WO2020075197A1

WO2020075197A1 - Inactivated poliomyelitis vaccine composition

Info

Publication number: WO2020075197A1
Application number: PCT/IN2019/050755
Authority: WO
Inventors: Sivakumar Ayyaswamy PERNAMALLUR; Vikram Madhusadan Paradkar; Narender Dev MANTENA; Mahima DATLA; Martin Reers
Original assignee: Biological E Limited
Priority date: 2018-10-10
Filing date: 2019-10-10
Publication date: 2020-04-16

Abstract

The present invention provides inactivated poliomyelitis vaccine compositions comprising at least one inactivated poliovirus S19 strain for prevention of poliomyelitis. The present invention provides safe, stable and effective vaccine formulations which can be manufactured at low containment level leading to large scale production of poliomyelitis vaccines.

Description

TITLE: INACTIVATED POLIOMYELITIS VACCINE COMPOSITION

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to Indian Patent Application number 201841038423, filed on October 10, 2018, which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to an immunogenic composition comprising inactivated poliomyelitis vaccine S 19 strain, and at least one pharmaceutically acceptable carrier. More particularly, the present invention provides a vaccine composition comprising IPV S 19 strain, a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid protein from wild type / Salk strain serotypes Mahoney, MEF and Saukett.

BACKGROUND OF THE INVENTION

Polioviruses are members of the Enterovirus genus of the family Picornaviridae. Polioviruses are small, non-enveloped viruses with capsids enclosing a single stranded, positive sense RNA genome. There are three antigenically distinct serotypes of polioviruses: types 1, 2 and 3. Infections of susceptible individuals by poliovirus can result in paralytic poliomyelitis. Poliomyelitis is highly contagious. Two different categories of polio-vaccines have been developed, the inactivated poliovirus vaccine (IPV) of Salk or Sabin and the live, attenuated oral poliovirus vaccine (OPV) of Sabin. Inactivated polio vaccine (IPV) was first developed in 1955 by Dr Jonas Salk. Also called the Salk vaccine IPV consists of inactivated (killed) poliovirus strain of all three poliovirus serotypes.

Oral polio vaccine (OPV) is relatively inexpensive to manufacture and convenient to administer and has been used widely for vaccination programs. However, occasionally recipients suffer from vaccine-associated paralytic poliomyelitis (VAPP) due to the reversion of the live virus to wild type attributes. Furthermore, the Sabin strain can evolve into transmissible viruses, causing outbreaks of paralytic disease. Accordingly, WHO has recognized the need to withdraw OPV and to replace it with IPV.

Several standalone trivalent IPV products are commercially available. Besides Sabin strain based IPV, several Salk strain based standalone trivalent IPV products are also available in the market. IPOL^® 3 / IMOVAX^® Polio, is an Inactivated Poliovirus Vaccine, produced by Sanofi Pasteur, is a sterile suspension of three types of poliovirus: Type 1 (Mahoney), Type 2 (MEF- 1), and Type 3 (Saukett). Poliorix™, another Inactivated Poliovirus Vaccine, manufactured and marketed by GlaxoSmithKline Biologicals, is a solution for injection of three serotypes of poliovirus: Type 1 (Mahoney), Type 2 (MEF-l), and Type 3 (Saukett). Each of the three serotypes of poliovirus is individually grown in Vero cells, a continuous cell line of monkey kidney origin cultivated on microcarriers. The cells are grown in Eagle MEM modified medium, supplemented with new-bom calf serum tested for adventitious agents prior to use, obtained from countries believed to be free of bovine spongiform encephalopathy. For viral growth the culture medium is replaced by M-199 without calf serum. VeroPol another brand of IPV vaccine manufactured and marketed by SSI (Statens Serum Institut) contains Poliovirus type 1 (Briinhilde), type 2 (MEF-l) and type 3 (Saukett) and the viruses are grown in Vero cells.

Each 0.5 ml dose of commercially available Salk based IPV vaccine contains 40 D antigen units of type 1 (Mahoney or Briinhilde), 8 D antigen units of type 2 (MEF-l) and 32 D antigen units of type 3 (Saukett) of the polio virus.

The surface antigens (capsid) of the Salk strain and S 19 strain are the same. It is assumed that IPV S 19 strain is equally immunogenic compared to commercially available Salk strain. The containment requirements for manufacturing of IPV S19 strain bulk antigens are less as compared to commercially available Salk and Sabin bulk antigens.

