OA21991A - Improved methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof. - Google Patents

Abstract

Improved methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof. The present invention relates to method for producing vaccine composition containing alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens selected from the group comprising of type 1, type 2 and type 3 serotypes and atleast one preservative from formaldehyde and 2phenoxyethanol (2-PE). The reduced-dose Inactivated Polio Virus (IPV) antigens shows noninferiority/equivalent protection against polio when compared to a standard dose of IPV antigen and the preservative improves the stability and preservative efficacy of the vaccine. More particularly, it provides improved Inactivated Polio Virus (IPV) vaccine formulation for prophylaxis and treatment of infections caused by Polio Virus type 2 serotype in humans, which is less likely to revert to neurovirulence than Live-attenuated Sabin oral polio vaccines & is an alternative to currently used nOPV2. Applicant provides a method for producing such monovalent vaccine composition containing alum adsorbed reduceddose Inactivated Polio Virus (IPV) antigens comprising of type 2 serotype at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units per 0.5 ml dose; more preferably 2D-antigen units per 0.5 ml dose. Further, Applicant recommends using such "Dose Reduced Monovalent Type 2 IPV" for campaign, as an alternative to nOPV2 for stockpiling & combating epidemics.

Description

IMPROVED METHODS FOR POLIOVIRUS INACTIVATION, ADJUVANT ABSORPTION AND DOSE REDUCED VACCINE COMPOSITIONS OBTAINED THEREOF

REFERENCE TO RELATED PATENT APPLICATION

The présent Application is a Patent of Addition filed under Section 54 of the Indian Patents Act, 1970, claiming priority to Indian Patent Application No. 3180/MUM/2014, filed on 07^th October 2014. The teachings of the above applications are incorporated herein in their entirety by reference.

FIELD

The présent disclosure relates to method of manufacturing vaccine compositions. Particularly, the présent disclosure is related to improved process of manufacturing an Inactivated Polio Virus (IPV) vaccine formulation for prophylaxis and treatment of infections caused by Polio Virus.

BACKGROUND

Ail publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individualiy indicated to be incorporated by reference. The following description includes information that may be useful in understanding the présent invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Thanks to major immunisation efforts since the 1950 60’s with the parenterally administered inactivated polio vaccine (IPV) developed by Jonas Salk and the live attenuated Sabin oral polio vaccine (OPV), wild fomns of two of the three serotypes of polioviruses hâve been declared as being globally eradicated, wild poliovirus type 2 in September, 2015, and wild poliovîrus type 3 in October, 2019. Wild poliovirus type 1 has also been eliminated from many WHO régions around the world, including the Americas, Europe, South-East Asia, Western Pacific, and most recently Africa in 2020.

The trivalent oral polio vaccine (OPV) has accelerated the éradication initiative because of its easy distribution, low cost, and oral applicability. However, due to low immunisation rates within certain communities, another form of poliovirus, namely, circulating vaccine-derived poliovirus (cVDPV), hâve been observed, with 959 cases occurring globally in 2020. This constitutes an inhérent risk for children to develop vaccine-associated paralytic polio. This low but constant risk prompted the WHO to promote the shift first from trivalent OPV to bivalent (types I and 3) OPV, and later to inactivated polio vaccines (IPVs), which hâve no risk of inducing cVDPV (Bandyopadhyay A.S et al 2015)

The recent détection of vaccine-derived poliovirus (VDPV) in London (UK) and a case of paralytîc polio in New York (USA) hâve highlighted how the scourge of poliomyelitis has not been totally overcome and remains an international/global problem, not confined to Afghanistan and Pakistan (where wild-type l poliovirus remains endemic) or as outbreaks of circulating VDPV in countries in Africa.

A scientific consortium was formed in 2011 with funding support and a mission from the Bill & Melinda Gates Foundation to more sustainably complété éradication of ail forms of polioviruses. They subsequently found that circulating VDPV2 was the prédominant strain for paralytic poliomyelitis outbreaks; in 2020-22 circulating VDPV2 was responsible for 9799% of cases of polio globally.

Outbreaks of type 2 cVDPV (circulating vaccine derived poliovirus)—which account for most of the cVDPV cases globally—are a major challenge to achieving poliovirus éradication. In 2021, 682 cases of cVDPV2 were confirmed from 22 countries, a réduction from 1,081 cases in 24 countries in 2020, but nearly double the 366 cases from 16 countries reported in 2019. Circulation of cVDPV2 constitutes a significant challenge to the éradication campaign and a major risk for global health, and has appropriately been designated as a Public Health Emergency of International Concern (Polio Global Eradication Initiative, 2020).

These outbreaks are driven by several factors, including low quality and delayed polio outbreak response; declining gut immunity in young children to the type 2 virus after countries switched from trivalent to bivalent oral polio vaccine (bOPV) for routine immunization in 2016; and insufficient routine immunization coverage.

To address the risk of circulating VDPVs, a global collaborative effort over the past decade has enabled the development of novel oral polio vaccine type 2 (nOPV2) that is as immunogenic as the current Sabin strain and equally effective, while being less likely to revert to neurovirulence than Sabin oral polio vaccines.

nOPV2 has introduced modifications within at the 5' untranslated région of the Sabin2 genome to stabilize atténuation déterminants, 2C coding région to prevent recombination, and 3D polymerase to limit viral adaptability. Specifically, genome of this polio vaccine candidate (herein nOPV2) carries five modifications of the Sabin2 genome, including two modifications within the 5'-untranslated région (UTR) (relocated cre and Sl5domV), synonymous mutations at eight nucléotide positions in the 2C coding région to inactivate the internai cre and two mutations in the 3D polymerase (D53N and K.38R) to limit viral adaptability. Each of these modifications, contributes to genetic stability and atténuation. Importantly, their combination prevents détectable reversion to neurovirulence by reducing the capacity of the virus to acquire mutations that increase réplication fitness in neuronal tissues. (Andrew Macadam et al 2020). nOPV2 is a modified version of the existing OPV2 vaccine (also known as the Sabin OPV type 2 vaccine, or mOPV2) that provides comparable protection against poliovirus type 2. The vaccine is more genetically stable than OPV2, which makes it less likely to revert into a form that could cause paralysis in children who hâve not been sufficiently immunized.

Development of a poliovirus type 2 vaccine strain (nOPV2) that is genetically more stable and less likely to regain virulence than the original Sabin2 strain. The successful development of nOPV2—the first such vaccine against type 2 poliovirus and the first vaccine ever authorised by the WHO Prequalification team through îts Emergency Use Listing procedure—has led to the deployment of approximately 450 million doses of nOPV2 for outbreak control in 21 countries. It also paved the way for the subséquent Emergency Use Listing approval of COV1D-I9 vaccines in the global pandémie.

nOPV2 is only available through a global stockpile, with the vaccine released by the WHO Director-General. Distribution of nOPV2 is guided by a prioritization framework developed by the Global Polio Eradication Initiative (GPEI) which factors in countries’ readiness to use the vaccine (i.e. vérification status), their unique epidemiological situations and vaccine usage history. Ample supply of effective type-2 containing OPV is available for countries not yet eligible to use nOPV2. To date, close to 600 million doses of nOPV2 hâve been administered across 28 countries globally, and the majority of countries hâve seen no further transmission of cVDPV2 aftertwo immunization rounds.

However, Global Polio Eradication Initiative (GPEI) recently reported seven children, six in the Démocratie Republic of the Congo (DRC) and one in neighbouring Burundi, had recently been paralyzed by poliovirus strains derived from a vaccine (nOPV2). The problem is that in areas where polio vaccination rates are low, in rare cases the vaccine virus can continue to spread among un- or under-immunized people for months, accumulating enough mutations to revert to its paralytic form. Thîs happens most often with poliovirus type 2, one of three serotypes. Global Polio Eradication Initiative (GPEI) reported on I6th Mar 2023 that they hâve received notification of the détection of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Burundi and the Démocratie Republic of the Congo (DRC) linked with the novel oral polio vaccine type 2 (nOPV2). The viruses were isolated frotn the stool samples of seven children with acute flaccid paralysis (AFP) - six in DRC (eastern Tanganyika and South Kivu provinces), one in Burundi (Bujumbura Rural province) — and from five 5 environmental samples coilected in Burundi (Bujumbura Mairie province). Ail reported isolâtes stem from two separate and new' émergences of cVDPV2 linked with nOPV2 that originated in Tanganyika and South Kivu provinces in DRC.

Thus, with due considération of the emerging nOPV2 related vaccine derived poliovirus /paralysis cases (in spite of global nOPV2 stockpile) , the continuons increasing global 10 poliovirus vaccine demand to control the increasing number and géographie scope of cVDPV2 outbreaks, pressure for cost effective manufacturing/access, there is an urgent need for a safe alternative monovalent Type 2 IPV vaccine approach (inclusive of using a monovalent dose reduced Salk or Sabin Type 2 IPV) & an efficient platform process for manufacturing an improved Inactîvated Polio Virus (IPV) vaccine formulation for 15 prophylaxis and treatment of infections caused by Polio Virus type 2 serotype in humans, whîle being less likely to revert to neurovirulence than Live-attenuated Sabin oral polio vaccines.

OBJECTS

Some of the objects of the présent disclosure, which at least one embodiment herein satisfies, are as follows:

An object of the présent disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.

lt is an object of the présent disclosure to provide a method for producing a vaccine composition comprising inactivated poliovirus partîcles

Yet another object of the présent disclosure is to provide a method for producing a vaccine composition containing various alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens which shows non-inferiority/equivalent protection against polio when compared to a standard dose of IPV antigen.

Yet another object of the présent disclosure is to provide a method for producing a monovalent vaccine composition containing alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens selected from the group comprising of type I, type 2 and type 3 serotypes.

Yet another object of the présent disclosure is to provide a method for producing a monovalent vaccine composition containing alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens comprising of type 2 serotype at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units per 0.5 ml dose.

Yet another object of the présent disclosure is to use “Salk/Sabin Dose Reduced Monovalent Type 2 IPV” for campaign, as an alternative to nOPV2 for stockpiling & combating épidémies.

Yet another object of the présent disclosure is to manufacture a bivalent vaccine (i.e. Type 2/Type l, Type 2/Type 3)

Yet another object of the présent disclosure is to provide a method for producing a vaccine composition containing alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens and preservative comprising formaldéhyde and at least one additional preservative to improve the stability and preservative efficacy of the vaccine.

Other objects and advantages of the présent disclosure will be more apparent from the following description, which is not intended to limit the scope of the présent disclosure.

SUMMARY

Applicant provides improved Inactivated Polio Virus (IPV) vaccine formulation for prophylaxis and treatment of infections caused by Polio Virus type 2 serotype in humans, 5 which is less likely to revert to neurovirulence than Live-attenuated Sabin oral polio vaccines & is an alternative to currently used nOPV2. Applicant has found a method for producing such monovalent vaccine composition containing alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens comprising of type 2 serotype at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units per 0.5 ml dose; more preferably 2D-antigen unîts per

0.5 ml dose and preservative comprising formaldéhyde and at least one additional preservative such as 2-phenoxyethanol (2-PE) to improve the stability and preservative efficacy of the vaccine.

Detailed Description:

Although the présent disclosure may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion, with the understanding that the présent disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description. Embodiments are provided so as to thoroughly and fully convey the scope of the présent disclosure to the person skilled in the art. Numerous details are set forth, relating to spécifie components, and methods, to provide a complété understanding of embodiments of the présent disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the présent disclosure. In some embodiments, well-known composition, well-known processes, and wellknown techniques are not described in detail.

The terminology used, in the présent disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the présent disclosure. As used in the présent disclosure, the forms a,” an, and the may be intended to include the plural forms as well, unless the context clearly suggests otherwise.

