FORM 2 THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
IMPROVED METHODS FOR POLIOVIRUS INACTIVATION, ADJUVANT ADSORPTION AND DOSE REDUCED VACCINE COMPOSITIONS OBTAINED
THEREOF
SERUM INSTITUTE OF INDIA PRIVATE LIMITED
an Indian Company
Of 212/2, Soli Poonawalla Road,
Hadapsar, Pune-411028
Maharashtra, India
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

REFERENCE TO RELATED PATENT APPLICATION
The present 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 07th October 2014. The teachings of the above applications are incorporated herein in their entirety by reference.
FIELD
The present disclosure relates to method of manufacturing vaccine compositions. Particularly, the present 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
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present 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 forms of two of the three serotypes of polioviruses have been declared as being globally eradicated, wild poliovirus type 2 in September, 2015, and wild poliovirus type 3 in October, 2019. Wild poliovirus type 1 has also been eliminated from many WHO regions 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 eradication 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), have been observed, with 959 cases occurring globally in 2020. This constitutes an inherent 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 1 and 3) OPV, and later to inactivated polio vaccines (IPVs), which have no risk of inducing cVDPV (Bandyopadhyay A.S et al 2015)
The recent detection of vaccine-derived poliovirus (VDPV) in London (UK) and a case of paralytic polio in New York (USA) have 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 1 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 complete eradication of all forms of polioviruses. They subsequently found that circulating VDPV2 was the predominant strain for paralytic poliomyelitis outbreaks; in 2020–22 circulating VDPV2 was responsible for 97– 99% 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 eradication. In 2021, 682 cases of cVDPV2 were confirmed from 22 countries, a reduction 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 eradication 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 region of the Sabin2 genome to stabilize attenuation determinants, 2C coding region 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 region (UTR) (relocated cre and S15domV), synonymous mutations at eight nucleotide positions in the 2C coding region to inactivate the internal cre and two mutations in the 3D polymerase (D53N and K38R) to limit viral adaptability. Each of these modifications, contributes to genetic stability and attenuation. Importantly, their combination prevents detectable reversion to neurovirulence by reducing the capacity of the virus to acquire mutations that increase replication 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 have 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 its 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 subsequent Emergency Use Listing approval of COVID-19 vaccines in the global pandemic.
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. verification 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 have been administered across 28 countries globally, and the majority of countries have seen no further transmission of cVDPV2 after two immunization rounds.
However, Global Polio Eradication Initiative (GPEI) recently reported seven children, six in the Democratic 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. This happens most often with poliovirus type 2, one of three serotypes. Global Polio Eradication Initiative (GPEI) reported on 16th Mar 2023 that they have received notification of the detection of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Burundi and the Democratic Republic of the Congo (DRC) linked with the novel oral polio vaccine type 2 (nOPV2). The viruses were isolated from 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 environmental samples collected in Burundi (Bujumbura Mairie province). All reported isolates stem from two separate and new emergences of cVDPV2 linked with nOPV2 that originated in Tanganyika and South Kivu provinces in DRC.
Thus, with due consideration of the emerging nOPV2 related vaccine derived poliovirus /paralysis cases (in spite of global nOPV2 stockpile) , the continuous increasing global poliovirus vaccine demand to control the increasing number and geographic 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 Inactivated Polio Virus (IPV) vaccine formulation for prophylaxis and treatment of infections caused by Polio Virus type 2 serotype in humans, while being less likely to revert to neurovirulence than Live-attenuated Sabin oral polio vaccines.

OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
It is an object of the present disclosure to provide a method for producing a vaccine composition comprising inactivated poliovirus particles
Yet another object of the present 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 present 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 1, type 2 and type 3 serotypes.
Yet another object of the present 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 present disclosure is to use “Salk/Sabin Dose Reduced Monovalent Type 2 IPV” for campaign, as an alternative to nOPV2 for stockpiling & combating epidemics.
Yet another object of the present disclosure is to manufacture a bivalent vaccine (i.e. Type 2/Type 1, Type 2/Type 3)
Yet another object of the present disclosure is to provide a method for producing a vaccine composition containing alum adsorbed reduced-dose Inactivated Polio Virus (IPV) antigens and preservative comprising formaldehyde and at least one additional preservative to improve the stability and preservative efficacy of the vaccine.

Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present 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, 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 units per 0.5 ml dose and preservative comprising formaldehyde 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 present disclosure may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion, with the understanding that the present 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 present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present 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 present disclosure. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present 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 first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure. The present disclosure provides an immunogenic composition and a process for preparing the same.
The term "vaccine" is optionally substitutable 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 Preparation - available from Ph. Eur. Secretariat, e.g. Code P 216 0000) for standardisation of such methods between manufacturers (Pharmeuropa Special Issue, Bio 96-2). Thus, the D-antigen unit value is well understood in the art.
The term "dose" herein is typically one administration of the vaccine of the invention, which 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 1, 2, 3 monovalent or bivalent or trivalent combination of either one, two or all three of these types. 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 (S15, S16, S17, S18 & S19), S19 Type 1 (Mahoney or Brunhilde), S19 Type 2 (MEF-1) and S19 Type 3 (Saukett) S19 Type 1 (Strain S19/MahP1/N18S), S19 Type 2 (S19/MEF2P1/N18S) and S19 Type 3 (S19/SktP1/N18S). Sabin based chimeric recombinant strains may be attenuated Poliovirus Strains (S15, S16, S17, S18 & S19) wherein the capsid protein coding regions (P1) of S15, S16, S17, S18 and S19 were replaced exactly with the P1 regions of the serotype 1, 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® (Serum Institute of India Pvt. Ltd.). Thus, where it is stated herein that one, two, three fold dose reduction (reduced) as compared to standard dose of Salk based IPV is present in an immunogenic composition of the invention it is meant D-antigen units equating to X% of reduction 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 present disclosure envisages method for producing vaccine composition comprising 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; and preservative comprising formaldehyde and at least one additional preservative to improve the stability and preservative efficacy of the vaccine.
An important aspect of the present 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 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 1x;
d) inactivating the poliovirus particles by incubation with 0.025 % formaldehyde at 37 °C for 5 to 13 days; and
e) adsorbing the poliovirus particles on aluminium hydroxide salt 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 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
f) blending alum adsorbed poliovirus particles obtained in step (e) with sterile dilution medium comprising preservative.
According to an embodiment of the present disclosure, said formaldehyde 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 present disclosure, post-incubation of the poliovirus particles with 0.025 % formaldehyde at 37 °C for 5 to 13 days the mixture may be subjected to intermediate 0.22µ filtration on day 7 and final filtration on day 13.
According to an embodiment of the present 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 present disclosure, post inactivation of the poliovirus particles the bulk may be subjected to D-Ag ELISA test for D-Antigen unit determination.
According to an embodiment of the present disclosure, the adsorption of poliovirus particles on aluminium salt adjuvant may comprise of following steps:
1. taking the desired volume of autoclaved Aluminium salt adjuvant in a blending vessel to get the final concentration of Alum (Al+++) less than 1.2 mg Al3+ per 0.5mL dose;
2. adding IPV bulk with adjusted D-Ag unit and making up the volume with a sterile 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 present disclosure, wherein adsorption of formalin inactivated IPV can be done on aluminium hydroxide salt adjuvant wherein final concentration of Alum(Al+++) less than 1.2 mg Al3+ per 0.5mL dose, preferably aluminium content per 0.5mL dose, is 0.4 mg Al3+ for Type 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ 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 present disclosure, wherein the said process of formalin inactivation and aluminium hydroxide adsorption can result in D- Antigen recovery post-inactivation between 50% and 80% and percent adsorption of aluminium hydroxide can be between 85 and 99%, preferably IPV Type 1, 2 and 3 antigens are adsorbed on to aluminium salt of hydroxide (Al(OH)3) having percentage adsorption of at least 95 %.
According to an embodiment of the present disclosure, the blending in step (f) may comprise of stirring at 200 to 250 rpm continuously for not less than 16 hrs at temperature of 2-8°C to form a homogeneous mixture.
According to an embodiment of the present disclosure, the sterile dilution medium may comprise of atleast one preservative selected from the group of 2-phenoxyethanol (2-PE), Benzethonium chloride (Phemerol), Phenol, m-cresol, p-chlor-m-cresol, chlorobutanol, Thiomersal, Formaldehyde, paraben esters comprising methyl- ethyl- butyl- or propyl-parabens, or benzyl alcohol or a combination thereof.
In a preferred embodiment, the sterile dilution medium may comprise of preservative selected from 2-phenoxyethanol or Formaldehyde or a combination of 2-PE and formaldehyde.

More preferably, the sterile dilution medium may comprise of a combination of 2-PE and formaldehyde 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 formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.
In an embodiment, the sterile dilution medium may additionally comprise of M-199 Medium containing 0.5 % glycine, Water for Injection (WFI) other than the preservative.
According to an embodiment of the present 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 present 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 present 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 all 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 all 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 (S15, S16, S17, S18 & S19), S19 Type 1 (Mahoney or Brunhilde), S19 Type 2 (MEF-1) and S19 Type 3 (Saukett) S19 Type 1 (Strain S19/MahP1/N18S), S19 Type 2 (S19/MEF2P1/N18S) and S19 Type 3 (S19/SktP1/N18S).
More preferably Salk poliovirus may be one of Mahoney, MEF-1 and Saukett strain.

