FIELD OF THE INVENTION:
The present invention pertains to the field of viral vaccine compositions. Particularly, the present invention relates to Japanese encephalitis vaccine composition and processes or methods of manufacture of vaccine composition against Japanese encephalitis infections. The present invention relates to processes for production and purification of virus bulk and inactivation techniques of the said Japanese encephalitis virus bulk thereof.
BACKGROUND OF THE INVENTION:
Japanese encephalitis virus (JEV) is a mosquito-borne infection, a leading cause of viral encephalitis throughout Asia and is spreading beyond its continental boundaries. JEV is classified under flavivirus family and genus flavivirus. It is a small enveloped virus (50nm) containing a 10.9 kb single-strand, negative sense RNA genome. Viron RNA codes for 11 proteins include four structural and seven non structural Proteins. Among the 4 structural protein the surface envelope protein (E protein) serves as the cell receptor binding Protein and fusion protein for virus attachment and entry into the host. So antibodies against E protein neutralize the virus and play an important role in protection. The surface structural E protein is an important component of the vaccine so it is very important to stabilize this protein molecule with suitable stabilizer for vaccine efficacy.
Due to the vast area distribution of vaccines and the diversity of ambient temperature, there has been a need to improve the bioprocess to manufacture Japanese encephalitis Virus bulk for manufacturing the micro-organisms for production and further use of the vaccine. The bioprocess of Japanese encephalitis involves either growing in roller bottle flasks, or cell factory units. Production of industrial amounts of the Japanese encephalitis vaccine through roller bottle cell culture, or through cell factories needs higher amounts of time, with comparatively lesser yields, and also occupying large amount of space, which makes it difficult to control and regulate experimental parameters at a regular and constant basis in each cell factories. The production of virus bulk therefore inherently lacks uniformity of operation. The present invention overcomes these difficulties through its new bio-process technique wherein the time required to yield high amounts of industrial Japanese encephalitis bulk is reduced to great extents. Commercial production of the vaccine bulk is enabled within much lesser time as compared to earlier culture techniques. Vero cells are

first grown in a disposable bioreactor followed by infection with JEV, wherein multiple viral harvests is accomplished as a novel feature in this invention. Production of the virus is followed by simultaneous inactivation and stabilization methods particularly applicable to Japanese encephalitis vaccine antigen. Stabilization of Japanese encephalitis vaccine in general require maintaining the vaccine vials or the bulk at prescribed temperature ranges at 2°-8°C for stabilization for larger periods of time. This requires added infrastructure facility to transport and store the vaccine thereby increasing cost of the vaccine. Alternative stabilization techniques are required for Japanese encephalitis for overcoming some of the most practical limitations. The vaccine so obtained from the virus bulk according to the methods disclosed in this invention do not require any special refrigeration facilities mentioned above and are stable at ambient temperature conditions i.e. at room temperatures (25°C). The stabilities obtained by the present invention are comparable to the stabilities observed in case of Japanese encephalitis vaccines stored at refrigeration temperatures (2°-8°C). The potency and the immunogenecity of the viral bulk and the vaccine to confer sufficient immunization to the subjects also are retained.
OBJECTS OF THE INVENTION:
One object of the present invention is to provide a stable inactivated vaccine composition that is capable to prevent as well as provide treatment from Japanese encephalitis infections caused by Indian Kolar strain of the Japanese encephalitis virus.
Another object of the invention is to provide for a method of adaptation of the Kolar strain of the Japanese encephalitis virus in Vero Cells.
Yet another object of the invention is to provide for a suitable method of commercial production of purified inactivated Japanese encephalitis bulk at specified experimental parameters, and techniques which enables multiple harvesting of Japanese encephalitis bulk at much higher yields using a disposable bioreactor.
One more object of the invention is to provide for novel purification techniques using specific matrix column during the downstream processing of the Japanese encephalitis virus bulk at a commercial scale.