The terms flrst, second, third, etc., should not be construed to limit the scope of the présent disclosure as the aforementioned terms may be only used to distinguish one element, component, région, layer or section from another component, région, layer or section. Terms such as first, second, third etc., when used herein do not imply a spécifie sequence or order unless clearly suggested by the présent disclosure. The présent disclosure provides an immunogenic composition and a process for preparing the same.

The term vaccine is optionally substîtutable with the term immunogenic composition, “vaccine formulation”, “vaccine compositions” and vice versa.

The term D-antigen units (also referred to as international units or IU): The D antigenic form of the poliovirus induces protective neutralising antibodies. D antigen units referred to herein (for instance in the vaccines of the invention) are the measured total D antigen units of each unadsorbed bulk IPV antigen type prior to formulation of the final vaccine which are added in each human dose of formulated vaccine (typically 0.5mL final volume). Reliable methods of measuring D-antigen units are well known in the art and are published, for instance, by the European Pharmacopoeia. For instance, D-antigen units may be measured using the ELISA test (D-antigen quantification by ELISA) below. European Pharmacopoeia provides a test sample (European Pharmacopoeia Biological Reference Préparation - available from Ph. Eur. Secrétariat, e.g. Code P 216 0000) for standardisation of such methods between manufacturers (Pharmeuropa Spécial Issue, Bio 96-2). Thus, the Dantigen unit value is well understood in the art.

The term dose herein is typically one administration of the vaccine of the invention, whîch is typically one injection. A typical human dose is 0.5mL. Of course, various doses may be administered in a vaccine administration schedule.

The term IPV or a vaccine composition comprising these components herein is intended to mean inactivated polio virus based on Salk or Sabin Serotype l, 2, 3 monovalent or bivalent or tri valent combination of either one, two or ail three of these types. Salk poliovirus may be one of Brunenders, Brunhilde, CHAT, Cox, Lansing strains, Mahoney, MEF-l, Saukett, Saukett H and G, Leon strains. Salk based chimeric recombinant strains may be attenuated Poliovirus Strains (Sl5, Sl6, Sl7, Sl8 & Sl9), Sl9 Type l (Mahoney or Brunhilde), SI9 Type 2 (MEF-l) and SI9 Type 3 (Saukett) SI9 Type l (Strain Sl9/MahPl/Nl8S), Sl9 Type 2 (S19/MEF2P1/N18S) and SI9 Type 3 (S19/SktPl/N18S). Sabin based chimeric recombinant strains may be attenuated Poliovirus Strains (S 15, S16, S17, S18 & S19) wherein the capsid protein coding régions (PI) of S15, S16, S17, SI8 and S19 were replaced exactly with the PI régions of the serotype I, serotype 2 and serotype 3 Sabin live-attenuated vaccine strains (Sabin 1 Sabin 2, and Sabin 3), S19 Type 1 (Sabin), S19 Type 2 (Sabin) and S19 Type 3 (Sabin).

An example of a full (or standard) dose (40-8-32 D antigen units of Salk based IPV types 1, 2 and 3 respectively) IPV immunogenic composition for the purposes of this invention could be Poliovac® (Sérum Institute of India Pvt. Ltd.). Thus, where it is stated herein that one, two, three fold dose réduction (reduced) as compared to standard dose of Salk based IPV is présent in an immunogenic composition of the invention it is meant D-antigen units equating to X% of réduction of dose of 40, 8, and/or 32 D-antigen units of IPV types 1, 2 and/or 3 respectively (as measured in each bulk IPV antigen type) are formulated within each dose of said vaccine.

The présent disclosure envisages method for producing vaccine composition comprising alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens which shows noninferiority/equîvalent protection against polio when compared to a standard dose of IPV antigen; and preservative comprising formaldéhyde and at least one additional preservative to improve the stability and preservative efficacy of the vaccine.

An important aspect of the présent disclosure, wherein the method for producing a vaccine composition comprising inactivated poliovirus particles may comprise of following steps:

a) purifying the poliovirus particles using method selected from ultrafiltration, diafiltration, and chromatography in the presence of a phosphate buffer;

b) exchanging the phosphate buffer of the poliovirus particles wîth a buffer selected from TRIS, TBS, MOPS, HEPES, BICARBONATE, having a pH of 6.8 to 7.2 and a concentration in the range of 30 mM to 50 mM, preferably 40 mM;

c) adding 10X concentrated M-I99 medium containing 0.5 % glycine to achieve final concentration of Ix;

d) inactivating the poliovirus particles by incubation with 0.025 % formaldéhyde at 37 °C for 5 to 13 days; and

e) adsorbing the poliovirus particles on aluminium hydroxide sait adjuvant whereby percentage adsorption on alum is at least 95%, and wherein aluminium content per 0.5mL dose, is 0.4 mg AI3+ for Type l, 0.2mg AI3+ for Type 2, and 0.2mg AI3+ for Type 3;

f) blending alum adsorbed poliovirus particles obtained in step (e) with stérile dilution medium comprising preservative.

According to an embodiment of the présent disclosure, said formaldéhyde inactivation can occur in presence of TRIS, TBS, MOPS, HEPES, and bicarbonate buffer having concentration selected from 30mM, 40mM and 50mM, preferably 40mM and at a pH selected from 6.8, 6.9, 7, 7.1 and 7.2, preferably between 6.8 and 7.2 wherein said inactivation does not utilize any phosphate buffer.

According to an embodiment of the présent disclosure, post-incubation of the poliovirus particles with 0.025 % formaldéhyde at 37 °C for 5 to 13 days the mixture may be subjected to intcrmediatc 0.22μ filtration on day 7 and final filtration on day 13.

According to an embodiment of the présent disclosure, post inactivation of the poliovirus particles bulk in step (d) the bulk may be stored at 2-8°C.

According to an embodiment of the présent disclosure, post inactivation of the poliovirus particles the bulk may be subjected to D-Ag ELISA test for D-Antigen unit détermination.

According to an embodiment of the présent disclosure, the adsorption of poliovirus particles on aluminium sait adjuvant may comprise of following steps:

l. taking the desired volume of autoclaved Aluminium sait adjuvant in a blending vessel to get the final concentration of Alum (Al⁺⁺⁺) less than l .2 mg Al³⁺ per 0.5mL dose;

2. adding IPV bulk with adjusted D-Ag unit and making up the volume with a stérile dilution medium comprising preservative;

3. Adjusting the final formulation pH and obtaining final formulation with pH between 6 and 7;

According to an embodiment of the présent disclosure, wherein adsorption of formalin inactivated IPV can be done on aluminium hydroxide sait adjuvant wherein final concentration of Alum(AI⁺⁺⁺) less than 1.2 mg Al³⁺ per 0.5mL dose, preferably aluminium content per 0.5mL dose, is 0.4 mg A13+ for Type 1,0.2mg A13+ for Type 2, and 0.2mg AI3+ for Type 3 and at a pH selected from 6.2, 6.3, 6.4 and 6.5, preferably 6.5.

According to an embodiment of the présent disclosure, wherein the said process of formalin inactivation and aluminium hydroxide adsorption can resuit in D- Antigen recovery postinactivation between 50% and 80% and percent adsorption of aluminium hydroxide can be between 85 and 99%, preferably IPV Type I, 2 and 3 antigens are adsorbed on to aluminium sait of hydroxide (A1(OH)3) having percentage adsorption of at least 95 %.

According to.an embodiment of the présent disclosure, the blending in step (f) may comprise of stirring at 200 to 250 rpm continuously for not less than 16 hrs at température of 2-8°C to form a homogeneous mixture.

According to an embodiment of the présent disclosure, the stérile dilution medium may comprise of atleast one preservative selected from the group of 2-phenoxyethanol (2-PE), 20 Benzéthonium chloride (Phemerol), Phénol, m-cresol, p-chlor-m-cresol, chlorobutanol, Thiomersal, Formaldéhyde, paraben esters comprising methyl- ethyl- butyl- or propylparabens, or benzyl alcohol or a combination thereof.

In a preferred embodiment, the stérile dilution medium may comprise of preservative selected from 2-phenoxyethanol or Formaldéhyde or a combination of 2-PE and formaldéhyde.

More preferably, the stérile dilution medium may comprise of a combination of 2-PE and formaldéhyde as preservative, wherein the concentration of preservative per 0.5 ml dose of the vaccine composition may comprise of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.

In an embodiment, the stérile dilution medium may additionally comprise of M-199 Medium 30 containing 0.5 % glycine, Water for Injection (WFI) other than the preservative.

According to an embodiment of the présent disclosure, post blending in step (f) the adjusted final pH of the vaccine formulation bulk may be between pH 6.0 - 7.0.

According to an embodiment of the présent disclosure, the vaccine formulation bulk may be filled in a container selected from group comprising of a bottle, a vial, an ampule, an IV bag, a wearable injector, a bolus injector, a pre-filled syringe, a pen, a pump, a multidose needle syringe, a multidose vial, a multidose pen, a syrette, an auto-injector or a Vaccine Microarray Patches.

In a preferred embodiment, the vaccine formulation bulk may be filled in a type-1 glass vials with bromo-butyl rubber stoppers and aluminium seal with coloured polypropylene flip cap.

According to an embodiment of the présent disclosure, the inactivated polio virus may be based on Salk or Sabin Serotype 1, 2, 3 monovalent or bivalent or trivalent combination of either one, two or ail three of these serotypes.

In a preferred embodiment, the inactivated polio virus may be based on Salk Serotype 1, 2 and 3 monovalent or bivalent or trivalent combination of either one, two or ail three of these serotypes.

Salk poliovirus may be one of Brunenders, Brunhilde, CHAT, Cox, Lansing strains, Mahoney, MEF-1, Saukett, Saukett H and G, Leon strains. Salk based chimeric recombinant strains may be attenuated Poliovirus Strains (S 15, S16, S17, SI8 & S19), SI9 Type 1 (Mahoney or Brunhilde), S19 Type 2 (MEF-1) and S19 Type 3 (Saukett) S19 Type 1 (Strain S19/MahP 1 /N 18S), S19 Type 2 (S 19/MEF2P1 /N 18S) and S19 Type 3 (S 19/SktP 1 /N 18S).

More preferably Salk poliovirus may be one of Mahoney, MEF-1 and Saukett strain.

According to an embodiment of the présent disclosure, wherein the vaccine composition obtained from the method may be a trivalent composition comprising:

a) inactivated poliovirus type 1 at a dose greater than 1 D-antigen units and less than 11 D-antigen units, per 0.5ml;

type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; and type 3 at a dose greater than 2 D-antigen units and less than 17 D-antigen units, per 0.5ml;

b) aluminium content per 0.5mL dose, is 0.4 mg A13+ for Type 1,0.2mg AI3+ for Type 2, and 0.2mg A13+ for Type 3;

c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 gg/0.5 ml dose.

In a preferred embodiment, wherein the vaccine composition obtained from the method may be a trivalent composition comprising:

a) inactivated poliovirus of Salk strain and the dose of individual Type 1, Type 2, or Type 3 of Salk strain based IPV may be selected from the group of dose composition comprising of 7.5-16-10, 8-2-5, 10-2-5, 10-2-10, 10-2-12, 10-2-16, 7.5-16-10, 5-2-5, 5-1-5, 2-0.5-2.5, (3 to 3.3)-(0.6 to 0.9)-(3.1 to 3.2) D antigen units; more particularly dose of individual Type 1, Type 2, or Type 3 of Salk strain based IPV is selected from group of 8-2-5 and 10 -2 -10 D antigen units;

b) aluminium content per 0.5mL dose, is 0.4 mg AI3+ for Type 1,0.2mg AI3+ for Type 2, and 0.2mg AI3+ for Type 3;

c) preservative comprising of 2-Phenoxyethanol in a range of 2.5 mg/0.5 ml dose and formaldéhyde in a range of 12.5 pg/0.5 ml dose.