According to an embodiment of the present 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 Al3+ for Type 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldehyde in a range of 5 µg to 12.5 µg/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 Al3+ for Type 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
c) preservative comprising of 2-Phenoxyethanol in a range of 2.5 mg/0.5 ml dose and formaldehyde in a range of 12.5 µg/0.5 ml dose.
According to an embodiment of the present 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-antigen units, per 0.5ml;
b) aluminium content per 0.5mL dose is 0.4 mg Al3+ for Type 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldehyde in a range of 5 µg to 12.5 µg/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 7.5-10, 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 1 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 1 and Type 2 of Salk strain based IPV may be selected from the group of dose composition comprising of 7.5-16, 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 1, 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 16-15

10, 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 1, 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 Al3+ for Type 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
c) preservative comprising of 2-Phenoxyethanol in an amount of 2.5 mg/0.5 ml dose and formaldehyde in an amount of 12.5 µg/0.5 ml dose.
According to an embodiment of the present 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 17 D-antigen units, per 0.5ml;
b) aluminium content per 0.5mL dose is 0.4 mg Al3+ for Type 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.
According to a preferred embodiment of the present disclosure, wherein the vaccine composition obtained 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 Al3+ for Type 2;
c) preservative comprising of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldehyde in a range of 5 µg to 12.5 µg/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 Al3+ for Type 2;
c) preservative comprising of 2-Phenoxyethanol - 2.5 mg/0.5 ml dose and formaldehyde -12.5 µg/0.5 ml dose.
According to another aspect of the present 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(HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugate(Ηib), Haemophilus influenzae(a, c, d, e, f serotypes and the unencapsulated 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 purified 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, modified 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 embodiment of the present disclosure, wherein the Combination Vaccines comprising dose reduced IPV may comprise of antigens selected from Diphtheria toxoid(D), Tetanus toxoid (T), Whole cell pertussis(wP), acellular pertussis, hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugate(Ηib) other than dose reduced IPV as disclosed in earlier embodiment.
According to a preferred embodiment of the present 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 1 to 30 Lf, the wP antigen in an amount of 1 to 50 IOU or acellular pertussis antigen, the HBsAg antigen in an amount of 1 to 20 µg, the Ηib antigen in an amount of 1 to 20 µg, the IPV Type 1 antigen in an amount of 1 -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 present 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 µg, the Ηib PRP-TT conjugate antigen in an amount of 8 or 10 or 13 µg of PRP, the inactivated polio virus (IPV) is a Salk strain 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, 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 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 present 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 µg, the Ηib PRP-TT conjugate antigen in an amount of 8 or 10 or 13 µg 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 1 and 2, or IPV type 1 and 3, or IPV type 2 and 3, or IPV type 1, 2 and 3.
According to an embodiment of the present 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 formaldehyde in presence of Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) resulting in maximum recovery of D-antigen. Subsequent adsorption of said IPV on aluminium hydroxide provides significantly dose reduced IPV compositions. Specification 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 b is composed of polyribosyl-ribitol-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 present invention may comprise of preparation 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, CRM197 (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 lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative 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 pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins or any equivalents thereof. Preferably the carrier protein in conjugate is selected from TT or CRM197.

An aspect of the present 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 temperature, 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 precipitation using a detergent N, N, N-trimethyl-1-hexadecanaminium bromide, followed by ethanol gradient precipitation and filtration. Final purified PRP polysaccharide was tested for meeting the specifications like endotoxin, nucleic acid and protein as per the WHO, BP, EP, IP etc.
Another aspect of the present invention, wherein the polysaccharide – protein conjugate 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 (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanate ester; coupling of activated cyanylated polysaccharide to amino group of carrier protein; purification of final conjugate using ultrafiltration.
More preferably, the optimal input ratio of reactants i.e. PRP, CDAP and CRM197 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 (1-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 CRM197. The degree of conversion of Hib conjugate was confirmed by the offline testing using HPLC. The conjugation reaction was quenched by achieving the desired level of conversion of conjugate with the specification 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 100kDa) to remove nonreactive reagents and byproducts. Final conjugate bulk is 0.22 µm filtered and stored at 2-8ºC.

