Title of Invention	"A PROCESS FOR THE PREPARATION OF POLIO VACCINE"
Abstract	The present invention relates to the the process of preparation of polio vaccine. Provided is a Polio vaccine derived from Sabin strain for immunization against Poliomyelitis characterized in that the Polio Vaccine prepared is stabilized by adding a stabilizer and then inactivated by adding Formaldehyde (Formalin). Also provided is process of preparation of such vaccine.

Full Text

The present invention relates to the Sabin strain derived inactivated Poliomyelitis Vaccine for immunization against Poliomyelitis. BACKGROUND OF THE INVENTION: Polio is caused by three types of quite stable viruses belonging to the family of enteroviruses. There are different polio vaccines available in the market. These vaccines are trivalent containing a mixture of all the three types of poliovirus so as to confer immunity against all of them. One type of Polio Vaccine available is the Oral Polio Vaccine (OPV) based on Sabin strain. This orally administered Polio vaccine consists of live attenuated strains of polio virus. The OPV is commonly used because of its following advantages: 1. Manufacture of OPV does not pose any risk of accidental release of virulent polio viruses in the environment. 2. Worldwide availability of production facilities & easily available Monovalent bulks. 3. Greater herd immunity because of stimulation of mucosal immunity and the resultant curtailment of spread of wild virus; displacement of wild virus in the community by development of more than 30 vaccine-related strains, and involuntary immunization of contacts by vaccine-related virus. 4. Its administration is easier. However the OPV has the following disadvantages: 1. The OPV is highly perishable and requires cold chain maintenance below minus 20 deg. C. 2. Vaccine derived Polio viruses (Reversion of attenuated strains to virulent strains). 3. Vaccine associated paralytic poliomyelitis. 4. Paralytic poliomyelitis in case of immuno-suppressed recipients of OPV. Another Polio Vaccine available is Inactivated Polio Vaccine based on Salk strain (Salk IPV). The vaccine contains all the three types of polio virus, inactivated bylformalin. The advantage of Salk IPV is: It can be incorporated with other childhood immunizations like DPT. However the Salk IPV has the following disadvantages: 1. It poses a risk of accidental release of live virulent polio viruses in the environment due to handling of virulent strain of polio viruses during its production process. 2. Requires stringent standards like BL-3 laboratory for growing the Salk strain of poliovirus. It is an object of the present invention to provide a Polio Vaccine derived from Sabin strains for immunization against Poliomyelitis which obviates the disadvantages, associated with known Polio vaccines OPV and Salk IPV. It is another object of the present invention to provide a polio vaccine, which can be administered through injection. Accordingly the present invention provides a Polio Vaccine derived from Sabin strain for immunization against Poliomyelitis prepared from Sabin seed strain or Sabin strain monovalent bulk suspensions used for blending of trivalent oral polio vaccine or by growing Sabin strain Polio viruses (from monovalent bulk used for trivalent OPV) in vero cell line or any suitable cell culture as per standard methods for the preparation of Oral Polio Vaccine characterized in that the Polio virus is first stabilized by adding a stabilizer, as herein described, and then inactivated by adding Formaldehyde (Formalin). The present invention also provides a process for the preparation of Polio Vaccine derived from Sabin strain for immunization against Poliomyelitis which comprises of preparing Polio Vaccine from Sabin seed strain or Sabin strain monovalent bulk suspensions used for blending of trivalent oral polio vaccine or by growing Sabin strain Polio viruses (from monovalent bulk used for trivalent OPV) in vero cell line or any suitable cell culture as per standard methods for the preparation of Oral Polio Vaccine characterized in that the Polio vaccine so prepared is first stabilized by adding a stabilizer, as herein described, and then inactivated by adding Formaldehyde (Formalin) according to a specific procedure as herein described. The Polio vaccine prepared from Sabin strain, as stated above, may be inactivated, according to present invention, by one of the following two processes. A) Process -I : 1) Stabilization process: The monovalent bulk of poliovirus is to be initially stabilized by adding stabilizer like Sucrose or Trehalose or Arginine hydrochloride to retain the antigenicity of the vaccine during inactivation process. 