According to an embodiment of the présent disclosure, wherein the vaccine composition obtained from the method may be a bivalent composition comprising:

a) inactivated poliovirus selected from the group comprising of:

type 1 at a dose greater than 1 D-antigen units and less than 11 D-antigen units, per 0.5ml; and type 3 at a dose greater than 2 D-antigen units and less than 17 D-antigen units, per 0.5ml;

or type 1 at a dose greater than 1 D-antigen units and less than 11 D-antigen units, per 0.5ml; and type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml;

or type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; and type 3 at a dose greater than 2 D-antigen units and less than 17 D-antîgen units, per 0.5ml;

b) aluminium content per 0.5mL dose is 0.4 mg A13+ for Type 1,0.2mg AI3+ for Type 2, and 0.2mg Al3+ for Type 3;

c) preservative comprising of 2-Phenoxyethanol in a range of I mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.

In a preferred embodiment, wherein the vaccine composition obtained from the method may be a bivalent composition comprising:

a) inactivated poliovirus of Salk strain and the dose of individual Type 1 and Type 3 of Salk strain based IPV may be selected from the group of dose composition comprising of 7.510, 8-5, 10-5, 10-10, 10-12, 10-16, 7.5-10, 5-5, 5-5, 2-2.5, (3 to 3,3)-(3 to 3.2) D antigen units; more particularly dose of individual Type I and Type 3 of Salk strain based IPV is selected from group of 8-5 and 10-10 D antigen units;

or

a) inactivated poliovirus of Salk strain and the dose of individual Type I and Type 2 of Salk strain based IPV may be selected from the group of dose composition comprising of 7.516, 8-2, 10-2, 10-2, 10-2, 10-2, 7.5-16, 5-2, 5-1, 2-0.5, (3 to 3.3) -(0.6 to 0.9) D antigen units; more particularly dose of individual Type I, Type 2, or Type 3 of Salk strain based IPV is selected from group of 8-2 and 10 -2 D antigen units;

or

a) inactivated poliovirus of Salk strain and the dose of individual Type 2 and Type 3 of Salk strain based IPV may be selected from the group of dose composition comprising of 1610, 2-5, 2-5, 2-10, 2-12, 2-16, 16-10, 2-5, 1-5, 0.5-2.5, (0.6 to 0.9) - (3 to 3.2) D antigen units; more particularly dose of individual Type I, Type 2, or Type 3 of Salk strain based IPV is selected from group of 2-5 and 2 -10 D antigen units;

b) aluminium content per 0.5mL dose is 0.4 mg A13+ for Type 1, 0.2mg A13+ for Type 2, and 0.2mg A13+ for Type 3;

c) preservative comprising of 2-Phenoxyethanol in an amount of 2.5 mg/0.5 ml dose and formaldéhyde in an amount of 12.5 pg/0.5 ml dose.

According to an embodiment of the présent disclosure, wherein the vaccine composition obtained from the method may be a monovalent composition comprising:

a) inactivated poliovirus selected from the group comprising of:

type 1 at a dose greater than 1 D-antigen units and less than 11 D-antigen units, per 0.5ml;

type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; or type 3 at a dose greater than 2 D-antigen units and less than 1 7 D-antigen units, per 0.5ml;

b) aluminium content per 0.5mL dose is 0.4 mg AI3+ for Type 1, 0.2mg A13+ for Type 2, and 0.2mg A13+ for Type 3;

c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.

According to a preferred embodiment of the présent disclosure, wherein the vaccine composition obtamed from the method may be a monovalent composition comprising:

a) inactivated poliovirus type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml;

b) aluminium content per 0.5mL dose is 0.2mg A13+ for Type 2;

c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.

More preferably, the vaccine composition obtained from the method may be a monovalent composition comprising:

a) inactivated poliovirus type 2 at a dose of 2 D-antigen units, per 0.5ml;

b) aluminium content per 0.5mL dose is 0.2mg A13+ for Type 2;

c) preservative comprising of 2-Phenoxyethanol - 2.5 mg/0.5 ml dose and formaldéhyde 12.5 pg/0.5 ml dose.

According to another aspect of the présent disclosure, the vaccine composition prepared by instant methods can be “Combination Vaccines containing reduced dose IPV” wherein said non-IPV antigens of combination vaccines can be selected from but not limited to Diphtheria toxoid(D), Tetanus toxoid (T), Whole cell pertussis(wP), hepatitis B virus surface antigen////A'4gZ Haemophilus influenzae b PRP-Carrier prolein c:O«/z/gü/e(l Hb), Haemophilus influenzae (a, c, d, e, f sera types and the unencapsulaled strains),Neisseria meningitidis A antigen(s), Neisseria meningitidis C antigen(s), Neisseria meningitidis W-135 antigen(s), Neisseria meningitidis Y antigen(s), Neisseria meningitidis X antigen(s), Streptococcus Pneumoniae antigen(s), Neisseria meningitidis B bleb or purifted antigen(s), Staphylococcus aureus antigen(s), Anthrax, BCG, Hepatitis (A, C, D, E, F and G strains) antigen(s), Human papilloma virus, HIV, Salmonella typhi antigen(s) , acellular pertussis, modifîed adenylate cyclase, Malaria Antigen (RTS,S), Measles, Mumps, Rubella, Dengue,

Zika, Ebola, Chikungunya, Japanese encephalitis, rotavirus, Diarrheal antigens, Flavivirus, smallpox, yellow fever, Shingles, Varicella virus antigens.

According to an embodîment of the présent disclosure, wherein the Combination Vaccines comprising dose reduced IPV may comprise of antigens selected from Diphtheria toxoicKO), Tetanus loxoid (T), Whole cell pertussis(wP), acellular pertussis, hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conywg«te(Hib) other than dose reduced IPV as disclosed in earlier embodiment.

According to a preferred embodiment of the présent disclosure, wherein the Combination Vaccines (per 0.5 ml dose) comprising dose reduced IPV may comprise of D antigen in an amount of 1 to 50 Lf, the T antigen in an amount of l to 30 Lf, the wP antigen in an amount of 1 to 50 1OU or acellular pertussis antigen, the HBsAg antigen in an amount of I to 20 gg, the Hib antigen in an amount of 1 to 20 gg, the IPV Type 1 antigen in an amount of I -11 DU, the IPV Type 2 antigen in an amount of 0.4 - 8 DU, and the IPV Type 3 antigen in an amount of 2 - 17 DU.

A more preferred embodiment of the présent disclosure, wherein the Combination Vaccines (per 0.5 ml dose) comprising dose reduced IPV may comprise of D antigen in an amount of 10 or 22.5 or 25 Lf, , the T antigen in an amount of 2 or 7.5 or 10 Lf, the wP antigen in an amount of 12 or 15 or 16 IOU or acellular pertussis antigen, the HBsAg antigen in an amount of 8 or 10 or 12.5 gg, the Hib PRP-TT conjugale antigen in an amount of 8 or 10 or 13 gg of PRP, the inactivated polio virus (IPV) is a Salk straîn and the concentration of individual Type 1, Type 2, and Type 3 of Salk strain based IPV may be selected from the group of dose composition comprising of 7.5-16-10, 8-2-5, 10-2-5, 10-2-10, 10-2-12, 10-2-16,7.5-16-10, 52-5, 5-1-5, 2-0.5-2.5, (3 to 3.3)-(0.6 to 0.9)-(3.1 to 3.2) D antigen units; more particularly concentration of individual Type 1, Type 2 and Type 3 of Salk strain based IPV is selected from group of 8-2-5 and 10 - 2 - 10 D antigen units.

A more preferred embodiment of the présent disclosure, wherein the Combination Vaccines (per 0.5 ml dose) comprising dose reduced IPV may comprise of D antigen in an amount of 10 or 22.5 or 25 Lf, , the T antigen in an amount of 2 or 7.5 or 10 Lf, the wP antigen in an amount of 12 or 15 or 16 IOU or acellular pertussis antigen. the HBsAg antigen in an amount of 8 or 10 or 12.5 gg, the Hib PRP-TT conjugale antigen in an amount of 8 or 10 or 13 gg of PRP, the inactivated polio virus is a Sabin strain and the concentration of individual Type 1, Type 2 and Type 3 of Sabin strain based IPV may be selected from the group of dose composition comprising of 5-16-10, 2.5-8-5, 5-8-10 D antigen units; more particularly concentration of individual Type 1, Type 2, and Type 3 of Sabin strain based IPV is 5-16-10 D antigen units.

Alternatively, the combination Vaccines (per 0.5 ml dose) comprising dose reduced IPV may comprise of IPV type 1 or IPV type 2 or IPV type 3, or IPV type I and 2, or IPV type 1 and 5 3, or IPV type 2 and 3, or IPV type 1, 2 and 3.

According to an embodiment of the présent disclosure, wherein the vaccine composition obtained from the method may be a monovalent, bivalent or trivalent composition comprising inactivated poliovirus comprising of Sabin type 2 at a dose greater than 0.4 D-antigen units and less than 18 D-antigen units, per 0.5ml.

Components of the Combination Vaccine composition:

• Inactivated Polio Virus (IPV)

Improved methods of Polio virus (Salk or Sabin strains) inactivation by formaldéhyde in presence of Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) resulting in maximum recovery of D-antigen. Subséquent adsorption of said IPV on aluminium 15 hydroxide provides significantly dose reduced IPV compositions. Spécification disclosed in detail in the earlier embodiments.

• Haemophilus influenzae b (Hib) PRP - Protein Conjugate:

Haemophilus influenzae type b is a Gram-negative bacterium that causes meningitis and acute respiratory infections, mainly in children. The outermost structure of H. influenzae type 20 b is composed of polyribosyl-rîbitol-phosphate (PRP), a polysaccharide that is responsible for virulence and immunity. PRP is a hapten which is considered as poor immunogenic in nature hence PRP is linked covalently with a carrier protein to make highly immunogenic Hib antigen. This process changes the polysaccharide from a T-independent to a T-dependent antigen and greatly improves immunogenicity, particularly in young children.

The présent invention may comprise of préparation of Hib PRP-protein conjugate. It may be noted that the carrier proteins used for the conjugation of the Hib antigen may be selected from group comprising of Tetanus toxoid, CRM 197 (Cross Reactive Material 197, a genetically detoxified form of diphtheria toxoid), Diphtheria toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H.

influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and léthal factor (LF) of Bacîllus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human sérum albumin, bovine sérum albumin (BSA) and purified protein dérivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogenderived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins or any équivalents thereof. Preferably the carrier protein in conjugale is selected from TT or CRM197.

An aspect of the présent invention, wherein the Hib antigen may be derived from the capsular polysaccharide of Haemophilus influenzae type b strain. To produce the PRP polysaccharide, H. Influenzae type-b bacteria was grown in semi synthetic media under certain conditions of température, agitation and optical density etc. PRP is an outer membrane bound polysaccharide, gets released into the medium during the fermentation under agitation condition. Fermented biomass separated broth contains crude PRP, which was again purified by précipitation using a detergent N, N, N-trimethyl-l-hexadecanaminium bromide, followed by éthanol gradient précipitation and filtration. Final purified PRP polysaccharide was tested for meeting the spécifications like endotoxin, nucleîc acid and protein as per the WHO, BP, EP, IP etc.

Another aspect of the présent invention, wherein the polysaccharide - protein conjugale may be prepared by coupling of polysaccharide (PRP) with a carrier protein. Hib PRP was conjugated to carrier protein using conjugation process comprising of steps including depolymerization of PRP using alkaline buffer to achieve size reduced PRP; treatment with cyanylation agent like CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanate ester; coupling of activated cyanylated polysaccharide to amino group of carrier protein; purification of final conjugale using ultrafiltration.