Yet another object of the invention is to provide a simultaneous method of inactivation and stabilization of the purified Japanese encephalitis virus bulk.
SUMMARY OF THE INVENTION:
According to one embodiment of the invention, various methods of culture of Vero cells in a disposable bioreactor is disclosed. Satisfactory levels of growth of Vero cells at various experimental parameters involving proper media composition and requirement of change in media is optimized, to obtain satisfactory levels of cell count within a desired amount of time. Vero cells are infected with the live Japanese encephalitis virus, Indian Kolar strain, and accordingly the embodiment describes, a method of adaptation of Japanese encephalitis virus to grow in Vero cells in a disposable bioreactor.
According to another embodiment of the invention, the adaptation of Kolar strain of Japanese encephalitis virus in Vero cells is scaled up from a disposable bioreactor to next subsequent sizes of the disposable bioreactor to generate production batch size yield of purified JEV bulk. The disposable bioreactors have been named as DB-1 to DB-100 to DB-500, depending upon cell capacity.
A further embodiment of the invention provides alternative methods of downstream purification of the live JEV bulk using various novel chromatographic techniques, which results in obtaining high amounts of purified JEV bulk.
One more embodiment of the invention discloses novel methods of simultaneous inactivation and stabilization of the purified JEV bulk.
The invention also provides experimental data of stability of the purified inactivated JEV bulk at 25 °C - 37°C, ready for vaccine formulation.
Yet a further embodiment of the invention provides a Japanese encephalitis vaccine composition comprising a vaccine antigen, wherein the antigen is Kolar strain of Japanese encephalitis virus.

DETAILED DESCRIPTION OF THE INVENTION: Definitions:
Initial cell seeding: Initial Quantity of Cells required to seed in the culture flask. DMEM: Dulbecco's Modified Eagle Medium EMEM: Eagle's minimal essential medium NBCS: New Born Calf serum
The present invention discloses a novel Indian strain of the Japanese encephalitis virus JEV821564-XY which is a Kolar strain of the Japanese encephalitis virus. This Kolar strain JEV821564 of the Japanese encephalitis virus (hereafter mentioned as 'Kolar strain') was transferred to Bharat Biotech International Limited through a Material Transfer Agreement from National Institute of Virology, Pune. At present there is no vaccine composition against the Indian strain (Kolar) of the Japanese encephalitis virus. Furthermore, this particular strain of the Japanese encephalitis virus is grown in a method wherein the strain is adapted in Vero cells for its culture. Virus culture is generally grown in liquid media. Cell culture in Vero cells of any biological product includes very high number of roller bottles or cell factories. Virus culture in Cell-Factories takes longer durations of time. Cell Factories occupy higher amounts of space, and it is also difficult to monitor all the experimental parameters during virus culture in cell factories individually. The produce of Japanese encephalitis through conventional cell factories is minimal.
Hence the present invention discloses a novel method of manufacture of this Kolar strain of the Japanese encephalitis virus in a disposable bioreactor. Japanese encephalitis virus is adapted to be grown in Vero cells, and this particular strain of the virus is adapted to propagate in Vero cells supplied with specific media. The same Vero cells are capable to give multiple harvests of the virus bulk, within single infection. This particular technique of multiple virus harvest of Japanese encephalitis virus (JEV) presented in this invention is unique and distinct, although propagation of JE virus in Vero cells is already known. Furthermore, obtaining desired amount of cell growth (of Vero Cells) in a new apparatus (other than roller bottles and cell factories) such as the one used in this invention which is a disposable bioreactor, and scaled up from smaller capacity disposable bioreactors to higher capacity production size batches of disposable bioreactors is not provided earlier. The