More preferably, Hib PRP may be conjugated to carrier protein wherein the saccharide: protein ratio (w/w) may be between 0.4 and 1; 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 present invention, wherein the Hib PRP may be conjugated to tetanus toxoid (TT) by CNBr chemistry, Reductive amination chemistry, Cyanylation chemistry or any other chemistry already discloses in Kniskern 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 present invention, wherein the carrier protein may be present in both free and conjugated form in a composition of the present 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 present at less than 5% by weight, more preferably is less than 2%.
Yet another aspect of the present invention, wherein the Hib antigen is not substantially adsorbed on to any adjuvant.
Yet another aspect of the present invention, wherein the Hib antigen may not be subjected to deliberate or intentional adsorption on any adjuvant.
Yet another aspect of the present invention, wherein the percentage of adsorption of Hib antigen on to any adjuvant may be less than 20%.
Yet another aspect of the present invention, wherein the Hib antigen used in the combination vaccine of the present 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 exotoxin secreted by Corynebacterium diphtheria, 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 /Formaldehyde, 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 diphtheria. Existence of dimers can lead to adverse reactions.
The present invention may comprise of preparation of Diphtheria toxoid as disclosed below.
In first aspect of the present invention, diphtheria toxin (exotoxin) may be obtained from Corynebacterium diphtheria and detoxified using a suitable inactivating agent. The example of suitable inactivating agent includes Formaldehyde.
In second aspect of the present invention, diphtheria 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, arginine) or polysorbate solution at a final concentration of 5 – 300 mM, and stored at temperature -20 to +40 °C till further use. The purified D thus obtained comprises of homogenous fraction devoid of undesirable aggregates (Refer Figure 1) with atleast 80 – 90 % monomeric diphtheria 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 permeation chromatography. The purified diphtheria toxoid is stabilized by addition of Histidine (200mM) amino acid buffer solution.
In third aspect of the present invention, diphtheria toxoid may be adsorbed on to adjuvant selected from the group of aluminium salt (Al3+) such as aluminium hydroxide (Al(OH)3) or aluminium phosphate (AlPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpG-containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water emulsion or a combination thereof.
Yet preferably diphtheria toxoid may be adsorbed on to Aluminium salt including Aluminium hydroxide and Aluminium phosphate, preferably on Aluminium phosphate.
Yet preferably the Diphtheria toxoid (D) antigen may be adsorbed on to aluminium phosphate having percentage adsorption of atleast 50%.
• Tetanus toxoid (T)
Tetanus is an acute infectious disease caused by toxigenic strains of the bacterium Clostridium tetani (C. tetani), a gram-positive, spore-forming, strictly anaerobic 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 /Formaldehyde, Acetylethyleneimine, etc. To increase 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 present invention may comprise of preparation of Tetanus toxoid as disclosed below.
In first aspect of the present invention, Tetanus toxin may be obtained from Clostridium tetani and detoxified using a suitable inactivating agent. The example of suitable inactivating agent includes Formaldehyde.
In second aspect of the present 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, arginine) or polysorbate solution at a final concentration of 5 – 300 mM, and stored at temperature -20 to +40 °C till 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 for the purpose of purification using Gel permeation chromatography. The purified tetanus toxoid is stabilized by addition of Histidine (200mM) amino acid buffer solution.
In third aspect of the present invention, Tetanus toxoid may be adsorbed on to adjuvant selected from the group of aluminium salt (Al3+) such as aluminium hydroxide (Al(OH)3) or aluminium phosphate (AlPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpG-containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water emulsion or a combination thereof.
Yet preferably tetanus toxoid may be adsorbed on to Aluminium salt including Aluminium hydroxide and Aluminium phosphate, preferably on Alum phosphate.
Yet preferably the tetanus toxoid (T) antigen may be adsorbed on to aluminium phosphate having percentage adsorption of atleast 40%.
• Pertussis Antigen

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 formalin. Immunization with wP vaccine is relatively inexpensive and 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.
The present invention may comprise of preparation of the pertussis vaccine comprising pertussis antigen selected from one or more of whole cell pertussis or acellular pertussis.
In first aspect of the present invention, pertussis vaccine may be an acellular pertussis antigen selected from one or more of 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 antigens may be expressed in suitable host using recombinant DNA technology.
Preferably acellular 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 8µg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8µg); - Pertactin: 1 to 20 micrograms (More particularly 2.5µg); -Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.
In second aspect of the present invention, pertussis vaccine may be a whole cell pertussis comprising of Bordetella pertussis strains 134, 509, 25525 and 6229 in a specific 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.
In third aspect of the present invention, wP inactivation process may include heat inactivation at 56±2°C for 10 to 15 minutes in presence of formaldehyde; wherein wP bulk remains non-clumpy 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 prevention of HBV infection. HBsAg protein can be recombinantly expressed in a suitable host microorganism; or can be isolated from the blood plasma of a chronic Hepatitis B patient/carrier.
The present invention may comprise of preparation of the Hepatitis B surface antigen (HBsAg) as disclosed below.
In one of the aspects of present invention, HBsAg may be expressed in Hansenula polymorpha yeast cells using recombinant DNA technology. Other yeasts such as Saccharomyces cerevisiae may also be used as host cell for recombinant expression of HBsAg.
In one of the aspect of present invention, Hepatitis B antigen (HBsAg) may be adsorbed on to adjuvant selected from the group of aluminium salt (Al3+) such as aluminium hydroxide (Al(OH)3) or aluminium phosphate (AlPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpG-containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water emulsion or a combination thereof.
Yet preferably Hepatitis B antigen (HBsAg) may be adsorbed on to Aluminium salt 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 present invention may comprise of the process for preparation of combination vaccine composition/formulation comprising Dose reduced IPV as disclosed below.