More preferably, the optimal input ratio of reactants i.e. PRP, CDAP and CRM 197 may be selected at 1:1.5:1 ratio for conjugation reaction. During conjugation, purified PRP polysaccharide was depolymerized using an alkaline buffer (0.4M Carb-Bicarbonate buffer, pH 10.5 ±0.1) to achieve size reduced PRP. Size reduced PRP was treated for cyanylation using CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoroborate) chemistry to form a cyanate ester. The activated cyanylated polysaccharide was coupled directly with amino group on the carrier protein CRM 197. The degree of conversion of Hib conjugale was confirmed by the offlîne testing using HPLC. The conjugation reaction was quenched by achieving the desired level of conversion of conjugate with the spécification of not less than 65% conversion of Hib conjugate, and then conjugate reaction was neutralized by Glycine (2M) addition. The Hib PRP-CRM197 Conjugate was further purified on ultra-filtration membrane filters (300kDa and lOOkDa) to remove nonreactive reagents and byproducts. Final conjugate bulk is 0.22 pm fîltered and stored at 2-8°C.

More preferably, Hib PRP may be conjugated to carrier protein wherein the saccharide: protein ratio (w/w) may be between 0.4 and I; and the free PRP content in final Hib PRP protein conjugate bulk may be not more than 5%, more preferably is less than 2%.

Yet another aspect of the présent invention, wherein the Hib PRP may be conjugated to teianus toxoid (TT) by CNBr chemistry, Reductive amination chemistry, Cyanylation chemistry or any other chemistry already discloses in Kniskem et al., “Conjugation: design, chemistry, and analysis” in Ellis et al., Development and clinical uses of Haemophilus influenzae type B conjugate vaccines. New York: Marcel Dekker, 1994: 37-69

Yet another aspect of the présent invention, wherein the carrier protein may be présent in both free and conjugated form in a composition of the présent disclosure, the unconjugated form is preferably no more than 20% of the total amount of the carrier protein in the composition as a whole, and more preferably présent at less than 5% by weight, more preferably is less than 2%.

Yet another aspect of the présent invention, wherein the Hib antigen is not substantially adsorbed on to any adjuvant.

Yet another aspect of the présent invention, wherein the Hib antigen may not be subjected to deliberate or intentional adsorption on any adjuvant.

Yet another aspect of the présent invention, wherein the percentage of adsorption of Hib antigen on to any adjuvant may be less than 20%.

Yet another aspect of the présent invention, wherein the Hib antigen used in the combination vaccine of the présent disclosure is derived from the capsular polysaccharide of Haemophilus influenzae type b (Hib) strain 760705.

• Diphtheria toxoid

Diphtheria is an infectious disease caused by the bacterium Corynebacterium diphtheria, which primarily infects the throat and upper airways, and produces a toxin affecting other organs. Diphtheria toxin is an exotoxîn secreted by Corynebacterium diphtheria, possesses antigenic properties and is toxîc in nature. To reduce toxicity, the toxin is converted to the inactive toxoid by subjecting it to inactivation. The inactivation process may be selected from one or more of treatment wîth Heat, UV, Formalin /Formaldéhyde, Acetylethyleneimine, etc, To increase immunogenicity, the toxoid is adsorbed to an adjuvant. The toxoid thus formed is able to induce anti toxin antibodies against C diphtherîa. Existence of dimers can lead to adverse reactions,

The présent invention may comprise of préparation of Diphtherîa toxoid as disclosed below.

In first aspect of the présent invention, diphtherîa toxin (exotoxin) may be obtained from Corynebacterium diphtherîa and detoxified using a suitable inactivating agent. The example of suitable inactivating agent includes Formaldéhyde.

In second aspect of the présent invention, diphtherîa toxoid obtained may be purified using Gel filtration chromatography with Sephacryl S-300 HR as resin with linear flow rate of 2 - 5 ml/min and stabilized by addition of an amîno acid buffer solution (Histidine, lysine, glycine, arginine) or polysorbate solution at a final concentration of 5 - 300 mM, and stored at température -20 to +40 °C till further use. The purified D thus obtained comprises of homogenous fraction devoîd of undesirable aggregates (Refer Figure 1) with atleast 80 - 90 % monomeric diphtherîa toxoid further used for formulation of multivalent vaccine(s). Further PLgel, Sephacryl S-200HR. Sephadex, Bio-Gel (cross linked polyacrylamide), agarose gel and/or Styragel may also be used for the purpose of purification using Gel perméation chromatography. The purified diphtherîa toxoid is stabilized by addition of Histidine (200mM) amîno acid buffer solution.

In third aspect of the présent invention, diphtherîa toxoid may be adsorbed on to adjuvant selected from the group of aluminium sait (Al³⁺⁾ such as aluminium hydroxide (AI(OH)s) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS2I, a CpGcontaining oligodeoxynucleotide adjuvant, liposome, or oil-in-water émulsion or a combination thereof.

Yet preferably diphtherîa toxoid may be adsorbed on to Aluminium sait including Aluminium hydroxide and Aluminium phosphate, preferably on Aluminium phosphate.

Yet preferably the Diphtherîa toxoid (D) antigen may be adsorbed on to aluminium phosphate having percentage absorption of atleast 50%.

• Tetanus toxoid (T)

Tetanus is an acute infectious disease caused by toxigenic straîns of the bacterium Clostridium tetani (C. tetani), a gram-positive, spore-forming, strictly anaérobie bacterium.

Tetanus toxin is an exotoxin secreted by Clostridium tetani, possesses antigenic properties and is toxic in nature. To reduce toxicity, the toxin is converted to the inactive toxoid by subjecting it to inactivation. The inactivation process may be selected from one or more of treatment with Heat. UV, Formalin /Formaldéhyde, Acetylethyleneimine, etc. To increase 5 immunogenicity, the toxoid is adsorbed to an adjuvant. The toxoid thus formed is able to induce anti toxin antibodies against Clostridium tetani. Existence of dimers can lead to adverse reactions.

. The présent invention may comprise of préparation of Tetanus toxoid as disclosed below.

In First aspect of the présent invention, Tetanus toxin may be obtained from Clostridium 10 tetani and detoxified using a suitable inactivating agent. The example of suitable inactivating agent includes Formaldéhyde.

In second aspect of the présent invention, Tetanus toxoid obtained may be purified using Gel > filtration chromatography with Sephacryl S-300 HR as resin with linear flow rate of 2 - 5 । ml/min and stabilized by addition of an amino acid buffer solution (Histidine, lysine, glycine, 15 arginine) or polysorbate solution at a final concentration of 5 - 300 mM, and stored at température -20 to +40 °C tîll further use. The purified T thus obtained was a homogenous fraction devoid of undesirable aggregates with atleast 80 - 90 % monomeric tetanus toxoid further'used for formulation of multivalent vaccine. Further PLgel, Sephacryl S-200HR, Sephadex, Bio-Gel (cross-linked polyacrylamide), agarose gel and Styragel may also be used 20 for the purpose of purification using Gel perméation chromatography. The purified tetanus toxoid is stabilized by addition of Histidine (200mM) amino acid buffer solution.

In third aspect of the présent invention, Tetanus toxoid may be adsorbed on to adjuvant selected from the group of aluminium sait (Al³⁺⁾ such as aluminium hydroxide (AI(OH)î) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS2I, a CpG25 containîng oligodeoxynucleotide adjuvant, liposome, or oil-in-water émulsion or a combination thereof.

Yet preferably tetanus toxoid may be adsorbed on to Aluminium sait including Aluminium hydroxide and Aluminium phosphate, preferably on Alum phosphate.

Yet preferably the tetanus toxoid (T) antigen may be adsorbed on to aluminium phosphate 30 having percentage adsorption of atleast 40%.

• Pertussis Antigen

I e ·! <15 i

.il··. .1 l

Pertussis (whooping cough) is caused by Bordetella pertussis, a small Gram-negative coccobacillus that infects the mucosal layers of the human respiratory tract. Two forms of vaccine are in use, the whole-cell pertussis vaccine (wP), and the acellular pertussis vaccine (aP). Whole-cell pertussis vaccines are suspensions of the entire B. pertussis organism that has been inactivated, usually with formai in. Immunization with wP vaccine is relatively inexpensivetand highly effective. Also, presence of wP in combination vaccines acts as an adjuvant for many other antigenic component.

Acellular pertussis (aP) vaccines contain purified components of B. pertussis such as inactivated pertussis toxin either alone or in combination with other B. pertussis components such as filamentous haemagglutinin, fimbrial antigens, pertactin, and modified adenylate cyclase more particularly a non-cytotoxic polypeptide, derived from the adenylate cyclase protein (CyaA-derived polypeptide) of a Bordetella pertussis. Acellular pertussis vaccine offers less adverse reaction as compared to wP vaccine.

t ' 1

The présent invention may comprise of préparation of the pertussis vaccine comprising pertussis antigen selected from one or more of whole cell pertussis or acellular pertussis.

In first aspect of the présent invention, pertussis vaccine may be an acellular pertussis antigen .i selected from one or more of filamentous haemagglutinin, fimbrial antigens, pertactin, and modified adenylate cyclaset more particularly a non-cytotoxic polypeptide, derived from the adenylate cyclase protein (CyaA-derived polypeptide) of a Bordetella pertussis. Acellular pertussis antigens may be expressed in suitable host using recombinant DNA technology.

Preferablyiacellular pertussis antigen may be selected from - Bordetella toxin in detoxified form (in particular either genetically or chemically detoxified), in particular Pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly Pertussis toxoid: 1 to 50 'micrograms (More particularly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8pg); - Pertactin: 1 to 20 micrograms (More particularly 2.5gg); Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

In second aspect of the présent invention, pertussis vaccine may be a whole cell pertussis comprising of Bordetella pertussis strains 134, 509, 25525 and 6229 in a spécifie ratio and subsequently inactivated by utilizing improved methods of inactivation devoid of thimerosal; hence leading to reduced reactogenicity and increased potency. Preferably, wP antigen is made from Bordetella pertussis strains 134, 509, 25525 and 6229 mixed in a ratio of 1:1:0.25:0.25.

ln third aspect of the présent invention, wP inactivation process may mclude heat inactivation at 56±2°C for I0 to I5 minutes in presence of formaldéhyde; wherein wP bulk remains nonclumpy and easily homogenized thereby leading to reduced reactogenicity and giving better wP potency for a longer duration.

• Hepatitis B surface antigen (HBsAg)

Hepatitis B is a potentially life-threatening liver infection caused by the Hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) is a surface protein that also acts as an immunogen in highly effective vaccines for prévention of HBV infection. HBsAg protein can ¹ Ί-.

be recombinantly expressed in a suitable host microorganism; or can be isolated from the blood plasma of a chronic Hepatitis B patient/carrier.

.1 . . j ;

The présent invention may comprise of préparation of the Hepatitis B surface antigen (HBsAg) as disclosed below. ., h j.- . .

In one of the aspects of présent invention, HBsAg may be expressed in, Hansenula, ,, r_f! . ,ί polymorpha ‘yeast cells using recombinant DNA technology. Other, yeasts such :as , _: ,,

Saccharomyces cerevisiae may also be used as host cell for recombinant expression of ‘il i ... o,. r . .·. _: .

HBsAg.

c:s-,i£.d 'J · >'·.· vu,, : !*' . !.

In one of the aspect of présent invention, Hepatitis B antigen (HBsAg) may be^adsorbcd οη,ΐο ;

adjuvant selected from the group of aluminium sait (AI³⁺⁾ such as aluminium hydroxide ...

(Al(OH)j) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, _t QS21, a CpG-containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water émulsion .V i Il:·.

or a combination thereof.

Yet preferably Hepatitis B antigen (HBsAg) may be adsorbed on to Aluminium sait including Aluminium hydroxide and Aluminium phosphate, preferably on Alum phosphate.