conventionally used method of propagation of the Japanese encephalitis virus in Vero cells are generally grown in Cell Factories or roller bottles in which the Vero cells are utilized in a layer formation for only once. The Vero cell line is utilized by infecting the Japanese encephalitis virus, and the cell line is exhausted after only one infection by the virus seed bank. But, in the present invention, the Vero cells are grown in such unique and novel technique and the experimental parameters are set in such manner that, a single infection of the seed Japanese encephalitis virus is capable of producing at least thrice to five time harvest of the virus bulk from the same Vero cell line culture involving the disposable bioreactor. At the same time, this reduces the number of infections as well as the amount of space and more quantity of cell factories required to infect and grow the same viral cell culture quantity. Additionally, multiple harvests from the cell culture, also automatically adds on the amount of yields, both in terms of quantity and quality of the viral harvest to very high amounts as compared to the processes involved in doing and attaining similar yields with Cell Factories.
Furthermore, the present invention is directed to stabilize the purified JEV bulk both during inactivation process and during storage of virus bulk. The composition of each stabilizer ranges from 0.5 to 1% w/v. The chemical stabilizers which are added to viral antigen are D-sorbitol and glycine. The viral antigen with suitable stabilizer was stored in the sufficient physiologically acceptable phosphate buffer saline to maintain pH at from 7.0 to about 7.4.
Example 1: Cell Culture and adaptation of Kolar strain of JE virus to Vero Cells.
Initial cell seeding of Vero cells are taken in a T-175 flask comprising of Vero Cells. Vero cell culture is done in T-175 flasks and grown in suitable media. Cell growth obtained from 1 T-175 flask is transferred to 5 T-175 flasks after 4-5 days. This is further transferred to 1 cell-factory-10 (CF10) after 4-5 days again, and then again transferred to 5 Cell Factory-40 (CF-40). CF-40 is used presently for commercial production of JEV bulk. One batch of JEV bulk is produced involving at least 30 CF-40s in a period of one month for production of one commercial batch of JEV bulk. The present invention teaches a particular method of propagation of the said Kolar strain of JE in Vero cells, useful for commercial scale-up of the bulk production and is capable to produce JEV bulk upto approximately 140 CF-40s within the same period of time i.e. approximately one month. This leads to higher yields of

the virus, as the total amount of bulk extracted is very high as compared to 30 CF40s. Such a high yield of the JEV bulk within the same period of time is a big advantage to increase the production capacity commercially. Furthermore it is practically impossible to monitor and control approximately 200 CF40s at a time for an amount of time i.e. approximately one month. According to current practices, for operation of a batch size of 30 CF-40s, a minimum requirement of a walk-in incubator room which can accommodate at least 3-4 people for operation and carrying out the experiments is needed as the culture facility. The incubator room shall retain a constant prescribed temperature at 37°C throughout the batch. Hence, it may be ascertained that the requirement of having run an incubator room constantly for a period of 5-6 months which would provide output of 200 cell factory-40s (200 CF40s) will definitely increase the production costs. Alternatively, for running approximately upto 150 CF-40s at a time within a period of one month will require a huge and an extra large sized incubator room maintained constantly at 37°C to provide sufficient space for at least 20 people to operate and control the experimental parameters. Building up such a facility is not economical at all. Such a huge incubator room will invite additional and huge investments thereby making the vaccine unaffordable to those who are in most need of JE vaccine. All the experimental parameters will be varying, and the experiment endpoints cannot be accomplished at the same time, for recovering the bulk Japanese encephalitis virus, meant for vaccine production. Cell culture in 150-200 CF40s will take approximately more than 6 months of time for equivalent amount of JEV bulk production. The important experimental parameters such as temperature, pH, dissolved oxygen, agitation and media supply is not possible to regulate simultaneously for 150-200 CF40s within one month of time. Moreover, operation of all the said experimental parameters of 30 CF40s in one batch of JEV bulk is also very tidy and requires highly developed skill and human resource and labour to ensure that the batch succeeds to produce the required quantity of JEV bulk. Unlike the situation with CF40s, the present invention discloses a novel method of manufacture of JEV bulk in a disposable bioreactor, and not in cell factories (CF40) wherein all the technical specifications in the disposable bioreactor can be regulated with required amount of accuracy and precision required for cell culture and virus culture which cannot be attained in the case of cell factories. The present invention replaces the use of CF40s in JEV bulk production, with such suitable disposable bioreactor. The given disposable bioreactor provides the necessary channels, probes and sensors through