IPV is produced from poliovirus strains of each serotype that have been inactivated (killed) with formalin. As an injectable vaccine, it can be administered alone or in combination with other vaccines (e.g., diphtheria, tetanus, pertussis, hepatitis B, and haemophilus influenza). Generally, three timely spaced doses are administered to generate adequate levels of seroconversion, and in most countries, a booster dose is added during late childhood. IPV has been used successfully in the polio eradication programs in few countries, but until recently most countries have used the oral polio vaccine. IPV provides serum immunity to all three types of poliovirus, resulting in protection against paralytic poliomyelitis. Most studies indicate that the degree of mucosal immunity in the intestine is significantly less than that provided by OPV, although this difference may be less pronounced in the pharyngeal mucosal lining. Adverse events following administration of IPV are very mild and transient. There is a growing consensus that inactivated poliovirus vaccine (IPV) may contribute to more rapid eradication of wild-type polio and control of emergent circulating vaccine-derived polioviruses (cVDPVs) when used in conjunction with existing OPV vaccine (Wright and Modlin, 2008; John, 2009).

However, current production of IPV is more expensive and may even be prohibitively expensive for least developed countries, where a strong need for polio vaccines still exists. The culture systems for producing bulk poliovirus material that can be used, in particular non- attenuated poliovirus, contribute to a large extent to the relatively high costs. Due to the risks associated with the handling of large quantities of poliovirus needed for IPV production, while global cessation of poliovirus transmission is occurring, a high-level containment (BSL- 3/GAPIII) for all manufacturing and quality control areas where live virus is handled must be implemented.

Both live attenuated (OPV) and inactivated (IPV) polio vaccines have been effective in controlling the polio disease worldwide. Polio vaccines are based on the Salk or the Sabin strains. The serotypes Mahoney or Brunhilde Type 1, MEF Type 2 and the Saukett Type 3 are the Salk strains that have been used in the vaccine against the poliomyelitis disease. The Sabin strain include the serotypes Sabin 1, Sabin 2 and Sabin 3.

Sarah et.al. (PLoS Pathog. 2015 Dec; 11(12)) discloses the understanding of the molecular virology of the Sabin vaccine strain to design viruses that are maximally genetically stable (resistant to reversion to wild-type) and minimally thermodynamically stable (hyper-attenuated growth under gut conditions) resulting into a new strain S 19 for the production of inactivated polio vaccine at low-containment level after eradication.

U.S. Patent No. 8,557,252 B2 discloses an attenuated poliovirus which does not have a base pair mismatch in stem (a) or (b) of domain V of the 5' non-coding region of its genome, wherein at least seven of the base pairs in stems (a) and (b) are U-A or A-U base pairs.

U.S. Patent No. 9,402,892 B2 discloses an attenuated polio virus having a 5' non-coding region consisting of the 5' non-coding region of serotype Sabin 3, modified so that it does not have a base pair mismatch in stem (a) or (b) of domain V, wherein seven or eight of the base pairs in stems (a) and (b) are U-A or A-U base pairs; and a capsid protein from the Sabin 1, Mahoney, MEF or Saukett strain. Due to a global shortage in the supply of inactivated polio vaccine (IPV), there is a need to produce large quantities of safe, stable and effective immunogenic composition of IPV at low containment requirements. Accordingly, the present invention provides means for manufacturing safe, stable and effective vaccine formulations at very low containment level.

OBJECTIVE OF THE INVENTION

It is an objective of the invention to provide safe, stable and effective immunogenic composition of IPV S 19 strain at very low containment levels.

Yet another objective of the present invention to provide a process for the preparation of an Inactivated Poliomyelitis vaccine derived from S 19 strain for immunization against Poliomyelitis.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic composition/vaccine formulation comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units.

The present invention provides an immunogenic composition / vaccine formulation comprising at least one inactivated poliomyelitis vaccine S 19 strain, and at least one pharmaceutically acceptable carrier. The S 19 strain in the immunogenic composition/vaccine formulation is selected from inactivated S 19 Type 1 (Mahoney), S 19 Type 2 (MEF-l) and S 19 Type 3 (Saukett) and at least one pharmaceutically acceptable carrier.

The S 19 strain in the immunogenic composition/vaccine formulation is selected from inactivated S 19 Type 1 (Strain S l9/MahPl/Nl8S), S 19 Type 2 (S 19/MEF2P1/N18S) and S 19 Type 3 (S l9/SktPl/Nl8S) and at least one pharmaceutically acceptable carrier.