2) Inactivation process I. i) Inactivation process is to be initiated immediately. ii) Inactivation of each of filtered & purified monovalent pool is to be carried out by adding formaldehyde (formalin) at 0.025% concentration and incubation at 37°C upto 48 hrs. & then at 2°C to 8°C up to 12 days in a single cycle before further processing. Either of the treatment could be used for inactivation. iii) The test for free formaldehyde content is to be performed after every 12 hrs. during the inactivation process and the desired concentration level is maintained by intermittent readjustments. iv) A second filtration is to be done after the process of inactivation. v) Consistent inactivation of the virus is monitored and verified. B) Process -II : 1) Stabilization process: The monovalent bulk vaccines are stabilized using the same procedure as described for Process-l. 2) Inactivation Process -II : i) Inactivation is carried out by adding formaldehyde (formalin) at 0.025% concentration and incubation at 37°C for 4 hrs followed by chilling at 2°C to 8°C for 20 hrs. This incubation & chilling cycle can be repeated up to 12 times so that total exposure at 37°C does not exceed 48 hrs during 12 days of incubation. ii) The test for free formaldehyde content is to be performed after every 12hrs during the inactivation process and the desired concentration level is maintained by intermittent readjustments. iii) A second filtration is to be done after the process of inactivation. iv) Consistent inactivation of the virus is monitored and verified. Maintenance of potency/ antigenic integrity of highly heat labile Sabin viruses: The inactivation processes are standardized in such a way that live attenuated virus of each type is completely inactivated and the process ensures intact antigenic structure of Sabin strain. Inactivated vaccine is able to impart adequate immunity in vaccines when compared with standard vaccine. The Sabin strain poliovirus, in present invention, is stabilized before inactivation using stabilizer like sucrose, trehalose, arginine hydrochloride to avoid degradation of nativeness and antigenic structure. The inactivated Polio Vaccine, prepared according to the present invention, in monovalent, bivalent or trivalent form has required amount of equivalent D - antigen or antigen form which could be compared with an established reference standard of inactivated polio vaccine and/or provide sero-conversion and required protection. The process for the preparation of Polio vaccine based on Sabin strain for immunization against Poliomyelitis, according to the present invention, is illustrated by the following Examples: Example 1: Seed strain of the same type and passage as used for poliomyelitis vaccine (oral); Oral Polio Vaccine (OPV), is propagated to produce monovalent suspension for inactivation. CELL CULTURE: i) Different cell substrates for propagation of virus: 1. Human diploid cell line 2. Monkey kidney cells 3. Vero cell line 4. Any other suitable cell culture ii) Large scale production of cells: 1. Preparation of the Manufacturer's working cell bank (MWCB): MWCB in case of primary cell cultures (cells derived from normal tissue and stored frozen at minus 70°C) or from any other permitted continuous cell line are prepared from the cells & the cells pooled after serial subculture within the specified number of cell cultures. 2. Any of the following systems can be used, which ever is suitable, for stepwise increase in the volume of cells. • Roller bottles - Growth in roller bottles differs from that in stationary cultures in distribution of cells on glass surface and maximum attainable cell density. Roller bottle cultures do not deteriorate rapidly as they can tolerate a density nearly two times higher than that of stationary cultures. Mammalian cells grown in monolayer culture will adapt best to roller bottle technique. • Cell factories - These are stack of culture trays that share a common inlet and outlet port. These provide cells with a growing surface as large as inside of roller bottle, but take up less space inside incubator. These are ideal for adherent cell cultures, have low contamination risk and are compact. • Roux flasks • Micro-carrier system 3. Samples for control cell cultures are incubated separately for at least two weeks and these are examined for evidence of cytopathic changes. PROPAGATION OF POLIO VIRUS IN CELL CULTURES: i) Seed lot system: a) Passage level as followed for Oral Polio Vaccine OR b) Its next passage level as derived from monovalent bulk suspension that could be used to manufacture oral polio vaccine. 1. The vaccine is manufactured on the basis of virus seed lot system. Virus seed lot is a quantity of virus processed together and of uniform composition prepared from seed lot. 2. Sub-culture of the seed virus should not be done more than 10 times, counted from a seed lot used for the production of the vaccine on which original laboratory and field tests were done. SINGLE HARVEST AND MONOVALENT POOLS: The virus is propagated in cell cultures and harvested from cell cultures derived from a single batch of cells and processed together. This is known as single harvest. The single harvests of one type of virus suspension are processed at the same time to get crude form of monovalent pool. FILTRATION OR CLARIFICATION OF MONOVALENT POOL: Crude virus suspension of each monovalent pool is purified stepwise through filters of decreasing porosity and finally through 0.22 µm filter. The purpose of filtration step is to remove particulate material and other substance that may affect inactivation process as such aggregates tend to increase