which it is possible to regulate the cell culture and virus culture process automatically. Hence, control of various experimental parameters like temperature, pH, dissolved oxygen, agitation and media supply are easily done in the given disposable bioreactor with the required accuracy and precision. Additionally, adaptation of the Kolar strain of the JE virus into Vero cells in a bioreactor is capable of production of JEV bulk to a quantity of at least 40 CF40s to 200 CF40s in one batch of produce at the same time as required in case of 30 CF40s in one batch. The yields obtained are also higher both in case of equivalent corresponding production capacity and output of 30 CF40s and obviously very very high in case of corresponding production capacity and produce that would have been obtained for up to 200 CF40s in 5-6 months. Also, the amount of loss is significantly minimized to considerable extents for use of bioreactors instead of CF40s. It is very evident that there must have been or it is bound to experience loss in the batch produce for a period of 5-6 months in case of production involving approximately 200 Cell Factories. This possibility is entirely omitted because of use of bioreactor for JEV bulk production according to the methods disclosed in this invention. From the following experiments, it is shown that, the present invention of adapting the particular Kolar strain of the JE virus in Vero cells grown in the given bioreactor increases the yield of the virus production both in cases of comparable amount of produce as in 30 CF40s and/or 200 CF40s. In addition to the above mentioned advantages for JE bulk production in a disposable bioreactor, one further advantage lies in the fact that, contamination risks are minimized to great extents, in case of disposable bioreactors as disclosed in the present invention. Since all the experimental parameters are controlled through electronic instructions, manual operation is minimal in case of JE bulk production in the disposable bioreactor. Therefore, unlike in case of batch production in cell factories, wherein there lies greater probabilities and possibilities of contamination of a batch, the same is not applicable in case of JE bulk production in disposable bioreactors of various sizes termed as DB, DB-100 and DB-500 as described below in the paragraphs to follow. One further advantage of the disposable bioreactor is that it can produce the consistent cells thereby resulting in consistent yields, whereas in case of cell factories, it is not possible to maintain consistent yields to such similar extents to that of disposable bioreactors due to manual handling for regulating the experimental parameters.

In general the total available surface area for cell growth in 30CF40s is 763200 cm which might be having the capacity of -75000-90000 x 106 cells, whereas the disposable bioreactor used in this invention has total surface area available for cell growth ranging from 26000 cm2 to 5000000 cm which supports the required amount of cells to grow and further availability of Vero cells as the substrate for the virus so that it can infect more and more number of Vero Cells, to suffice the required amounts of increased bulk production of JE in the bioreactor. The total cell count capacity ranges from approximately 7800 x 106 to 1500000 x 10 cells. Increase in number of availability of cells signifies increase in available substrate of the virus to infect more and more number of cells, and hence, larger amount of JE bulk productions can be achieved.

Cell culture is a widely practiced technique in biotechnological processes. However, while a given cell is capable to grow in more than one given medium, their characteristics may alter when the medium is changed. It is also crucial to note that, respective to specific cell line, the time and duration of cell growth also varies. Cell growth depends upon a few important parameters such as, medium, growth factors, concentration of glucose, oxygen availability, and the amount of initial seeding and multiplicity of infection (MOI). Furthermore, growths of cells are also prone to irregular pH changes, which require constant monitoring at different phases of cell culture. Hence, it is already established that growing of cells is specific to the purpose and the surrounding environmental, and physico-chemical factors. Growing Vero cell line in the mentioned bioreactor, instead of cell factories unlike the earlier practice is a separate hurdle that has been overcome in the present invention. Additionally, growing the Vero cell line in the bioreactor may need frequent media changes at specific and appropriate time intervals for optimum cell growth. Changing in media also requires particular calculated cost-effective measures to corroborate maximum cell growth. Control of temperature fluctuations in the bioreactor also is an important issue to support good cell growth. At the same time, very high amount of cell growth may result into poor yields, whereas in case of very or low cell growth below the desired levels also results into failure of the experiments. Therefore, it is desirable to enhance and hone all the above mentioned parameters in a new environment (i.e. the disposable bioreactor) to get the required amount of healthy Vero cells that would produce the best yields of the JE bulk. Accordingly, numbers of experiments were performed to grow the Vero cells in micro-carrier based cell culture in the given suitable disposable bioreactor and the cell growth in respective medium were observed and tabulated according to corresponding number of days. The experiments that succeeded in reaching greater than 100000 cells/cm2 and the corresponding day from initial seeding was noted down. The results were further utilized to infect Japanese encephalitis virus in the corresponding experiments with multiplicity of infection (MOI) of 0.005.
Adaptation of a specific strain of a virus to a particular cell substrate is again a challenge. Although adaptation of JE virus on Vero cells is known, yet it is always strain specific. Each individual strain of the virus involves specific conditions to grow and infect Vero cells in a given manner which ensures high growth and quick multiplication of the virus.