Yet preferably the Hepatitis B surface antigen (HBsAg) may be adsorbed on to aluminium phosphate having percentage adsorption of atleast 70%.

The présent invention may comprise of the process for préparation of combination vaccine composition/formulation comprising Dose reduced IPV as disclosed below.

According to an embodiment of the présent disclosure, wherein the process for préparation of Combination Vaccine Compositions comprising Dose reduced IPV, HBs, D, T, wP, and Hib PRP - Protein conjugate may be accordingly as given below:

a) adsorbing IPV (Sabîn/Salk strain) bulk individually on Aluminium hydroxide, followed by pH adjustment to 6.2 - 6.6, more preferably 6.5.

b) adsorbing D on Aluminium phosphate, followed by pH adjustment to 5.5 - 6.5

c) adsorbing T on Aluminium phosphate, followed by pH adjustment to 5.5 - 6.5

d) adsorbing HBsAg on Aluminium phosphate, followed by pH adjustment to 6.0 - 6.5.

e) blending the mixture as obtained in step (b), (c), (d) by agitation at room température for 18 - 24 hours.

f) Blending the mixtures as obtained in step (a) and (e), followed by pH adjustment to 6.4 6.6 and agitation at room température for 60 minutes.

g) adding inactivated wP antigen / acellular pertussis antigen and a stabilizer more preferably aminoacid buffer solution (Histidine 100 -300 mM) to the above mixture in step (f), followed by agitation for 60 minutes and left in static condition for overnight at 2 - 8 °C.

h) adding Hib antigen to the mixture obtained in step (g) at 2 - 8 °C, followed by pH adjustment to 6.4 - 6.6.

i) Adjusting pH to 6.0 to 7.0 with Sodium Hydroxide / Sodium Carbonate and adding normal saline (0.9% bJaCl) or WFI (q.s.) to make up the volume of the mixture obtained in step h, followed by agitation for 2 hours.

According to an embodiment of the présent disclosure, the pH of the composition/formulation may be in the range of pH 6.0 to pH 8.0; more preferably in the range of pH 6.0 to pH 7.5; still more preferably in the range of pH 6.2 to pH 7.2; and most preferably in the range of pH 6.3 to pH 6.8.

According to an embodiment of the présent disclosure, WFI or 0.9% saline (NaCI) may be added to the final combination vaccine composition to make up the volume.

According to another aspect of the présent disclosure, wherein the vaccine compositions obtained from the instant method may additionally comprise of a buffering agent selected from the group consisting of carbonate, phosphate, acetate, succînate, borate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents including a phosphate buffering agent that contains sodium phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH. In another example, the buffering agent contains Tris (hydroxymethyl) aminomethane, or Tris, formulated to achieve the desired pH. Yet in another example, the buffering agent could be the minimum essential medium with Hanks salts. Other buffets, such as HEPES, piperazine-N, Ν'-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged by the présent disclosure. The buffer aids in stabmzing the immunogenic composition of the présent disclosure. The amount of the buffer may be in the range of O.l mM to I00 mM, preferably selected from 5mM, 6mM, 7mM, 22 mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM and 30mM.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may additionally comprise of pharmaceutically acceptable excipients selected from the group consisting of surfactants, polymers and salts. Examples of Surfactants may include non-ionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of the polymers may include dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin, etc. Examples of the salts may include NaCl, KCI. KH2PO4, Na2HPO4.2H2O, CaCh, MgCh, etc. Preferably, the sait may be NaCl. Typically, the amount of the sait may be in the range of 100 mM to 200 mM.

Amino acids, such as Histidine, glycine, arginine and lysine may be added to stabilize the vaccine composition.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may additionally comprise of an immunostimulatory component selected from the group consisting of an oil and water émulsion, MF-59,a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A dérivatives, Monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, ASOl, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complété adjuvant, Freund’s incomplète adjuvant, polymers, co-polymers such as polyoxyethylenepolyoxypropylene copolymers, încluding block co-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram négative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-l), Quil A, choiera toxin B subunit, polyphosphazene and dérivatives, mycobacterium cell wall préparations, mycolic acid dérivatives, non-ionic block copolymer surfactants, OMV, fHbp, saponin combination with sterols and lipids.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may additionally comprise of preservative selected from the group consisting of methylparaben, propylparaben, Benzéthonium chloride (Phemerol), Phénol, m-cresol, Thiomersal, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, or benzyl alcohol or a combination thereof. A vaccine composition may include preservative for a single immunîzation, or may include preservative for multiple immunizations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptie adaptor for removal of material. Typically, the amount of the preservative may be in the range of O.l mg to 50 mg.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may additionally comprise of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, flavoring agents, colors, and the like, depending upon the route of administration and the préparation desired.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may be fully liquid but is not limîted thereto. Suîtable forms of liquid préparation may include solutions, suspensions, émulsions, syrups, isotonie aqueous solutions, viscous compositions and élixirs that are buffered to a desired pH.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may be formulated for use in a method for reducing the onset of or preventing a health condition comprising polio virus infection involving administration of an immunologically effective amount of the immunogenic composition to a human subject via parentéral or subeutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injectable administration or sustained release from implants or administration by eye drops or nasal or rectal or buccal or vaginal, pérorai or intragastric or mucosal or perlinqual, alveolar or gingival or olfactory or respiratory mucosa administration or any other routes of immunization.

Alternatively, the IPV vaccine compositions may be formulated in the form of transdermal préparations including lotions, gels, sprays, ointments or other suîtable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aérosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aérosol dispenser. Aérosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size. When in the form of solutions, suspensions and gels, in some embodiments, the immunogenic compositions contain a major amount of water (preferably purified water) in addition to the active ingredient(s).

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may be stable at 2-8 deg C for 12 to 36 months; at 25 deg C for 2 to 6 months; at 37 deg C for l week to 4 weeks.

Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may be formulated as single dose vials or multidose vials (2 Dose or 5 Dose or 10 Dose vials) or multidose kit or as pre-filled syringes wherein the said immunogenic composition may be given in a single dose schedule, or preferably a multiple dose schedule în which a primary course of vaccination is followed by l-3 separate doses given at subséquent time intervals after l-3 years if needed. The dosage regimen will also, at least in part, be determined on the need of a booster dose required to confer protective immunity.

Yet alternatively the composition may be formulated for administration to a human subject or children 2 years of âge or below according to two dose regimens consisting of a First dose, and second dose at subséquent time intervals after l -3 years.

Yet alternatively the composition may be admînîstered concomitantly with other drugs or any other vaccine.

Compositions may be presented în vials, or they may be presented in ready filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses (e.g. 2 doses). In one embodiment the dose is for human. In a further embodiment the dose is for an adult, adolescent, toddler, infant or less than one-year old human and may be administered by injection.

Vaccines of the invention may be packaged in unit dose form or in multiple dose form (e.g. 2 doses). The said multidose composition can be selected from a group consisting of 2 dose, 5 dose and 10 doses. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5mL.

Biological Source of Strains used in Vaccine composition:

DIPHTHERIA TOXOID:

The strain Corynebacterium diphlheriae PW8 CN2000 was obtained from the Wellcome Research Laboratory, London, United Kingdom by the National Control Authority Central Research Institute (C.R.L) Kasauli, Himachal Pradesh, India in lyophilized form in the year !973.The strain was revived and further lyophilized under Master Seed Lot- C. diphtheriae !

CN2000 Al atC.R.I. Kasauli. ^!

TET ANUS TOXOID:

The strain Clostridium tetani Harvard Strain No.49205 was obtained from The Rijks Institute Voor de Volksgezondheid (Netherlands) by the National Control Authority C.R.I. Kasauli, in Lyophilized form.

PERTUSSIS:

Manufacturing of Pertussis vaccine bulk at SIIPL involves usage of four strains of Bordetella pertussis viz. Strains 134, 509, 6229 and 25525.The Master Seed of Strains 134 and 509 are originally from Rijks Institute, The Netherlands, obtained through National Control Authority, Central Research Institute, Kasauli, Himachal Pradesh, India. The Master Seed of Strains 6229 and 25525 are originally from Lister Institute, England.

HEPATITIS B:

Rheîn Bîotech (Germany) constructed the recombinant Hansenula polymorpha strain containing the HBsAg surface antigen gene. Rhein Biotech also made the Master Cell Bank (MCB Hansenula polymorpha K3/8-I strain ADW, 12/94) and performed ail the characterîzation tests on this bank.

HAEMOPHILUS INFLUENZAE TYPE b:

The source organism for génération of cell substrate is Haemophilus influenzae type b, strain 760705. The strain was originally isolated from a 2 year and 2 months old baby boy (born on 14-8-74) in November l976.Three passages of the strain took place before storage at -70 °C at the Academie Medical Centre (AMC), LJniversity of Amsterdam. This strain was transferred to SIIPL as a part of collaboration between SIIPL and Netherlands Vaccines

Institute (NVI, The Netherlands).

IPV:

The strain and source of Salk poliovirus is given below.

Poliovirus type 1:

Salk

Strain: Mahoney or Brunhilde

Source: Bilthoven Biologicals, Netherlands

Sabin

Source: PT Bio Farma, Indonesia

Poliovirus type 2:

Salk

Strain: MEFI

Source: Bilthoven Biologicals, Netherlands

Sabin

Source: PT Bio Farma, Indonesia

Poliovirus type 3:

Salk

Strain: Saukett

Source: Bilthoven Biologicals, Netherlands

Sabin

Source: PT Bio Farma, Indonesia

Throughout this spécification the word “comprise”, or variations such as “comprises” or 20 “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of éléments, integers or steps, but not the exclusion of any other element, integer or step, or group of éléments, integers or steps.

The use of the expression “at least” or “at ieast one” suggests the use of one or more éléments or ingrédients or quantifies, as the use may be in the embodiment of the invention to achieve 25 one or more of the desired objects or results. While certain embodiments of the inventions hâve been described, these embodiments hâve been presented by way of exampie only, and are not intended to limit the scope of the inventions. Variations or modifications to the formulation of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within 30 the spirit of this invention.

The numerical values given for various physical parameters. dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assignée! to the physical parameters, dimensions and quantifies fall within the scope of the invention unless there is a statement in the spécification to the contrary.

While considérable emphasis has been placed herein on the spécifie features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Salk is a wild strain, handlîng of these live Salk viruses require BSL-4 facility. There is no such facility to handle wild Salk strain in India. The facility is available only with Bilthoven Biologicals, affiliate company of Sérum Institute. So, the Salk strains were inactivated in Bilthoven Biologicals and then these inactivated Salk strains were further researched to generate inactivation related experimental data.

Examples:

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventer to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments which are disclosed and still obtain a like or similar resuit without departing from the spirit and scope of the invention.

Example 1 - Purification of Polio Virus

I. Tangential flow filtration (TFF):

Clarîfied harvest pool was concentrated to 10X using tangential flow filtration System with lOOKda cassettes(O.5m²) and then diafiltered 3 times of harvest volume with phosphate buffer (30mM-70mM, pH: 7.0)

2. Column Chromatography: Ion Exchange Chromatography (IEC).

lOX TFF concentrate was passed through DEAE Sepharose fast flow (Weak- Anion exchanger) packed in column xk-26 using Akta explorer (GE Healthcare). Negatively charged impurities was found to bind to the column whereas polio virus was collected in flow through with phosphate buffer (30mM-70mM, pH: 7.0)

3. Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) exchange:

To minimize the loss of antigen in a quite cumbersome inactivation procédure! I3days), purified virus pool was buffer exchanged from phosphate buffer to Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) (30mM-70mM. pH: 7) with TFF System (100 KDa, O.l m2). The purified virus pool was exchanged with three volumes of Non-Phosphate

Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate).