According to an embodiment of the present disclosure, wherein the process for preparation 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 (Sabin/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 temperature 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 temperature 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% NaCl) 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 present 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 present disclosure, WFI or 0.9% saline (NaCl) may be added to the final combination vaccine composition to make up the volume.
According to another aspect of the present 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, succinate, 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 buffers, such as HEPES, piperazine-N, N′-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged by the present disclosure. The buffer aids in stabilizing the immunogenic composition of the present disclosure. The amount of the buffer may be in the range of 0.1 mM to 100 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, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, MgC12, etc. Preferably, the salt may be NaCl. Typically, the amount of the salt 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 emulsion, MF-59,a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, 3–deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram negative 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-1), Quil A, cholera toxin B subunit, polyphosphazene and derivatives,

mycobacterium cell wall preparations, mycolic acid derivatives, 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, Benzethonium chloride (Phemerol), Phenol, 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 immunization, 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 aseptic adaptor for removal of material. Typically, the amount of the preservative may be in the range of 0.1 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 preparation desired.
Yet another aspect of the disclosure, wherein the vaccine compositions obtained from the instant method may be fully liquid but is not limited thereto. Suitable forms of liquid preparation may include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions and elixirs 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 parenteral or subcutaneous 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, peroral 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 preparations including lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols 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 1 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 in which a primary course of vaccination is followed by 1-3 separate doses given at subsequent time intervals after 1-3 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 age or below according to two dose regimens consisting of a first dose, and second dose at subsequent time intervals after 1-3 years.
Yet alternatively the composition may be administered concomitantly with other drugs or any other vaccine.
Compositions may be presented in 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 diphtheriae PW8 CN2000 was obtained from the Wellcome Research Laboratory, London, United Kingdom by the National Control Authority Central Research Institute (C.R.I.) Kasauli, Himachal Pradesh, India in lyophilized form in the year 1973.The strain was revived and further lyophilized under Master Seed Lot- C. diphtheriae CN2000 A1 at C.R.I. Kasauli.
TETANUS 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:
Rhein Biotech (Germany) constructed the recombinant Hansenula polymorpha strain containing the HBsAg surface antigen gene. Rhein Biotech also made the Master Cell Bank (MCB Hansenula polymorpha K3/8-1 strain ADW, 12/94) and performed all the characterization tests on this bank.
HAEMOPHILUS INFLUENZAE TYPE b:

The source organism for generation 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 1976.Three passages of the strain took place before storage at -70 °C at the Academic Medical Centre (AMC), University 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: MEF1
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 specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example 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 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 assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
While considerable emphasis has been placed herein on the specific 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, handling 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 Serum 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 inventor 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 present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1 - Purification of Polio Virus
1. Tangential flow filtration (TFF):
Clarified harvest pool was concentrated to 10X using tangential flow filtration system with 100Kda cassettes(0.5m2) and then diafiltered 3 times of harvest volume with phosphate buffer (30mM-70mM, pH: 7.0)
2. Column Chromatography: Ion Exchange Chromatography (IEC).
10X 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 procedure(13days), 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, 0.1 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 1x. Inactivation agent formalin (0.025%) was added into purified virus bulk

while constant mixing. Inactivation was carried out at 37°C with continuous 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
Ferric Nitrate • 9H2O 0.00072
Magnesium 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 • 2HCl 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-Isoleucine 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
Folic Acid 0.00001
Menadione (sodium bisulfite) 0.000016
myo-Inositol 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-α-Tocopherol Phosphate • Na 0.00001
Thiamine • HCl 0.00001
Adenine Sulfate 0.01

Adenosine Triphosphate • 2Na 0.001
Adenosine Monophosphate • Na 0.0002385
Cholesterol 0.0002
Deoxyribose 0.0005
Glucose 1
Glutathione (reduced) 0.00005
Guanine • HCl 0.0003
Hypoxanthine 0.0003
Phenol 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 Formalin inactivation and after Formalin inactivation in presence of TRIS buffer (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 preservation for sIPV 1,2 and 3.
B. Effect of non-phosphate buffers other than TRIS (TBS, MOPS, HEPES, and 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 formaldehyde inactivation methods were particularly carried out in presence of phosphate buffer, significant D-antigen losses were observed, whereas it was found that formaldehyde 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 determination by ELISA. Day 1: Plate coating:
1. 100µl of specific bovine anti polio was pipetted in PBS per well

2. Microtiter plate was sealed and incubated overnight at room temperature.
Day 2: Blocking:
1. The plates were washed (Washing/dilution buffer -0.05% tween 20 in 1x PBS)3 times.
2. 300µl block buffer (1% BSA in PBS) was pipetted per well.
3. The plate was sealed and incubated for 45minutes at 37±1°C.
Sample addition:
1. The plate was washed 3 times.
2. 100µl of sample diluent was added in all wells except well of row A.
3. 100µl standard was added to first two wells of column 2 and 3.
4. 100µl sample was added to first two wells of column 4-12.
5. Pre-diluting sample to a suitable concentration.
6. 100µl sample diluents was added to first two wells of column 1.
7. Serial two fold dilution were made down the column by transferring 100ul from each well to adjacent well of the same column and discarding 100ul 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:
1. The plate was washed 3 times.
2. 100µl diluted (1:240) type specific monoclonal antibodies were added.
3. The plates were sealed and incubated for 2 hours at 37°C.
Conjugate:
1. The plate was washed 3 times