The invention as presented in this application refers to adaptation of Kolar strain of the Japanese encephalitis virus in Vero Cells. Characterization of a laboratory adapted strain of JE virus may exhibit different characteristics from those of its earlier isolated strains in terms of rapid growth in Vero cells, viral titer and their resultant potency. Virus characterization and genetic stability can differ the yield, and the necessary processes required for commercial production of the JE bulk. The difficulties associated with difference in virus strains cannot be directly correlated to earlier adapted processes. Therefore, this present invention discloses, adaptation of a new strain of Japanese encephalitis virus which is Kolar strain of JE virus isolated and endemic to the Indian subcontinent in Vero cells in a specific disposable bioreactor as mentioned in the above examples, which is capable of high yield of JE bulk with high potency sufficient to elicit protective immune response in the given subjects against Japanese encephalitis virus infections. The examples as presented in this invention discloses the unique method of adaptation of the novel Kolar strain of the Japanese encephalitis virus in Vero cells, and its multiple harvests obtained from the initial cell line. Experiments detailing multiple viral harvests are enumerated in the below mentioned experiments and tabulated as follows (Table 1.2) in the Disposable Bioreactor (DB) and further scaling up in DB-100 (Experiments I-II) and DB-500. While cell culture are performed in cell factories, the cell culture is gradually, sequentially and chronologically scaled up from T-175 flasks to cell-factories in the given order: 1 T-175 to 5 T-175s to 1 CF10 to 5 CFlOs to 5CF40s to 30CF40s. Transferring of live Vero cell lines from one platform to the next platform requires trypsinisation. Trypsinisation is a process by which the adhered cells are made to detach from the monolayer so that they may be transferred into the next level called as Cell expansion. Such trypsinisation is a critical process and requires skill to execute it, as well as trypsinisation at each particular intermediate step is also time taking. Initial experiments performed with initial cell seeding of 26000 cells/cm2 in the disposable bioreactor of initial size volume referred as DB meant that despite using DB100 or DB500, trypsinisation will still be required several times prior to initially seed the cells in the disposable bioreactor. Therefore we need to minimize the cell seeding in the disposable bioreactor since it is directly proportional to the large scale bioreactor seeding. For example, if we optimize the seeding of 26000 cells/cm2 for the initial sized disposable bioreactor (DB), then DB-100 seeding will require the 10 CF-40 grown cells (26000x106 cells) where as for DB-500 will

require the 50 CF-40 cells (130000x106 cells). It is highly difficult to handle these many CF to trypsinise for collecting the cells from it. With the modified or minimized 3000 cells/cm seeding DB-100 requires the 1 CF-40 grown cells (3000xl06 cells) where as for DB-500 will require the 6 CF-40 cells (15000x106 cells).
Experiments were carried out with disposable bioreactor with an initial cell seeding of 3000 cells/cm with EMEM, glucose lg/L. For the cell growth multiple times medium were exchanged and at approximate glucose concentration about lgm/L. There is a gradual increase in the cell multiplication but the growth rate was slower and time taking delaying infection with virus, further resulting in poor viral harvests after first and second harvests. Therefore, subsequent experiments were conducted to attain the objective to minimize the initial cell seeding with approximately 3000 cells/cm2 in the disposable bioreactor so that considerable growth of Vero cells is achieved along with appropriate medium supplied such as DMEM and high glucose concentration (4.5g/L). Faster growth rate was obtained, compared to previous experiments with optimized media requiring no need for media exchange as well.