The present invention also provides a process of preparing a vaccine formulation comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the present invention provides an immunogenic composition based on at least one IPV S 19 strain, a chimeric recombinant poliovirus comprising non-coding region from Sabin strain and the coding region from Salk strain. In yet another embodiment, the present invention provides an immunogenic composition / vaccine formulation based on at least one IPV S 19 strain, a chimeric recombinant poliovirus comprising the non-coding region based on Sabin strain with the capsid protein from wild type / Salk strain serotypes Mahoney, MEF and Saukett.

In yet another embodiment, the present invention provides an immunogenic composition / vaccine formulation based on at least one IPV S 19 strain, a chimeric recombinant poliovirus comprising the non-coding region based on Sabin strain with the capsid protein from wild type / Salk strain from Salk strain poliovirus Type 1 (Mahoney), Type 2 (MEF-l), and Type 3 (Saukett).

In an embodiment, the present invention provides an immunogenic composition based on at least one IPV S 19 strain a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid from wild type / Salk strain serotypes Mahoney, MEF and Saukett.

In an embodiment, the present invention provides IPV S 19 strain Type 1, a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid from poliovirus Type 1 (Mahoney) or Brunhilde strain (Type-l).

In an embodiment, the present invention provides IPV S 19 strain Type 2, a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid from poliovirus Type 2 (MEF-l).

In an embodiment, the present invention provides S 19 strain Type 3, a chimeric recombinant poliovirus, comprising non-coding region based on serotype 3 of a Sabin strain and the coding region based on Sabin strain with the capsid from poliovirus Type 3 (Saukett).

In an embodiment, the present invention provides trivalent vaccine comprising IPV S 19 strain Type 1, Type 2 and Type 3.

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

The term“D-antigen units” (DU, also referred to as“international units” or IU): The D antigen units referred to herein are the measured total D-antigen units of each un-adsorbed bulk IPV antigen prior to formulation of the final vaccine which is added in each human dose of formulated vaccine. The methods of measuring D-antigen units are well known in the art.

The terms“S19 Type-l” refers to S 19 serotype 1 (Mahoney orBrunhilde),“S19 Type-2” refers to S 19 serotype 2 (MEF-l), and“S19 Type-3” refers to S19 serotype 3 (Saukett).

In one embodiment, the present invention provides a vaccine comprising IPV S 19 strain Type 1, Type 2 and Type 3 at pharmaceutically acceptable doses.

In another embodiment, the present invention provides a vaccine comprising IPV S 19 Type 1 at a dose of 60 D-antigen units, S 19 Type 2 at a dose of 12 D-antigen units and S 19 Type 3 at a dose of 48 D-antigen units.

In another embodiment, the present invention provides a vaccine comprising IPV S 19 Type 1 at a dose of 50 D-antigen units, S 19 Type 2 at a dose of 10 D-antigen units and S 19 Type 3 at a dose of 40 D-antigen units.

In another embodiment, the present invention provides a vaccine comprising IPV S 19 Type 1 at a dose of 40 D-antigen units, S 19 Type 2 at a dose of 8 D-antigen units and S 19 Type 3 at a dose of 32 D-antigen units.

In yet another embodiment, the present invention provides a vaccine comprising IPV S 19 Type 1 at a dose of 20 D-antigen units, S 19 Type 2 at a dose of 4 D-antigen units and S 19 Type 3 at a dose of 16 D-antigen units.

In another embodiment, the present invention provides a vaccine comprising IPV S 19 Type 1 at a dose of 13.33 D-antigen units, S 19 Type 2 at a dose of 2.66 D-antigen units and S 19 Type 3 at a dose of 10.66 D-antigen units.

In another embodiment, the present invention provides a vaccine comprising IPV S 19 Type 1 at a dose of 4 D-antigen units, S 19 Type 2 at a dose of 0.8 D-antigen units and S 19 Type 3 at a dose of 3.2 D-antigen units.

In another embodiment, the present invention provides a vaccine formulation comprising IPV S 19 Type 1 at a dose of 40 D-antigen units, S 19 Type 2 at a dose of 8 D-antigen units and S 19 Type 3 at a dose of 32 D-antigen units and at least one pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides a vaccine formulation comprising IPV S 19 Type 1 at a dose of 20 D-antigen units, S 19 Type 2 at a dose of 4 D-antigen units and S 19 Type 3 at a dose of 16 D-antigen units and at least one pharmaceutically acceptable carrier. In another embodiment, the present invention provides a vaccine formulation comprising IPV S 19 Type 1 at a dose of 13.33 D-antigen units, S 19 Type 2 at a dose of 2.66 D-antigen units and S 19 Type 3 at a dose of 10.66 D-antigen units and at least one pharmaceutically acceptable carrier.