Example 2 - Inactivation of Polio Virus in presence of Non-Phosphate Buffers (TRIS, TBS, MOPS, HEPES, and bicarbonate) and Phosphate buffer

10X concentrate diluted 10 times with M - 199 (with 0.05% glycine) so as to achieve final concentration Ix. Inactivation agent formalin (0.025%) was added into purified virus bulk while constant mîxing. Inactivation was carried out at 37°C with continuons stirring for 13 days containing 0.22μ filtration on 7th day and 13th day.

Table 1: M — 199 composition
Component	Concentration (g/L)
Calcium Chloride	0.2
Ferrie Nitrate · 9H2O	0.00072
Magnésium Sulfate (anhydrous)	0.09767
Potassium Chloride	0.4
Sodium Acetate (anhydrous)	0.05
Sodium Chloride	6.8
Sodium Phosphate Monobasic (anhydrous)	0.122
L-Alanine	0.025
L-Arginine · HCl	0.07
L-Aspartic Acid	0.03
L-Cysteine · HCl · H2O	0.00011
L-Cystine · 2HC1	0.026
L-Glutamic Acid	0.0668
L-Glutamine	0.1
Glycine	0.05
L-Histidine · HCl · H2O	0.02188
Hydroxy-L-Proline	0.01
L-lsoleucine	0.02
L-Leucine	0.06
L-Lysine · HCl	0.07
L-Methionine	0.015
L-Phenylalanine	0.025
L-Proline	0.04

L-Serine	0.025
L-Threonine	0.03
L-Tryptophan	0.01
L-Tyrosine · 2Na · 2H2O	0.05766
L-Valine	0.025
Ascorbic Acid · Na	0.0000566
D-Biotin	0.00001
Calciferol	0.0001
Choline Chloride	0.0005
Folie Acid	0.00001
Menadione (sodium bisulfite)	0.000016
myo-lnositol	0.00005
Niacinamide	0.000025
Nicotinic Acid	0.000025
p-Aminobenzoic Acid	0.00005
D-Pantothenic Acid (hemicalcium)	0.00001
Pyridoxal · HCl	0.000025
Pyridoxine · HCl	0.000025
Retinol Acetate	0.00014
Riboflavin	0.00001
DL-a-Tocopherol Phosphate · Na	0.00001
Thiamine · HCl	0.00001
Adenine Sulfate	0.01
Adenosine Triphosphate · 2Na	0.001
Adenosine Monophosphate · Na	0.0002385
Cholestérol	0.0002
Deoxyribose	0.0005
Glucose	1
Glutathione (reduced)	0.00005
Guanine · HCl	0.0003
Hypoxanthine	0.0003
Phénol Red · Na	0.0213
PolyoxyethylenesorbitanMonooleate (TWEEN 80)	0.02
Ribose	0.0005
Thymine	0.0003
Uracil	0.0003
Xanthine · Na	0.000344
Sodium Bicarbonate	2.2

A. Effect of non-phosphate buffer (TRIS) on D antigen loss

Table 2: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of TRIS buffer (30 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	408.1	181.4	34.1

Table 3: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of TRIS (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7

After Formalin inactivation

487.1

185.2

37.9

Table 4: D-Antigen Units (DU/ml) before F< Formalin inactivation in presence of TRIS buffer	jrmalin inactivation and after 50 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	451.9	175.9	31.0

Further, TRIS Buffer at a concentration of 40mM was found to be most efficient in terms of D-Antigen content préservation for sIPV l,2 and 3.

B. Effect of non-phosphate buffers other than TRIS (TBS, MOPS, HEPES, and 5 bicarbonate) on D antigen loss compared to phosphate buffer:

Table 5: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of phosphate buffer (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	52.7	22.63	4.21

Table 6: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of TBS (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	399.15	170.5	22

Table 7: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of MOPS (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	400.21	165	20.1

Table 8: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of HEPES (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	385.45	172	19.9

Table 9: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of bicarbonate (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	395.6	179	21.3

When formaldéhyde inactivation methods were particulariy carried out in presence of phosphate buffer, sîgnîficant D-antigen losses were observed, whereas it was found that formaldéhyde inactivation in presence of Non-Phosphate Buffer other than TRIS (TBS, MOPS, HEPES, and bicarbonate) resulted in minimum loss of D-antigen.

Method: D-antigen content détermination by ELISA. Day 1: Plate coating:

1. 100μI of spécifie bovine anti polio was pipetted in PBS per well

2. Microtiter plate was sealed and incubated ovemight at room température.

Day 2: Blocking:

1. The plates were washed (Washing/dilution buffer -0.05% tween 20 in Ix PBS)3 times.

2. 300μΙ block buffer (1% BSA in PBS) was pipetted per well.

3. The plate was sealed and incubated for 45minutes at 37±1°C.

Sam pie addition:

1. The plate was washed 3 times.

2. 100μΙ of sample diluent was added in ail wells except well of row A.

3. 100μΙ standard was added to first two wells of column 2 and 3.

4. 100μΙ sample was added to first two wells of column 4-12.

5. Pre-diluting sample to a suitable concentration.

6. 100μ 1 sample dîluents was added to first two wells of column 1.

7. Serial two fold dilution were made down the column by transferrîng lOOul from each well to adjacent well of the same column and dîscarding lOOul from the last well.

8. Incubating at 37°c for 2 hr.

9. Plates were kept overnight at 4°C.

Day 3: Monoclonal antibody addition:

l. The plate was washed 3 times.

2. 100μI diluted ( l :240) type spécifie monoclonal antibodies were added.

3. The plates were sealed and incubated for 2 hours at 37°C.

Conjugate:

l. The plate was washed 3 times

2. ΙΟΟμΙ diluted conjugate (Typel- 1:2400, Type2- 1:1500, Type3 - 1: 4800) was added.

3. The plate was sealed and incubated for 1 hour at 37°C.

Substrate addition:

1. 100μΙ TMB substrate was added to ail wells.

2. Mixture incubated at room température for 10 minutes.

3. Reaction was stopped by adding ΙΟΟμΙ 2M H2SO4.

4. Plate was read at 450/630nm.

5. D antigen concentration was calculated using KC4 software.

Example 3 - Adsorption of Inactivated Polio Virus (IPV):

1. Autoclaved 1 % stock of Al(OH)j was used for the préparation of formulations.

2. Desired volume of A1(OH)3 was taken to get the required concentration of alum in a 100 ml glass bottle/blending vessel.

3. Inactivated polio virus bulk with known D-Ag Unit was added and volume make up was done with a stérile dilution medium comprising 2-phenoxyethanol and Formaldéhyde as preservative.

4. Final formulation pH was adjusted to 6.0 - 7.0 with I N HCl / NaOH.

5. The formulation bulk was kept on magnetic stirrer or blending is performed by stirring at 200 to 250 rpm continuously overnight or not less than 16 hours at 2-8°C to form a homogeneous mixture.

6. The vaccine formulation bulk was filled in a type-l glass vials with bromo-butyl rubber 5 stoppers and aluminium seal with coloured polypropylene flip cap.

Stérile dilution medium is made up of M-199 Medium containing 0.5 % glycine, 2Phenoxyethanol, Formaldéhyde, Water for Injection (WFI)

Example 4 - IPV Vaccine Composition

Inactivated Polio Virus (IPV) Vaccine (Adsorbed) is a yellowish to orange turbid liquid in which the minerai carrier tends to settle upon keeping.

Each dose of vaccine (0.5 mL) contains inactivated poliovirus (IPV):

Table 10: Monovalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.	Ingrédients	Concentration/ 0.5 mL	Function
a)	Inactivated Type 1. Mahoney Type 1	10 D antigen units	Active ingrédient (drug substance)
b)	Aluminium hydroxide (as Al+++), Adjuvant	0.4 mg	Adjuvant (Absorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg 12.5 pg	Stabilizer/ Preservative

Table 11: Monovalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.	Ingrédients	Concentration/ 0.5 mL	Function
a)	Inactivated Type 2. MEF -1 strain	2 D antigen units	Active ingrédient (drug substance)
b)	Aluminium hydroxide (as Al·^). Adjuvant	0.2 mg	Adjuvant (Absorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg 12.5 gg	Stabilizer/ Preservative

Table 12: Monovalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. | Ingrédients	Concentration/ 0.5 mL	Function

No.
a)	Inactivated Type 3, Saukett	10 D antigen units	Active ingrédient (drug substance)
b)	Aluminium hydroxide (as AIH+). Adjuvant	0.2 mg	Adjuvant (Adsorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg 12.5 pg	Stabiliser/ Préservâtive

Table 13: Bivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.	Ingrédients	Concentration/ 0.5 mL	Function
a)	Inactivated Type 1, Mahoney Type 1 Inactivated Type 3, Saukett	10 D antigen units 10 D antigen units	Active ingrédient (drug substance)
b)	Aluminium hydroxide (as AI***), Adjuvant	0.4 mg A13+ for Type 1 0.2mg AI3+ for Type 3	Adjuvant (Adsorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg 12.5 pg	Stabilizer/ Preservative

Table 14: Bivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.	Ingrédients	Concentration/ 0.5 mL	Function
a)	Inactivated Type 2, MEF -1 strain Inactivated Type 3. Saukett	2 D antigen units 10 or 16 D antigen units	Active ingrédient (drug substance)
b)	Aluminium hydroxide (as Al^). Adjuvant	0.2 mg AI3+ for Type 1 0.2mg A13+ for Type 3	Adjuvant (Adsorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg 12.5 pg	Stabilizer/ Preservative

Table 15: Bivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.	Ingrédients	Concentration/ 0.5 mL	Function
a)	Inactivated Type 1, Mahoney Type 1 inactivated Type 2, MEF -1 strain	10 D antigen units 2 D antigen units	Active ingrédient (drug substance)

b)	Aluminium hydroxide (as Al’1}. Adjuvant	0.4 mg AI3+ for Type l 0.2mg AI3+ for Type 2	Adjuvant (Adsorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg 12.5 pg	Stabiliser/ Preservative

Table 16: Trivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.	Ingrédients	Concentration/ 0.5 mL	Function
a)	Inactivated Type 1, Mahoney Type 1 Inactivated Type 2, MEF -1 strain Inactivated Type 3, Saukett	10 D antigen units 2 D antigen units 10 antigen units	Active ingrédient (drug substance)
b)	Aluminium hydroxide (as Al414), Adjuvant	0.4 mg AI3+ for Type 1 0.2mg A13+ for Type 2 0.2mg A13+ for Type 3	Adjuvant (Adsorbent)
c)	2-Phenoxyethanol Formaldéhyde	2.5 mg •2.5 pg	Stabilizer/ Preservative

Example 4 - Real time Stability data of IPV vaccine (Salk, Adsorbed) at 2-8°C for 12 Months (M), 25°C for 6 month and 37°C for 7 days (D).

Table 17 - IPV 1 dose Real time Stability data at 2-8°C for 12 Months (M)

Sr. No	Batch No	Initial	3M	6M	9M	12 M	Test parameters
1	323003	Compiles	Compiles	Compiles	Compiles	Compiles	Sterility, description, D antigen content, degree of adsorption, pH, efficacy (rat potency), bacterial endotoxin, aluminium content, formaldéhyde content, 2PE content, particulate matter, container closure integrity.
2	323004	Compiles	Compiles	Compiles	Compiles	Compiles
3	323005	Compiles	Compiles	Compiles	Compiles	Compiles

Conclusion: At exposure to 2-8°C, it was found that trivalent Inactivated Polio vaccine (Salk, Adsorbed) Idose batches are stable for 12 months for ail the test parameters mentioned above.