Experiment wise objective and results:
Experiment I - Objective: Scale up from Disposable Bioreactor to Commercial Batch size Disposable Bioreactor 100 (DB-100).
This experiment was carried out with disposable bioreactor-100 (DB100) with an initial cell seeding of 3000 cells/cm2 with EMEM, glucose 1.5 g/L. For the cell growth no medium were exchanged and maintained glucose concentration about l.Ogm/L was maintained. The media also contained 5% NBCS (moregate) There was a gradual increase in the cell multiplication. On 5th day it reached maximum of 100000 cells/cm2. JE virus Kolar strain was made to infect Vero cells on 5th day with 0.005 MOI (multiplicity of infection). Viral harvest 1 (VHl) has been collected using serum free medium on the 4th day of the infection and VH2, VH3 and VH4 were collected with 3 day intervals respectively starting from the day of infection. The viral harvests were clarified by microfiltration or depth filters. The clarified harvests were concentrated by using tangential flow filtration 300 kDa cassette and the concentrated harvest further subjected for column purification.
Results: Individual viral harvest titer values are mentioned in the above table. Desirable growths of the virus titer are obtained. No medium exchange is needed. VHl showed titer value 106'88 PFUs and VH2 showed titer value of 10702PFUs. VH3 and VH4 virus titers showed 106-49 PFUs and 10584 PFUs respectively.

Experiment II - Objective: Scale up from Disposable Bioreactor to Commercial Batch size Disposable Bioreactor 100.
This experiment was carried out with disposable bioreactor-100 (DB100) with an initial cell seeding of 3000 cells/cm2 with DMEM, glucose 4.5 g/L. For the cell growth no medium were exchanged and maintained glucose concentration about l.Ogm/L was maintained. The media also contained 5% NBCS (moregate) There was a gradual increase in the cell multiplication. On 5th day it reached maximum of 100000 cells/cmz. Cells were infected on 5th day with 0.005 MOI. Viral harvest 1 has been collected using serum free medium on the 4th day following the infection and VH2 and VH3 were collected with 3 day intervals respectively starting from the day of infection. The viral harvests were clarified by microfiltration or depth filters. The clarified harvests were concentrated by using tangential flow filtration 300 kDa cassette and the concentrated harvest further subjected for column purification.
Results: Individual viral harvest titers values are mentioned in the above table. Desirable growths of the titer are obtained. No medium exchange is needed. VH1 showed titer value lO6'75 PFUs and VH2 showed titer value of 10716PFUs. VH3 and VH4 virus titers showed less than 107PFUs.
Experiment III - Objective: Scale up from Disposable Bioreactor-100 (DB100) to Commercial Batch size Disposable Bioreactor-500 (DB500).
This experiment was carried out with disposable bioreactor-100 (DB100) with an initial cell seeding of 3000 cells/cm with EMEM, glucose 1.5 g/L. For the cell growth no medium were exchanged and maintained glucose concentration about l.Ogm/L was maintained. The media also contained 5% NBCS (moregate) There was a gradual increase in the cell multiplication. Cells were infected on 7th day with 0.005 MOI. Viral harvest 1 has been collected using serum free medium on the 4th day following the infection and VH2 and VH3 were collected with 3 day intervals respectively starting from the day of infection. The viral harvests were clarified by microfiltration or depth filters. The clarified