In another embodiment, the present invention provides a vaccine formulation comprising IPV S 19 Type 1 at a dose of 4 D-antigen units, S 19 Type 2 at a dose of 0.8 D-antigen units and S 19 Type 3 at a dose of 3.2 D-antigen units and at least one pharmaceutically acceptable carrier.

In yet another embodiment, the pharmaceutically acceptable carrier includes adjuvants, excipients, preservatives, stabilizers, buffers, pH indicators, surfactants, solvents, residuals, diluents and the like. Suitable stabilizer for the vaccine formulation includes Ml 99 medium, amino acids like alanine, histidine, proline, glutamine, glycine and the like, and combinations of pharmaceutically acceptable stabilizers. Suitable preservatives include 2-phenoxyethnaol, m-cresol, benzyl alcohol and benzoic acid and the like or a mixture thereof. pH indicators such as phenol red, phenolphthalein and the like. The concentration of the preservative may range from 0.001% to 2.0 % w/v. Suitable buffers include phosphate or histidine.

In a preferred embodiment, the present invention provides a vaccine composition comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units and at least one pharmaceutically acceptable carrier and 2 -phenoxy ethanol (2-PE) as preservative, wherein the concentration of 2-PE may range from 0.001% to 2.0 % w/v.

In a preferred embodiment, the present invention provides a vaccine composition comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 50 D-antigen units, S 19 Type 2 at a dose of 0.8 to 10 D-antigen units and S 19 Type 3 at a dose of 3.2 to 40 D-antigen units and at least one pharmaceutically acceptable carrier and 2-phenoxyethanol (2-PE) as preservative, wherein the concentration of 2-PE may range from 0.001% to 2.0 % w/v.

In a more preferred embodiment, the present invention provides a vaccine composition comprising IPV S19 strain selected from S 19 Type 1 at a dose of 4 to 40 D-antigen units, S 19 Type 2 at a dose of 0.8 to 8 D-antigen units and S 19 Type 3 at a dose of 3.2 to 32 D-antigen units and at least one pharmaceutically acceptable carrier and 2-phenoxyethanol (2-PE) as preservative, wherein the concentration of 2-PE may range from 0.001% to 2.0 % w/v. In one embodiment, the IPV of the invention may be adsorbed or unadsorbed and manufactured as monovalent poliovirus bulk antigen or trivalent poliovirus (TPV) bulk antigen containing S 19 Type 1, S 19 Type 2 and S 19 Type 3 strains. TPV may be prepared in concentrated solution such as 240 DU of Type-l; 48DU for Type-2 and 184 DU of Type-3. This TPV may be subsequently diluted to make IPV vaccine.

In another embodiment, the vaccine formulation comprises IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 40 D-antigen units, S 19 Type 2 at a dose of 0.8 to 8 D-antigen units and S 19 Type 3 at a dose of 3.2 to 32 D-antigen units.

In an embodiment, the present invention provides a process for the preparation of trivalent poliovirus (TPV) comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 60 D- antigen units, S19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units, comprises the steps of: a) aseptically transferring S 19 strain Typel, followed by Type 2 and Type 3 to the vessel, b) adding stabilizer and optionally preservative into the vessel to make the trivalent polio virus under stirring condition at a temperature in the range from 10 to 30 °C, c) optionally sterile filtering the bulk solution and adjusting the pH of the solution and d) filling the bulk composition into a container.

The TPV prepared according to the invention may be used in making the IPV vaccine with or without adjuvantation or adsorption.

In one embodiment, the preparation of IPV vaccine of the present invention may be carried out in suitable stainless steel container or vessel, siliconized container or plastic container such as High Density Polyethylene (HDPE), Polyethylene terephthalate (PETG), Polypropylene (PP) and the like.

In one embodiment of the present invention, process for preparation of IPV vaccine composition involves addition of stabilizer and preservative separately or simultaneously.