2. 100µl diluted conjugate (Type1- 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. 100µl TMB substrate was added to all wells.
2. Mixture incubated at room temperature for 10 minutes.
3. Reaction was stopped by adding 100µl 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)3 was used for the preparation of formulations.
2. Desired volume of Al(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 sterile dilution medium comprising 2-phenoxyethanol and Formaldehyde as preservative.
4. Final formulation pH was adjusted to 6.0 – 7.0 with 1 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-1 glass vials with bromo-butyl rubber stoppers and aluminium seal with coloured polypropylene flip cap.
Sterile dilution medium is made up of M-199 Medium containing 0.5 % glycine, 2-Phenoxyethanol, Formaldehyde, 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 mineral 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.
a) b)
c) Ingredients Concentration/ 0.5 mL Function

Inactivated Type 1, Mahoney Type 1 10 D antigen units Active ingredient (drug substance)

Aluminium hydroxide (as Al+++), Adjuvant 0.4 mg Adjuvant (Adsorbent)

2-Phenoxyethanol Formaldehyde 2.5 mg 12.5 µg Stabilizer/ Preservative

Table 11: Monovalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.
a) b)
c) Ingredients Concentration/ 0.5 mL Function

Inactivated Type 2, MEF -1 strain 2 D antigen units Active ingredient (drug substance)

Aluminium hydroxide (as Al+++), Adjuvant 0.2 mg Adjuvant (Adsorbent)

2-Phenoxyethanol Formaldehyde 2.5 mg 12.5 µg Stabilizer/ Preservative

Table 12: Monovalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.
a) b)
c) Ingredients Concentration/ 0.5 mL Function

Inactivated Type 3, Saukett 10 D antigen units Active ingredient (drug substance)

Aluminium hydroxide (as Al+++), Adjuvant 0.2 mg Adjuvant (Adsorbent)

2-Phenoxyethanol Formaldehyde 2.5 mg 12.5 µg Stabilizer/ Preservative

Table 13: Bivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.
a)
b) c) Ingredients Concentration/ 0.5 mL Function

Inactivated Type 1, Mahoney Type 1 Inactivated Type 3, Saukett 10 D antigen units 10 D antigen units Active ingredient (drug substance)

Aluminium hydroxide (as Al+++), Adjuvant 0.4 mg Al3+ for Type 1 0.2mg Al3+ for Type 3 Adjuvant (Adsorbent)

2-Phenoxyethanol Formaldehyde 2.5 mg 12.5 µg Stabilizer/ Preservative

Table 14: Bivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. Ingredients Concentration/ 0.5 mL Function
No.
a)

Inactivated Type 2, MEF -1 strain 2 D antigen units Active ingredient (drug
b) Inactivated Type 3, Saukett 10 or 16 D antigen units substance)

Aluminium hydroxide (as Al+++), 0.2 mg Al3+ for Type 1 Adjuvant (Adsorbent)
c) Adjuvant 0.2mg Al3+ for Type 3

2-Phenoxyethanol 2.5 mg Stabilizer/ Preservative
Formaldehyde 12.5 µg

Table 15: Bivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. No.
a)
b) c) Ingredients Concentration/ 0.5 mL Function

Inactivated Type 1, Mahoney Type 1 Inactivated Type 2, MEF -1 strain 10 D antigen units 2 D antigen units Active ingredient (drug substance)

Aluminium hydroxide (as Al+++), Adjuvant 0.4 mg Al3+ for Type 1 0.2mg Al3+ for Type 2 Adjuvant (Adsorbent)

2-Phenoxyethanol Formaldehyde 2.5 mg 12.5 µg Stabilizer/ Preservative

Table 16: Trivalent IPV Vaccine Composition (Salk strain) (Adsorbed)
Sr. Ingredients Concentration/ 0.5 mL Function
No.
a)

Inactivated Type 1, Mahoney Type 1 10 D antigen units Active ingredient (drug
Inactivated Type 2, MEF -1 strain 2 D antigen units substance)
b) Inactivated Type 3, Saukett 10 antigen units

Aluminium hydroxide (as Al+++), 0.4 mg Al3+ for Type 1 Adjuvant (Adsorbent)
Adjuvant 0.2mg Al3+ for Type 2
c) 0.2mg Al3+ for Type 3

2-Phenoxyethanol 2.5 mg Stabilizer/ Preservative
Formaldehyde 12.5 µg

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 3 M 6 M 9 M 12 M Test parameters
1 323003 Complies Complies Complies Complies Complies Sterility, description, D antigen content, degree of adsorption,
pH, efficacy (rat potency), bacterial endotoxin, aluminium
content, formaldehyde content, 2PE content, particulate matter,
container closure integrity.
2 323004 Complies Complies Complies Complies Complies