on standing. CONCENTRATION OF THE MONOVALENT POOL: Each filtered monovalent pool is concentrated by Ultra-filtration. PURIFICATION OF THE MONOVALENT POOL: Chromatography with DEAE sepharose / immobilized DNA-ase column / immuno-adsorption column. The purification process should reduce consistently the level of cellular DNA from that of initial virus harvest by a factor of at least 108. The D-antigen concentration is to be determined by ELISA and display of comparable immunogenicity in rats. Accordingly, potency is to be adjusted. The monovalent pools of Poliovirus are to be blended to form the final trivalent bulk product. STABILISING THE SABIN VIRUS BEFORE INACTIVATION: The antigenicity of the vaccine is closely associated with the stability of the native virus antigens during inactivation process. The poliovirus antigenic structure is to be initially stabilized by adding stabilizer like sucrose or Trehalose or arginine hydrochloride. INACTIVATION USING FORMALDEHYDE: i) Inactivation process is initiated preferably within 24 hrs. and not later than 72 hrs after filtration. ii) Inactivation of each of filtered and purified monovalent pool is carried out by adding formaldehyde (formalin) at 0.025% and incubation at 37°C for specific time period as described earlier. The test for free formaldehyde is performed at intervals and the desired concentration level is maintained by intermittent readjustments. iii) A second filtration is to be done after the process of inactivation. iv) Consistent inactivation of the virus is monitored and verified. v) Formaldehyde is neutralized by adding sodium bisulfite and subsequently dialyzing it out. FORMULATION AND MANUFACTURING OF THE VACCINE: The inactivated monovalent viruses may be mixed with stabilizer or/and preservative to make monovalent, bivalent or trivalent inactivated vaccine. The final vaccine may be spray dried & resuspended in ready-to-inject liquids like perfluorocarbons, requiring no refrigeration for storage or preparation before injection. TESTING THE EFFICACY OF THE VACCINE: The vaccine (as manufactured above) efficacy as compared with established reference vaccine is found to be equal in in-vitro and/or in-vivo method. Example 2 : The monovalent bulk suspensions obtained for the purpose of oral polio vaccine are inactivated using the same method as described in example 1 and then used for the purpose of manufacturing of Sabin based inactivated polio vaccine. The Polio Vaccine SIPV, prepared according to the present invention, has the following advantages: 1. The SIPV does not require growing of virulent strain of polio virus and hence its production process does not involve accidental release of live virulent polio viruses in the environment. 2. The SIPV does not have disadvantages like vaccine associated paralytic poliomyelitis and vaccine derived virus. 3. The SIPV has the combined advantages of both OPV and Salk IPV. 4. The monovalent bulk is available easily. 5. Its production facilities are available worldwide. We claim: 1. A process for the preparation of Polio vaccine derived from Sabin strain which comprises of the following steps a. preparing Polio Vaccine from Sabin Seed strain or Sabin strain monovalent bulk suspensions used for blending of trivalent oral polio vaccine or by growing Sabin strain polio viruses from monovalent bulk used for trivalent OPV in vero cell line or any suitable cell culture for the preparation of Oral Polio Vaccine, b. stabilizing the Polio Vaccine so prepared by adding a stabilizer and c. inactivating the stabilized vaccine wherein the inactivation process comprises of the following steps: i. Adding formaldehyde (formalin) at 0.025% concentration to each of filtered & purified monovalent pool and incubating at 37°C upto 48 hrs followed by inactivating at 2°C to 8°C up to 12 days in a single cycle, or adding formaldehyde (formalin) at 0.025% concentration and incubating at 37°C for 4 hrs followed by chilling at 2°C to 8°C for 20 hrs wherein the incubation & chilling cycle can be repeated up to 12 times so that total exposure at 37°C does not exceed 48 hrs during 12 days of incubation, and ii. Testing for free formaldehyde content after every 12 hrs during the inactivation process and maintaining the desired concentration level of formaldehyde by intermittent readjustments to obtain the desired inactivated vaccine. 2. A process as claimed in claim 1, wherein the stabilizer used is Sucrose. 3. A process as claimed in claim 1, wherein the stabilizer used is Trehalose. 4 A process as claimed in claim 1, wherein the stabilizer used is Arginine hydrocloride. 5. A process as claimed in claim 1, wherein the stabilizer used is Gelatin. 6. A Polio vaccine prepared by the process as claimed in claim 1.

Documents:

1213-del-2001-abstract.pdf

1213-del-2001-claims.pdf

1213-del-2001-correspondence-others.pdf

1213-del-2001-correspondence-po.pdf

1213-del-2001-description (complete).pdf

1213-del-2001-form-1.pdf

1213-del-2001-form-13.pdf

1213-del-2001-form-19.pdf

1213-del-2001-form-2.pdf

1213-del-2001-form-3.pdf

1213-del-2001-form-4.pdf

1213-del-2001-form-5.pdf

1213-del-2001-gpa.pdf

1213-del-2001-petition-137.pdf