In another embodiment, IPV components either TPV or monovalent polio bulk antigens may be adsorbed separately or together as a mixture onto an aluminium salt such as aluminium hydroxide, aluminium phosphate and the like or mixture of both aluminium hydroxide and aluminium phosphate. The adsorbent may be prepared in situ or may be added during the manufacturing process. In an embodiment, the present invention provides a process for the preparation of Inactivated poliomyelitis vaccine composition comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units and at least one pharmaceutically acceptable carrier, which comprises the steps of: a) transferring a sterile filtered stabilizer to the blending vessel,

b) optionally adding one or more preservative to the above vessel,

c) aseptically transferring IPV S 19 strain Type 1, followed by Type 2 and Type 3 monovalent bulk antigens or TPV to the blending vessel under stirring condition at a temperature in the range from 10 to 30 °C to make bulk vaccine composition, d) optionally sterile filtering the bulk solution and adjusting the pH of the solution and e) filling the bulk composition into unit dose container.

In an embodiment, the present invention provides a process for the preparation of Inactivated poliomyelitis vaccine composition comprising IPV S 19 strain selected from S 19 Type 1 at a dose of 4 to 60 D-antigen units, S 19 Type 2 at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 at a dose of 3.2 to 48 D-antigen units and at least one pharmaceutically acceptable carrier, which comprises the steps of: a) aseptically transferring adjuvant to the blending vessel, followed by transferring the monovalent bulk of IPV S 19 strain Type 1, Type 2, and Type 3 antigens or TPV to the blending vessel or adsorbing the monovalent bulk of IPV S 19 strain Type 1, Type 2, and Type 3 antigens or TPV to an adjuvant and transferring to the blending vessel, b) adding stabilizer to the adsorbed IPV obtained in step (a) under stirring condition at a temperature in the range from 10 to 30 °C,

c) optionally adding a preservative,

d) optionally sterile filtering the bulk solution and adjusting the pH of the solution and e) filling the final bulk vaccine into unit dose container.

In a preferred embodiment, the vaccine formulation of the present invention is free of thiomersal.

In an embodiment, antigens in the vaccine composition of the present invention will be present in "immunologically effective amounts" i.e. the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention of disease. The dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).

The vaccine compositions of the present invention may be in the form of lyophilised powder or in aqueous form. The aqueous form may be in the form of solutions or suspension. The vaccine composition may be filled in a vial, ampoule or they may be presented as pre-filled syringes. The vaccine of the present invention may be a single dose vial or multi-dose vial. The single dose vial may preferably be a 0.5ml vial or ampoule or prefilled syringe.

In one embodiment, the vaccine of the present invention is administered to an adult, adolescent, toddler, infant or less than one-year-old human and may be administered by injection.

In another embodiment, the vaccine of the present invention may be available as a multivalent vaccine comprising one or more other antigens conferring protection in a host against disease caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheria, Haemophilus influenza type B, Neisseria meningitidis type A, C, W, Y; Salmonella typhi, Streptococcus pneumonia, Malaria and / or Hepatitis A or Hepatitis B.

The present disclosure provides a high level production of inactivated poliomyelitis S 19 virus strain, which comprises the steps of:

a) revival of adherent cells into static containers;

b) amplification of adherent cells in a stirred tank bioreactor containing microcarriers in a culture medium,

c) detachment of cells inside the bioreactor and transferring the cell culture to another bioreactor,

d) repeating the steps (b) and (c) at least twice,

e) transferring the cells to production bioreactor having a volume of at least 100 liters, f) infecting the adherent cells in production bioreactor with S 19 polio virus,

g) incubating the infected cell culture to propagate S 19 virus for a period of at least 3 days, h) harvesting the S 19 polio virus, followed by inactivation and purification to produce inactivated polio virus in high yield.

The cell culture media used for amplification is a conventional culture media and is free of any protein of animal origin or even of any product of animal origin. The cell culture media may contain low-molecular-weight proteins at very low concentrations. Suitable media include VP SFM medium, Opti Pro™ serum-free (InVitrogen), Episerf (InVitrogen), Ex-Cell® MDCK (Sigma- Aldrich), Ex-Cell™ Vero (SAFC biosciences), MP-BHK® serum free (MP Biomedicals), SFC-10 BHK express serum free (Promo cell), SFC-20 BHK express protein free (Promo cell), HyQ PF Vero (Hyclone Ref. SH30352.02), Hyclone SFM4 Megavir, MDSS2 medium (Axcell biotechnology), Iscove's modified DMEM medium (Hyclone), Ham's nutritive media (Ham's F10, Ham's F12), Leibovitz L-15 medium (Hyclone), ProVero medium (Lonza) and Power MDCK medium (Lonza) that are free of any product of animal origin and which contain little or no proteins.