Table 18- IPV 5 dose Real time Stability data at 2-8°C for 12 Months (M)

Sr. No	Batch No	Initial	3 M	6M	9M	12 M	Test parameters
1	324003	Compiles	Compiles	Compiles	Compiles	Compiles	Sterility, description, D antigen content, degree of adsorption, pH, efficacy (rat potency), bacterial endotoxin, aluminium content, formaldéhyde content, 2PE content, particulate matter, container closure integrity.
2	324004	Compiles	Compiles	Compiles	Compiles	Compiles
3	324005	Compiles	Compiles	Compiles	Compiles	Compiles

Conclusion: At exposure to 2-8°C, it was found that trivalent Inactivated Polio vaccine (Salk, Adsorbed) 5 dose batches are stable for 12 months for ail the test parameters mentioned above.

Table 19 — IPV 1 dose Real time Stability data at 25°C for 6 Months (M)

Sr. No	Batch No	Initial	1 M	2 M	3 M	6M	Test parameters
1	323003	Compiles	Compiles	Compiles	Compiles	Compiles	Sterility, description, D antigen content, degree of adsorption, pH, efficacy (rat potency), bacterial endotoxin, aluminium content, formaldéhyde content, 2PE content, particulate matter, container closure integrity.
2	323004	Compiles	Compiles	Compiles	Compiles	Compiles
3	323005	Compiles	Compiles	Compiles	Compiles	Compiles

Conclusion: At exposure to 25±2°C,60±5%RH, it was found that trivalent Inactivated Polio vaccine (Salk ,Adsorbed) Idose batches are stable for 6 months for ail the test parameters mentioned above.

Table 20 - IPV 5 dose Real time Stability data at 25°C for 6 Months (M)

Sr. No	Batch No	Initial	1 M	2 M	3 M	6 M	Test parameters
1	324003	Complies	Complies	Complies	Complies	Compiles	Sterility, description, D antigen content, degree of adsorption, pH, efficacy (rat potency), bacterial endotoxin, aluminium content, formaldéhyde content, 2PE content, particulate matter, container closure integrity.
2	324004	Compiles	Complies	Complies	Complies	Complies
3	324005	Complies	Complies	Complies	Complies	Complies

Conclusion: At exposure to 25±2°C,60±5%RH, it was found that trivalent Inactivated Polio vaccine (Salk ,Adsorbed) 5 dose batches are stable for 6 months for ail the test parameters mentioned above.

Table 21 - IPV 1 dose Real time Stability data at 37°C for 7 days (D)

Sr. No	Batch No	Initial	1 D	2 D	3 D	5D	7D	Test parameters
1	323003	Compiles	Compiles	Compiles	Compiles	Compiles	Compiles	Sterility, description, D antigen content, degree of adsorption, pH. efficacy (rat potency), bacterial endotoxin, aluminium content, formaldéhyde content. 2PE content, particulate matter, container closure integrity.
2	323004	Compiles	Compiles	Compiles	Compiles	Compiles	Compiles
3	323005	Compiles	Compiles	Compiles	Compiles	Compiles	Compiles

Conclusion: At exposure to 37±1°C, it was found that trivalent Inactivated Polio vaccine (Salk ,Adsorbed) Idose batches are stable for 7 days for ail the test parameters mentioned above.

Table 22- IPV 5 dose Real time Stability data at 37°C for 7 days (D)

Sr. No	Batch No	Initial	1 D	2 D	3 D	SD	7D	Test parameters
1	324003	Compiles	Compiles	Compiles	Compiles	Compiles	Compiles	Sterility, description, D antigen content, degree of adsorption, pH, efficacy (rat potency), bacterial endotoxin, aluminium content, formaldéhyde content, 2PE content, particulate matter, container closure integrity.
2	324004	Compiles	Compiles	Compiles	Compiles	Compiles	Compiles
3	324005	Compiles	Compiles	Compiles	Compiles	Compiles	Compiles

Conclusion: At exposure to 37±1°C, it was found that trivalent Inactivated Polio vaccine (Salk ,Adsorbed) 5 dose batches are stable for 7 days for ail the test parameters mentioned above.

Example 5 - Immunogenicity studies of Alum Adsorbed IPV

CCID50 for Polio virus titration:

To test the number of live infective virus particles, an equally sensitive cell line that is Hep-2C is used as substrate and virus content in given sample can be determined. The CCID50 titration was performed in 96 well plates. 12,000 to 15,000 cells in 2% CM were seeded per well (ΙΟΟμΙ). Each dilution was added in 10 wells. After 6-8 days of incubation in CO2 incubator at 37°C, wells were screened for CPE positive and CPE négative.

Table 23: Sera Neutralization Test (SNT) was carried out to study immune response elicited by Dose sparing Alum adjuvanted Salk IPV Type-I in comparison to Standard dose Salk IPV Type-1 (nonadjuvanted) in rat. Animal Model: Wistar rat (8 weeks, approx 200 gm) 50% male and 50 % female per group. Route of Inoculation: Intra Muscular. Dose Volume: 0.5 ml Blood withdrawal: on day 21. Site ofbleeding: Cardiac puncture Sera was separated and used to test the presence of neutralizing antibodies for type spécifie polio virus. Control sera used to validate the test. Virus back-titration was also performed to get the number of challenge virus particles added.
Sera titre	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
Standard Dose 40 DU (Without adjuvant)	10 DU 0.4 mg AI(OH)3 as Al·1*	5 DU 0.4 mg AI(OH)3 as AI3*	2 DU 0.4 mg AI(OH)3 as AI3*	1 DU 0.4 mg AI(OH)3 as Al3*	-ve control
Rats - SNT	Rats - SNT	Rats - SNT	Rats - SNT	Rats - SNT	Rats
1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8
(<1:2)	4-	+	+	4-		+	4-	+	4-	4-	4-	+	+	4-	4-	+	4-	+	+	+	+	+	+	+	4-	+	4-	+	4-	+	+	+	+	+	+	4-	+	4-	4-	4-	-
(1:2)	+	+	-	+	+	+	+	+	4-	4-	4-	+	4-	4-	4-	4-	4-	4-	4-	-	4-	4-	4-	-	4-	4-	-	4-	4-	4-	+	4-	4-	4-	-	-	4-	+	4-	+	-
(1:4)	+	-	-	+	+	+	+	+	4·	4-	4-	4-	4-	4-	4-	4-	4-	4-	4-	-	4·	4-	4-	-	+	4-	-	4-	4-	4-	-	4-	4-	-	-	-	-	4-	4-	+	-
(1:8)	4-	-	-	+	4-	+	4-	4-	4-	4-	4-	4-	4-	4-	-	4-	4-	4-	4-	-	4-	+	4-	-	+	+	-	4·	+	+	-	+	-	-	-	-	-	4-	+	-	-
(1:16)	+	-	-	+	+	+	4-	+	··	+	+	+	4-	4-	-	+	4-	4-	4-	-	4*	+	-	-	4-	4-	-	4-	4-	-	-	4-	-	-	-	-	-	-	4-	-	-
(1:32)	+	-	-	+		-		4-		4-	4-	-	4-	4-	-	4-	4-	4-	4-	-	4-	-	-	-	4-	4-	-	-	4-	-	-	-	-	-	-	-	-	-	-	-	-
(1:64)	-	-	-	+		-	-	4-	-	4-	-	-	4-	4-	-	4-	4-	4-	-	-	4-	-	-	-	+	-	-	-	4-	-	-	-	-	-	-	-	-	-	-	-	-
(1:128)	-	-	-	+	-	-	-	-	-	4-	-	*	-	4-	-	4-		4-	-	-	4-	-	-	-	+	-	-	-	4-	-	-	-	-	-	-	-	-	-	-	-	-
(1:256)														4-							-											-
(1:512)														4-

(+) — SNT positive; (-) =SNT négative;

Interprétation: Salk type I IPV with aluminium hydroxide adjuvant having 10 DU/dose gives équivalent sero conversion as compared to commercial IPV standalone with 40 DU/ dose. Addition of aluminium hydroxide to IPV led to 4 fold dose sparing effect in rats compared to IPV standalone.

Table 24: Sera Neutralization Test (SNT) was carried out to study immune response elicited by Dose sparing Alum adjuvanted Salk IPV Type-2 in comparison to Standard dose Salk IPV Type-2 (non-adjuvanted) in rat. Animal Model: Wistar rat (8 weeks, approx 200 gm) 50% male and 50 % female per group. Route of Inoculation: Intra Muscular. Volume: 0.5 ml Blood withdrawal: on day 21. Site of bleeding: Cardiac puncture Sera was separated and used to test the presence of neutralizing antibodies for type spécifie polio virus. Control sera used to validate the test. Virus back-titration was also performed to get the number of challenge virus particles added.
Sera titre	Group 1	Group 2	Group 3	Group 4	Group 5
Standard dose 8 DU (Without adjuvant)	2 DU 0.2 mg AI(OH)3 asAP*	1 DU 0.2 mg AI(OH)3 as AP*	0.5 DU 0.2 mg AI(OH)3 as AP*	-ve control
Rats - SNT	Rats - SNT	Rats - SNT	Rats-SNT	Rats - SNT
1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8
(=1:2)	+	+	+	+	+	+	+	+	4-	4-	+	+	+	+	+	+	4*	+	+	+	+	+	+	+	+	4·	4-	4-	4-	4-	4-	+	-
(1:2)	4-	+	+	4-	4-	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	4-	4-	+	4-	+	4-	4-	4-	+	+	+	-
(1:4)	+	+	+	+	+	+	4-	+	+	+	+	+	+	+	+	+	+	+	+	+	4-	+	+	4-	+	+	+	4-	4-	4-	4-	4-	-
(1:8)	+	+		+	+	+	+	+	+	+	+	+	4-	+	+	+	4-	+	+	4*	+	+	4-	+	+	+	4-	4-	4-	4-	4-	4-	-
(1:16)	+	+	+	+	+	+	+	-	+	4-	+	+	+	+	+	+	+	+	+	4-	+	+	4-	+	+	+	+	-	4-	4-	4-	+	-
(1:32)	-	+	-	+	+	-	+	-	+	+	-	+	+	+	+	+	4-	+	-	+	-	+	+	-	-	-	4-	-	-	4-	-	-	-
(1:64)	-	-	-	+	+	-	-	-	-	+	-	+	+	+	+	+	-	-	-	+	-	4-	+	-	-	-	+	-	-	4-	-	-	-
(1:128)	-	-	-	-		-	-	-	-	+		+	+	+	+	+	-	-	-	+	-	+	+						-
(1:256)	-	-	-	-	-		-	-	-	+	-	4-	-	-	-	-	-	-	-	-	-		-	-	-	-	-	-	-	-	-	-
(1512)				-

(+) — SNT positive; (-) =SNT négative;

Interprétation: Salk type 2 IPV with alum hydroxide adjuvant having 2 DU/dose gives équivalent sero-conversion as compared to commercial IPV standalone with 8 DU/ dose. Addition of aluminium hydroxide to IPV led to 4-fold dose sparing effect in rats compared to IPV standalone.