3 323005 Complies Complies Complies Complies Complies

Conclusion: At exposure to 2-8°C, it was found that trivalent Inactivated Polio vaccine (Salk, Adsorbed) 1dose batches are stable for 12 months for all 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 6 M 9 M 12 M Test parameters
1 324003 Complies Complies Complies Complies Complies Sterility, description, D antigen content, degree of adsorption,
pH, efficacy (rat potency), bacterial endotoxin, aluminium
content, formaldehyde content, 2PE content, particulate matter,
container closure integrity.
2 324004 Complies Complies Complies Complies Complies

3 324005 Complies Complies Complies Complies Complies

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 all 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 6 M Test parameters
1 323003 Complies Complies Complies Complies Complies Sterility, description, D antigen content, degree of adsorption,
pH, efficacy (rat potency), bacterial endotoxin, aluminium
content, formaldehyde content, 2PE content, particulate matter,
container closure integrity.
2 323004 Complies Complies Complies Complies Complies

3 323005 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) 1dose batches are stable for 6 months for all 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 Complies Sterility, description, D antigen content, degree of adsorption,
pH, efficacy (rat potency), bacterial endotoxin, aluminium
content, formaldehyde content, 2PE content, particulate matter,
container closure integrity.
2 324004 Complies 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 all 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 5 D 7 D Test parameters
1 323003 Complies Complies Complies Complies Complies Complies Sterility, description, D antigen content, degree of
adsorption, pH, efficacy (rat potency), bacterial
endotoxin, aluminium content, formaldehyde
content, 2PE content, particulate matter, container
closure integrity.
2 323004 Complies Complies Complies Complies Complies Complies

3 323005 Complies Complies Complies Complies Complies Complies

Conclusion: At exposure to 37±1°C, it was found that trivalent Inactivated Polio vaccine (Salk ,Adsorbed) 1dose batches are stable for 7 days for all 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 5 D 7 D Test parameters
1 324003 Complies Complies Complies Complies Complies Complies Sterility, description, D antigen content, degree of
adsorption, pH, efficacy (rat potency), bacterial
endotoxin, aluminium content, formaldehyde
content, 2PE content, particulate matter, container
closure integrity.
2 324004 Complies Complies Complies Complies Complies Complies

3 324005 Complies Complies Complies Complies Complies Complies

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 all 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 (100µl). 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 negative.

Table 23: Sera Neutralization Test (SNT) was carried out to study immune response elicited by Dose sparing Alum adjuvanted Salk IPV Type-1 in comparison to Standard dose Salk IPV Type-1 (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.
Dose 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 specific polio virus.
Control sera used to validate the test. Virus back-titration was also performed to get the number of challenge virus particles added.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Standard Dose 40 DU 10 DU 5 DU 2 DU 1 DU -ve
titre (Without adjuvant) 0.4 mg Al(OH)3 as Al3+ 0.4 mg Al(OH)3 as Al3+ 0.4 mg Al(OH)3 as Al3+ 0.4 mg Al(OH)3 as Al3+ 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) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -
(1:2) + + - + + + + + + + + + + + + + + + + - + + + - + + - + + + + + + + - - + + + + -
(1:4) + - - + + + + + + + + + + + + + + + + - + + + - + + - + + + - + + - - - - + + + -
(1:8) + - - + + + + + + + + + + + - + + + + - + + + - + + - + + + - + - - - - - + + - -
(1:16) + - - + + + + + - + + + + + - + + + + - + + - - + + - + + - - + - - - - - - + - -
(1:32) + - - + - - - + - + + - + + - + + + + - + - - - + + - - + - - - - - - - - - - - -
(1:64) - - - + - - - + - + - - + + - + + + - - + - - - + - - - + - - - - - - - - - - - -
(1:128) - - - + - - - - - + - - - + - + + - - + - - - + - - - + - - - - - - - - - - - -
(1:256) - - - - - - - - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - -
(1:512) - - - - - - - - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - -

(+) = SNT positive; (-) =SNT negative;
Interpretation: Salk type 1 IPV with aluminium hydroxide adjuvant having 10 DU/dose gives equivalent 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 specific 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 Group 1 Group 2 Group 3 Group 4 Group 5
titre Standard dose 8 DU 2 DU 1 DU 0.5 DU -ve control
(Without adjuvant) 0.2 mg Al(OH)3 as Al3+ 0.2 mg Al(OH)3 as Al3+ 0.2 mg Al(OH)3 as Al3+
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) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -
(1:2) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -
(1:4) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -
(1:8) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -
(1:16) + + + + + + + - + + + + + + + + + + + + + + + + + + + - + + + + -
(1:32) - + - + + - + - + + - + + + + + + + - + - + + - - - + - - + - - -
(1:64) - - - + + - - - - + - + + + + + - - - + - + + - - - + - - + - - -
(1:128) - - - - - - - - + - + + + + + - - - + - + + - - - - - - - - - -
(1:256) - - - - - - - - - + - + - - - - - - - - - - - - - - - - - - - - -
(1:512) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