A cell protection agent is usually added, when the protein content in culture medium is low (e.g. < 15 mg/l according to the Bradford method). The cell protection agent protects the cells against the shear forces that are exerted when the medium is subjected to stirring. Suitable cell protection agents that can be used in the process of the present invention may include polyvinyl alcohol polymers, polyethylene glycol, polyvinylpyrrolidone and poloxamers and the like.

The propagation of poliovirus is carried out in adherent Vero cell, PER.C6 cells, HEK293 cell or MRC-5 cells, preferably vero cells, which are obtained from WHO 10-87 cell bank. The Adherent cells are passaged in a culture medium, one or more times to amplify viral copy number, as well as to introduce virus into a living system for replication efficiency. In order to amplify cells and form industrial cell batches, a plurality of cell passages are carried out in separate bioreactors.

The“cell passage” begins contacting a suspension of adherent cells with microcarriers in a culture medium and detachment of cells from microcarriers and transferring into a new bioreactor. This is done in a gradual manner, starting from small number of adherent cells in a smaller bioreactor. The microcarriers are made up of dextran polymers and commercially available under the brand name CYTODEX®. The microcarriers used are preferably spherical balls having an average diameter of about 50 to 300m in the dry state and a density slightly greater than 1. The concentration of microcarrier is in the range between 1 and 6 grams of microcarrier per liter of medium lgram of dry balls contains approximately 5xl0⁶ balls, corresponding to a total surface area per gram of about 0.6 m².

The detachment of cells inside the bioreactor and transferring to another bioreactor, i.e. passaging may be carried out multiple times. Each new cell passage is carried out in the different bioreactor or culture vessel. The cells are detached from the microcarrier by enzymatic treatment with trypsin after reaching confluency with a final cell density of at least 1.0 x 10⁶ and the cells are transferred to higher volume bioreactor. Further, in order to facilitate the detachment of the cells, a chelating agent such as Ethylenediaminetetraacetic acid ( EDTA ), Phosphate Buffered Saline (PBS)-EDTA, EGTA (ethylene glycol-bis(P-aminoethyl ether)- N,N,N',N'- tetraacetic acid) and sodium citrate may be added to the trypsin enzyme solution. Alternatively, the adherent cells are treated with a chelating agent before the enzyme treatment. Preferably, a highly diluted solution of purified trypsin, between 0.005 percent and 0.05 percent, is used.

The process for each type of poliomyelitis S 19 virus, for example Type 1, Type 2 and Type 3, consists of multiplying a cell strain, preferably the VERO cell, from a cell stock by culturing on microcarriers in a suspension containing suitable nutritive / culture medium by means of successive passages into increasing volumes of bioreactors. The last passage is carried out in a bioreactor whose tank holds at least 100 liters, into a suitable nutritive medium. Inoculating the bioreactor of the last virus passage, maintaining the temperature in the vicinity of 30° C to 40° C, the pH in the range of 7.4 to 7.8 and the partial oxygen pressure in the range of 5 to 15 percent.

The same culture medium is generally used to carry out the successive cell passages, into increasing volumes of bioreactors, although it may be advantageous to add nutritive supplements such as glutamine, glucose and pluronic F68 and the like during the successive cell passages.

The clarification of the cell harvest is carried out by means of 0.01 to 0.3 pm bioburden filtration to remove cell debris, aggregates and remaining micro-carriers. The clarified harvest is subjected to anion-exchange chromatography for the removal of host cells nucleic acids (DNA) and negatively charged host cell proteins (HCP), followed by ultrafiltration/diafiltration for the removal of host cell proteins and thereby resulting in the increased 50-200 X concentration of S 19 strain poliovirus. The concentrated S 19 poliovirus is subjected to 0.01 to 0.3 pm filtration by means of size exclusion chromatography (SEC) for removal of aggregates, host cell proteins and trace contaminants, followed by inactivation. The inactivation of S 19 poliovirus is carried out using dilution buffer which includes Ml 99 in the concentration range of 0.5 to 2 X and glutamine in the concentration range of 0.01 to 0.2 g/L and treatment with 0.001 to 1 % of formaldehyde (HCHO).