Table 25: Sera Neutralization Test (SNT) was carried out to sludy immune response elicited by Dose sparing Alum adjuvanted Salk IPV Type-3 in comparison to Standard dose Salk IPV Type3 (non-adjuvanted) in rat. Animal Model: Wistar rat (8 week s. approx 200 gm) 50% male and 50 % female per group. Route of Inoculation: Intra Muscular. Volume: 0.5 ml Blood withdrawal: on day 21. Site of bleeding: Cardiac puncture Sera was separated and used to test the presence of neutralizing antibodies for type spécifie polio virus. Control sera used to validate the test. Virus back-titration was also performed to get the number of challenge virus particles added.
Sera titre	Group 1	Group 2	Group 3	Group 4	Group 5
32 DU (Without adjuvant)	10 DU 0.2 mg AI(OH)3 as Al”	5 DU 0.2 mg Al(OH)j as Al”	2.5 DU 0.2 mg Al(OH)3 as A!”	-ve control
Rats - SNT	Rats - SNT	Rats - SNT	Rats - SNT	Rats - SNT
1	2	3	4	5	6	7	8	l	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8	1	2	3	4	5	6	7	8
(<1:2)	+	+	+	4-	+	4-	4-	4-	+		+	+	+	4-	4-	+	+	+	+	4-	4-	4	4-	4-	4-	4-	4-	4-	4-	+	4-	4-	-
(1:2)	+	+	+-	-	4-	4-	+	+	+	+	+	+	4-	4	+	+	4-	4-	+	4-	4-	+	4-	+	4-	4-	+	4-	4-	-	4-	4-	-
(1:4)	+	+	+	-	+	4-	4-	+	+	4-	+	4-	+	4-	4-	4-	4-	4-	+	4-	+	4-	4-	4-	4-	+	-	+	4-	-	4-	4-	-
(1:8)	+	+	+-	-	+	-F	+	4-	4-	4-	+	4-	4-	4-	4-	4-	4-	+	+	-	-		4-	4-	4-	4-	-	4-	4-	-	+	+	-
(1:16)	+	+	+	-	-	+	4-	+	4-	4-	+	4-	-	+	+	4-	+	+	4-	*	-	+	+	4-	4-	+	-	-	+	-	+	-	-
(1:32)	-	-	4-	-	-	-	4-	4-	-	4-		-	-	-b	+	+	4-	+	*	-	-	-	+	4-					-
(1-64)	-	-	+	-	-	-	-	-	-	-	+	-	-	+	+	4-	+	+	-	-	-	-	-	4-	-	-	-	-	-	-	-	-	-
(1128)	-	-	4-	-	-	-	-	-	-	-	+	-	-	4-	+	4-									-
(1:256)	-	-	-	-	-	-	-	-	-	-	+	-	-	4-	+									-
(1:512)														4			-

(+) = SNT positive; (-) =SNT négative;

interprétation: Salk type 3 IPV with alum hydroxide adjuvant having 10 DU/dose gives équivalent sero-conversion as compared to commercial IPV standalone with 32 DU/ dose. Addition of aluminium hydroxide to IPV led to 3-fold dose sparing effect in rats compared to IPV standalone.

Table 26: Potency study of adjuvanted trivalent single dose Salk and Sabin inactivated polio virus vaccine (IPV) formulation
Antigen	Formulation	Type 1 (μΙ)	Type 2 (μΐ)	Type 3 (μΙ)
Ag	AIOH3	dil	Total	Ag	A1OH3	dil	Total	Ag	A1OH3	dil	Total
Salk	Salk 10-2-10	196	1777	4027	6000	88	888	6024	7000	198	888	5910	7000
Sabin	Sabin 5-16-10	627	1777	3597	6000	5423	888	689	7000	4938	888	1174	7000
	Positive Control (Non-adjuvanted)	Polio standard dose IPV Salk (40-8-32)

Example 6 - Potency study of adjuvanted trivalent single dose Salk and Sabin inactivated polio virus vaccine (IPV)

20ml formulation was prepared.

> Antigen volume is dépendent on stock conc.

> For AI(OH)3, stock conc. was 9mg/mL

For Type l : Required conc is 0.4mg/dose i.e 0.8mg/ml

For Type 2: Required conc is 0.2mg/dose i.e 0.4mg/ml

For Type 3: Required conc is 0.2mg/dose i.e 0.4mg/ml

Total 1.6mg/ml

Formulation volume is 20ml i.e. 20X1.6=32mg.

Therefore, from stock of 9mg/ml, 3552ml was required. Hence 1777ml used for Type 1 and 888ml for Type 2 and 3 each.

Table 27: Salk 10-2-10 Eflïcacy (Rat potency) results: Spécification: Lower limit >25% relative to assignée! potency of sample. * Note: Values are calcuîated as considering dose of DU (40:8:32)
Poliovirus Type	Efficacy resuit	Lower limit	Upper limit
Type 1	253.9 %	130.8%	646.4 %
Type 2	242.9 %	125.0%	634.0 %
Type 3	213.0%	112.1 %	530.9 %
Tahle 28: Sabin 5-16-10 Efficac	v (Rat notenevt results: Snecifïc	atinn· I nwer limit >25% relative	to assiened notenev of samnle.
* Note: Values are calcuîated as considering dose of DU (40:8:32)
Poliovirus Type	Efficacy resuit	Lower limit	Upper limit
Type 1	90.3 %	44.3 %	175.4%
Type 2	74.2 %	34.4 %	148.3 %
Type 3	513.4%	297.0 %	1128.5%

Claims (16)

1. We Claim,

   l. A method for producing a vaccine composition comprising inactivated poliovirus particles, wherein the method comprises the steps of:

   a) purifying the poliovirus particles using method selected from ultrafiltration, diafiltration, and chromatography in the presence of a phosphate buffer;

   b) exchanging the phosphate buffer of the poliovirus particles with a buffer selected from TRIS, TBS, MOPS, HEPES, BICARBONATE, having a pH of 6.8 to 7.2 and a concentration in the range of 30 mM to 50 mM, preferably 40 mM;

   c) adding 10X concentrated M-199 medium containing 0,5 % glycine to achieve final concentration of Ix;

   d) inactivating the poliovirus particles by incubation with 0.025 % formaldéhyde at 37 °C for 5 to 13 days; and

   e) adsorbing the poliovirus particles on aluminium hydroxide sait adjuvant whereby percentage adsorption on alum is at least 95%, and wherein aluminium content per 0.5mL dose, is 0.4 mg Al³⁺ for Type l, 0.2mg Al³' for Type 2, and 0.2mg AP⁺ for Type 3;

   f) blending alum adsorbed poliovirus particles obtained in step (e) with stérile dilution medium comprising preservative.
2. 2. The method as claimed in claim l, wherein the blending in step (f) comprises of stirring at 200 to 250 rpm continuously for not less than 16 hrs at température of2-8°C.
3. 3. The method as claimed in claim l, wherein the stérile dilution medium comprises atleast one preservative selected from the group of 2-phenoxyethanol (2-PE), Benzéthonium chloride (Phemerol), Phénol, m-cresol, p-chlor-m-cresol, chlorobutanol, Thiomersal, Formaldéhyde, paraben esters comprising methyl- ethyl- butyl- or propyl- parabens, benzyl alcohol and a combination thereof.
4. 4. The method as claimed in claim 3, wherein the stérile dilution medium comprises preservative selected from 2-phenoxyethanol, Formaldéhyde or a combination of 2-PE and formaldéhyde.
5. 5. The method as claimed in in claim 3, wherein the stérile dilution medium comprises a combination of 2-PE and formaldéhyde.
6. 6. The method as claimed in claim l, wherein post blending in step (f), the final pH of the vaccine composition is between pH 6.0 - 7.0.
7. 7. The method as claimed in claim 1, wherein the vaccine composition is filled in a container selected from a bottle, a vial, an ampule, an IV bag, a wearable injector, a bolus injector, a prefilled syringe, a pen, a pump, a multidose needle syringe, a multidose vial, a multidose pen, a syrette, an auto-injector and a Vaccine Microarray Patches.
8. 8. The method as claimed in claim l, wherein the pohovirus is Sabin based inactivated pohovirus of serotypes l, 2 and 3.
9. 9. The method as claimed in claim 1, wherein the poliovirus is Salk based inactivated pohovirus of serotypes 1,2 and 3.
10. 10. The method as claimed in claim 9, wherein the Salk poliovirus is one of MEF-1, Mahoney, Saukett, Brunenders, Brunhilde, CHAT, Cox, Lansing, Saukett H and G, Leon strains, and chimeric recombinant strains.
11. 11. A vaccine composition obtained from the method as claimed in claim 1, wherein the vaccine composition is a monovalent composition comprising:

    a) inactivated poliovirus selected from the group comprising of:

    type 1 at a dose greater than 1 D-antigen units and less than 11 D-antigen units, per 0.5ml;

    type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; or type 3 at a dose greater than 2 D-antigen units and less than 17 D-antigen units, per 0.5ml;

    b) Adjuvant Aluminium hydroxide (as Al ) having aluminium content of 0.4 mg Al³⁺ for Type 1, 0.2mg Al³⁺for Type 2, and 0.2mg Al³⁺ for Type 3 per 0.5mL and;

    c) Preservative comprising 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.
12. 12. The vaccine composition as claimed in claim 11, wherein the vaccine composition is a monovalent composition comprising:

    a) Inactivated poliovirus selected from the group comprising of:

    type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml;

    b) Adjuvant Aluminium hydroxide (as Al’) having aluminium content of 0.2mg Al³⁺ per 0.5mL; and

    c) Preservative comprising 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.
13. 13. The vaccine composition as claimed in claim 11, wherein the vaccine composition is a monovalent composition comprising:

    a) inactivated poliovirus selected from the group comprising of:

    type 2 at a dose of 2 D-antigen units, per 0.5ml;

    b) Adjuvant Aluminium hydroxide (as Al ) having aluminium content of 0.2mg Al³⁺ per 0.5mL; and

    c) preservative comprising 2-Phenoxyethanol in an amount of 2.5 mg/0.5 ml and formaldéhyde in an amount of 12.5 pg/0.5 ml.
14. 14. The vaccine composition obtained from the method as claimed in claim 1, wherein the vaccine composition is a bivalent composition comprising:

    a) inactivated poliovirus selected from the group comprising of:

    type l at a dose greater than l D-antigen units and less than 11 D-antigen units, per 0.5ml; and type 3 at a dose greater than 2 D-antigen units and less than 17 D-antigen units, per 0.5ml; or type l at a dose greater than l D-antigen units and less than 11 D-antigen units, per 0.5ml; and type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; or type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; and type 3 at a dose greater than 2 D-antigen units and less than 17 D-antigen units, per 0.5ml;

    b) Adjuvant Aluminium hydroxide (as Al ' *') having aluminium content of 0.4 mg Al³⁺ for Type l, 0.2mg Al³⁺for Type 2, and 0.2mg Al³⁺ for Type 3 per 0.5ml; and

    c) preservative comprising of 2-Phenoxyethanol in a range of l mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.
15. 15. The vaccine composition obtained from the method as claimed in claim l, wherein the vaccine composition is atrivalent composition comprising: a) inactivated poliovirus type l at a dose greater than l D-antigen units and less than 11 D-antigen units, per 0.5ml;

    type 2 at a dose greater than 0.4 D-antigen units and less than 8 D-antigen units, per 0.5ml; and type 3 at a dose greater than 2 D-antigen units and less than 17 D-antigen units, per 0.5ml;

    b) Adjuvant Aluminium hydroxide (as Al· ) having aluminium content of 0.4 mg Al³⁺ for Type l, 0.2mg Al³⁺for Type 2, and 0.2mg Al³' for Type 3 per 0.5ml; and

    c) preservative comprising 2-Phenoxyethanol in a range of l mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to 12.5 pg/0.5 ml dose.
16. 16. The vaccine composition as claimed in claim 15, wherein the vaccine composition is a trivalent composition comprising:

    a) inactivated poliovirus type I at a dose of 10 D-antigen units, per 0.5ml;

    type 2 at a dose of 2 D-antigen units, per 0.5ml; and type 3 at a dose of 10 D or 16 D-antigen units, per 0.5ml;

    b) Adjuvant Aluminium hydroxide (as Al having aluminium content of 0.4 mg Al³⁺ for Type l, 0.2mg Al³⁺for Type 2, and 0.2mg Al^î+ for Type 3 per 0.5ml; and

    c) preservative comprising 2-Phenoxyethanol in a range of l mg to 4 mg/0.5 ml dose and formaldéhyde in a range of 5 pg to I2.5 pg/0.5 ml dose.