antigenic composition with adjuvant Aluminium Phosphate and preservative wherein the composition is active at 37°C for one month.
3. A combined multivalent vaccine composition as claimed in claimed 1, wherein a combined vaccine composition as Diphtheria-Tetanus-Pertussis (DTP) -Hepatitis B and Hib-TT.
4. A combined multivalent vaccine composition as claimed in claimed 1, wherein A multivalent vaccine in which the antigens HBsAg, Diphtheria toxoid and Tetanus toxoid is adsorbed to aluminium phosphate.
5. A combined multivalent vaccine composition as claimed in claimed 1, wherein the stability of the vaccine is such that the vaccine can be kept at 37° C for 1 month without substantial loss of immunogenicity of all the antigens.