EXAMPLES The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for the purposes of illustration and are not intended to limit the scope of the invention. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Example 1 : IPV Vaccine formulation I

Given below is the manufacturing process for preparing trivalent IPV S19 vaccine: a. sterile aluminum hydroxide gel, was aseptically transferred into HDPE container, b. aseptically transferred inactivated monovalent bulk of Poliovirus S 19 Type 1 into the above container and stirred at 200 ± 50 RPM for 10 -15 minutes at room temperature, c. aseptically transferred inactivated monovalent bulk of Poliovirus Type 2 into the above container and continued stirring at 200 ± 50 RPM for 10 -15 minutes at room temperature,

d. aseptically transferred inactivated monovalent bulk of Poliovirus Type 3 into the above container and continued stirring at 200 ± 50 RPM for 60 - 75 minutes at room temperature,

e. sterile filtered IX Ml 99 solution with 2-Phenoxy ethanol was added to the above container and continued stirring at 200 ± 50 RPM for 20-30 minutes at room temperature and

f. filled the required quantity of above solution into unit dose vials and stored the vials at 2-8°C.

Example 2 : IPV Vaccine formulation II

Given below is the manufacturing process for preparing a trivalent IPV S19 vaccine: a. sterile filtered 1 X Ml 99 solution with 2-Phenoxy ethanol was taken into 5L HDPC container and stirred at 200 ± 50 RPM at room temperature,

b. aseptically transferred inactivated monovalent bulk of Poliovirus S 19 Type 1 into the above container and stirred at 200 ± 50 RPM for 5 -10 minutes at room temperature, c. aseptically transferred inactivated monovalent bulk of Poliovirus Type 2 into the above container and continued stirring at 200 ± 50 RPM for 5 -10 minutes at room temperature,

d. aseptically transferred inactivated monovalent bulk of Poliovirus Type 3 into the above container and continued stirring at 200 ± 50 RPM for 15 - 20 minutes at room temperature,

e. filled the required quantity of above solution into unit dose vials and stored the vials at 2-8°C.

Example 3 : IPV Vaccine formulation III

Given below is the manufacturing process for preparing trivalent IPV S 19 vaccine: a. sterile aluminum hydroxide gel, was aseptically transferred into HDPE container, b. aseptically transferred inactivated monovalent bulk of Poliovirus S19 Type 1 into the above container and stirred at 200 ± 50 RPM for 10 -15 minutes at room temperature, c. aseptically transferred inactivated monovalent bulk of Poliovirus Type 2 into the above container and continued stirring at 200 ± 50 RPM for 10 -15 minutes at room temperature,

Example 4: Stability of vaccine formulations

The stability of the formulation prepared according to the example 1 (IPV Vaccine formulation I) was studied under real time and accelerated conditions upto 6 months. The initial, real time (2-8°C) and the accelerated (25°C±2°C) stability study results are given below in table 1: Table 1: The initial, real time (2-8°C) and accelerated storage stability results for example 1.

The stability of the formulation prepared according to the example 2 (IPV Vaccine formulation II) was studied under real time and accelerated conditions upto 6 months. The initial, real time (2-8°C) and the accelerated (25°C±2°C) stability study results are given below in table 2: Table 2: The initial, real time (2-8°C) and accelerated storage stability results for example 2.

The stability of the formulation prepared according to example 3 (IPV Vaccine formulation III) was studied under real time conditions. The initial and real time (2-8°C) storage stability study results are given below in table 3:

Table 3: The initial and real time (2-8°C) storage stability results for example 3.

It was found that the D antigen content of inactivated poliovirus (IPV) S 19 strains Type-l, Type-2 and Type-3 remained stable over the study period indicating the suitability of the formulations to be used as vaccine formulations. Example 5: Immunogenicity of the vaccine formulations

An open label, single center Phase-I study to evaluate safety, reactogenicity and immunogenicity was conducted on 24 healthy adults ( 18-45 year old) by administrating 0.5 mL dose of formulations disclosed in Example 1 and Example 2, through Intramuscular route. 10 mE blood sample was collected at Day 0, and Day 28 and it was found that the formulations were safe and immunogenic. ADVANTAGES OF THE INVENTION

Global shortage in the supply of inactivated polio vaccine (IPV) has led to re-strategizing use of IPV in the form of fractional doses for vaccinating a larger population. The present invention provides means of recouping with this shortage as the containment requirements for manufacturing S19 IPV bulk antigen are less as compared to presently used Salk and Sabin bulk antigens. The present invention thus provides a safe, stable and effective immunogenic composition of IPV S19 strains IPV that can be manufactured at low containment level independent of the herd immunization status of the country., The IPV S19 strain is handled pre- and post-polio eradication, which would potentially lead to large scale production, economies of scale, and hence availability of immunization for all.

Claims

The claims:

1. A vaccine formulation comprising Inactivated Poliovirus S 19 strain Type 1, S 19 Type 2 and S 19 Type 3 and at least one pharmaceutically acceptable carrier.