(+) = SNT positive; (-) =SNT negative;
Interpretation: Salk type 2 IPV with alum hydroxide adjuvant having 2 DU/dose gives equivalent 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 study immune response elicited by Dose sparing Alum adjuvanted Salk IPV Type-3 in comparison to Standard dose Salk IPV Type-
3 (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 specific polio virus.
Control sera used to validate the test. Virus back-titration was also performed to get the number of challenge virus particles added.
Group 1 Group 2 Group 3 Group 4 Group 5

32 DU 10 DU 5 DU 2.5 DU
Sera titre (Without adjuvant) 0.2 mg Al(OH)3 as Al3+ 0.2 mg Al(OH)3 as Al3+ 0.2 mg Al(OH)3 as Al3+ -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) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -
(1:2) + + + - + + + + + + + + + + + + + + + + + + + + + + + + + - + + -
(1:4) + + + - + + + + + + + + + + + + + + + + + + + + + + - + + - + + -
(1:8) + + + - + + + + + + + + + + + + + + + - - + + + + + - + + - + + -
(1:16) + + + - - + + + + + + + - + + + + + + - - + + + + + - - + - + - -
(1:32) - - + - - - + + - + + - - + + + + + - - - - + + - - - - - - - - -
(1:64) - - + - - - - - - - + - - + + + + + - - - - - + - - - - - - - - -
(1:128) - - + - - - - - - - + - - + + + - - - - - - - - - - - - - - - - -
(1:256) - - - - - - - - - - + - - + + - - - - - - - - - - - - - - - - - -
(1:512) - - - - - - - - - - - - - + - - - - - - - - - - - - - - - - - - -
(+) = SNT positive; (-) =SNT negative;
Interpretation: Salk type 3 IPV with alum hydroxide adjuvant having 10 DU/dose gives equivalent 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.

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

Table 26: Potency study of adjuvanted trivalent single dose Salk and Sabin inactivated polio virus vaccine (IPV) formulation
Antigen Formulation Type 1 (µl) Type 2 (µl) Type 3 (µl)

Ag AlOH3 dil Total Ag AlOH3 dil Total Ag AlOH3 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)
20ml formulation was prepared.
➢ Antigen volume is dependent on stock conc.
➢ For Al(OH)3, stock conc. was 9mg/ml. For Type 1: 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 Efficacy (Rat potency) results: Specification: Lower limit ≥25% relative to assigned potency of sample. * Note: Values are calculated as considering dose of DU (40:8:32)
Poliovirus Type Efficacy result 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 %

Table 28: Sabin 5-16-10 Efficacy (Rat potency) results: Specification: Lower limit ≥25% relative to assigned potency of sample. * Note: Values are calculated as considering dose of DU (40:8:32)
Poliovirus Type Efficacy result 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
We Claim,
1. 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 1x;
d) inactivating the poliovirus particles by incubation with 0.025 % formaldehyde at 37 °C for 5 to 13 days; and
e) adsorbing the poliovirus particles on aluminium hydroxide salt 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 1, 0.2mg Al3+ for Type 2, and 0.2mg Al3+ for Type 3;
f) blending alum adsorbed poliovirus particles obtained in step (e) with sterile dilution medium comprising preservative.

2. The method as claimed in claim 1, wherein the blending in step (f) comprises of stirring at 200 to 250 rpm continuously for not less than 16 hrs at temperature of 2-8°C.
3. The method as claimed in claim 1, wherein the sterile dilution medium comprises atleast one preservative selected from the group of 2-phenoxyethanol (2-PE), Benzethonium chloride (Phemerol), Phenol, m-cresol, p-chlor-m-cresol, chlorobutanol, Thiomersal, Formaldehyde, paraben esters comprising methyl- ethyl- butyl- or propyl-parabens, benzyl alcohol and a combination thereof.
4. The method as claimed in claim 3, wherein the sterile dilution medium comprises preservative selected from 2-phenoxyethanol, Formaldehyde or a combination of 2-PE and formaldehyde.
5. The method as claimed in in claim 3, wherein the sterile dilution medium comprises a combination of 2-PE and formaldehyde.
6. The method as claimed in claim 1, wherein post blending in step (f), the final pH of the vaccine composition is between pH 6.0 – 7.0.
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 pre-filled 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. The method as claimed in claim 1, wherein the poliovirus is Sabin based inactivated poliovirus of serotypes 1, 2 and 3.
9. The method as claimed in claim 1, wherein the poliovirus is Salk based inactivated poliovirus of serotypes 1, 2 and 3.

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. 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 formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.
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 formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.
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 formaldehyde in an amount of 12.5 µg/0.5 ml.
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 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-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 of 2-Phenoxyethanol in a range of 1 mg to 4 mg/0.5 ml dose and formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.
15. The vaccine composition obtained from the method as claimed in claim 1, wherein the vaccine composition is 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) 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 formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.
16. The vaccine composition as claimed in claim 15, wherein the vaccine composition is a trivalent composition
comprising:
a) inactivated poliovirus
type 1 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 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 formaldehyde in a range of 5 µg to 12.5 µg/0.5 ml dose.