2. The vaccine formulation as claimed in claim 1, wherein IPV S-19 Type-l is a Mahoney strain or a Brunhilde strain.

3. The vaccine formulation as claimed in claim 1 , wherein IPV S-19 Type-2 is a MEF-I strain.

4. The vaccine formulation as claimed in claim 1, wherein IPV S-19 Type-3 is a Saukett strain.

5. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises inactivated poliovirus (IPV) S 19 Type-1 at a dose of 4 to 60 D-antigen units.

6. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises inactivated poliovirus (IPV) S 19 Type-2 at a dose of 0.8 to 12 D-antigen units.

7. The vaccine formulation as claimed in claim 1, wherein the vaccine comprises inactivated poliovirus (IPV) S 19 Type-3 at a dose of 3.2 to 48 D-antigen units.

8. A vaccine formulation comprising Inactivated Poliovirus S 19 strain selected from S 19 Type 1 (Mahoney), at a dose of 4 to 60 D-antigen units, S 19 Type 2 (MEF-l) at a dose of 0.8 to 12 D-antigen units and S 19 Type 3 (Saukett) at a dose of 3.2 to 48 D-antigen units and at least one pharmaceutically acceptable carrier.

9. The vaccine formulation as claimed in any one of claims 1 to 8, comprising pharmaceutically acceptable adjuvants, excipients, preservatives, stabilizers, buffers, pH indicators, surfactants, solvents, residuals or diluents.

10. The vaccine formulation as claimed in any one of claims 1 to 8, wherein the inactivated polioviruses are optionally adsorbed onto an aluminium salt selected from a group comprising aluminium hydroxide, aluminium phosphate and combinations thereof.

11. The vaccine formulation as claimed in any one of claims 1 to 8, wherein stabilizers are selected from a group comprising Ml 99 medium and amino acids.

12. The vaccine formulation as claimed in any one of claims 1 to 8, wherein preservatives are selected from a group comprising 2-phenoxyethnaol, m-cresol, benzyl alcohol, benzoic acid and combinations thereof.

13. A vaccine formulation comprising Inactivated Poliovirus S 19 strain selected from S 19 Type 1 (Mahoney), at a dose of 40 D-antigen units, S 19 Type 2 (MEF-l) at a dose of 8 D- antigen units and S 19 Type 3 (Saukett) at a dose of 32 D-antigen units and at least one pharmaceutically acceptable carrier.

14. A vaccine formulation comprising Inactivated Poliovirus S 19 strain selected from S19 Type 1 (Mahoney), at a dose of 20 D-antigen units, S19 Type 2 (MEF-l) at a dose of 4 D- antigen units and S19 Type 3 (Saukett) at a dose of 16 D-antigen units and at least one pharmaceutically acceptable carrier.

15. A vaccine formulation comprising Inactivated Poliovirus S 19 strain selected from S19 Type 1 (Mahoney), at a dose of 13.33 D-antigen units, S19 Type 2 (MEF-l) at a dose of 2.66 D-antigen units and S19 Type 3 (Saukett) at a dose of 10.66 D-antigen units and at least one pharmaceutically acceptable carrier.

16. A vaccine formulation comprising Inactivated Poliovirus S 19 strain selected from S19 Type 1 (Mahoney), at a dose of 4.0 D-antigen units, S19 Type 2 (MEF-l) at a dose of 8.0 D-antigen units and S19 Type 3 (Saukett) at a dose of 3.2 D-antigen units and at least one pharmaceutically acceptable carrier.

17. A process of preparing the vaccine formulation as claimed in any of the claims 1 to 16, comprising:

a. aseptically transferring inactivated poliovirus (IPV) S19 strain S19 Type-l, S19 Type-2 and S19 Type-3 to a vessel;

b. optionally, adding one or more components selected from pharmaceutically acceptable adjuvants, excipients, preservatives, stabilizer, buffers, pH indicator, surfactants, solvents, residuals or diluents to the vessel under stirring conditions; c. stirring;

d. optionally, subjecting the formulation to sterile filtration;

e. optionally, adjusting the pH of the formulation to obtain the vaccine formulation.

18. A method of preventing or treating poliomyelitis, comprising administering an effective amount vaccine formulations as claimed in any one of claims 1 to 16.

19. Vaccine formulations as claimed in any one of claims 1 to 16, wherein the vaccine formulations are useful for prophylaxis against poliomyelitis.