harvests were concentrated by using tangential flow filtration 300 kDa cassette and the concentrated harvest further subjected for column purification.
Results: Individual viral harvest titers values are mentioned in the above table. Desirable growths of the titer are obtained. No medium exchange is needed. VHl showed high titer value lO7'22 PFUs and VH2 showed titer value of 10692PFUs. VH3 and VH4 virus titers showed less than 10 PFUs.
Experiment IV - Objective: Scale up from Disposable Bioreactor-100 (DB100) to Commercial Batch size Disposable Bioreactor-500 (DB500).
This experiment was carried out with disposable bioreactor-100 (DB100) with an initial cell seeding of 3000 cells/cm2 with DMEM, glucose 4.5 g/L. For the cell growth no medium were exchanged and maintained glucose concentration about l.Ogm/L was maintained. The media also contained 5% NBCS (moregate) There was a gradual increase in the cell multiplication. Cells were infected with JE virus Kolar strain was on 7th day with 0.005 MOI. Viral harvest 1 has been collected using serum free medium on the 4th day following the infection and VH2, VH3 and VH4 were collected with 3 day intervals respectively starting from the day of infection. The viral harvests were clarified by microfiltration or depth filters. The clarified harvests were concentrated by using tangential flow filtration 300 kDa cassette and the concentrated harvest further subjected for column purification.
Results: Individual viral harvest titers values are mentioned in the above table. Desirable growths of the titer are obtained. No medium exchange is needed. VHl showed high titer value lO741 PFUs and VH2 showed titer value of 10711 PFUs. VH3 and VH4 virus titers showed 106'47PFUs and 105'78 PFUs respectively.
Example 2: Purification of JE bulk using celufine sulfate ester matrix and Inactivation of the JE bulk.
The purification of the JE bulk produced in the disposable bioreactor is carried out using celufine sulfate column based on affinity chromatography. Celufine sulphate column

purification comprises a chromatographic technique wherein the said celufine sulphate is a column matrix made of cellulose support matrix with the presence of an activated group sulphate ester. The total harvest volume is first concentrated with 300 kDa membrane from 300 liter to 50 liter to obtain the retentate containing live JEV. The permeate is discarded.. The retentate contains the JE virus. The retentate is subjected to column purification using the said celufine sulphate matrix. The concentrated harvest volume of 50 liter is passed through the celufine sulphate column and the fiowfhrough is discarded since it contains unwanted protein and other genetic material. The virus which is bound to the column will be eluted out by using elution buffer containing high amount of salts. The elute is quantified through optical density values measured giving a sharp peak, which signifies the presence of purified live Japanese encephalitis virus. The virus collected in the elution is diafiltered to remove the salts, certain unwanted proteins and other genetic material by buffer exchange against phosphate buffered saline repeatedly for at least 5-6 times. The retentate is collected and passed through 0.45|_i PVDF (polyvinylidene fluoride) membrane filtration. Subsequently, the live JE bulk is inactivated with formalin at the ratio between 1:1500 v/v to 1:2500 v/v for 7 days at 22°C. along with the stabilizer mixture containing 0.1-1% sorbitol, and 0.1-1% Glycine added during the inactivation process only. Stabilizers at specific concentration of 0.5% w/v glycine and 1% w/v sorbitol are added to the live JE virus along with inactivating agent either formalin or BPL. The stabilizers are added to prevent degradation of the virus during the inactivation incubation period of 7 days only, so mentioned above due to addition of the inactivating agent formalin or betapropio lactone (BPL). After inactivation the JE bulk is further diafiltered through buffer exchange at least for 5-6 times against phosphate buffered saline to remove the formalin from the purified inactivated JE bulk. The diafiltered JE bulk is sterile filtered by using 0.22u PVDF (polyvinylidene fluoride) membrane filtration. The inactivated JE bulk is stored for stability studies at 5°C+ 3°C for at least 24 months to 36 months. The purification and inactivation steps of Japanese encephalitis (Kolar strain) bulk according to one of the methods disclosed in this invention can be summarized below:
(a) clarification of the viral harvest with 0.45 u membrane to remove cell debris from the viral harvest;
(b) concentration of the harvest volume to at least 4 times with 300kDa polyethersulfone (PESU) membrane cassette to obtain the concentrated retentate;

(c) column purification of the concentrated harvest retentate of step (b) through celufine sulphate matrix column, wherein the said matrix consists of a cellulose support matrix with the presence of an activated group sulphate ester to collect the elute containing the desired virus, high amount of salts cellular proteins and nucleic acid material;
(d) diafiltration and buffer exchange using 300kDa PESU membrane cassette against phosphate buffered saline of the elute of step (c) for at least 5-6 times to collect the retentate to remove the undesirable high amount of salts cellular proteins and nucleic acid material from the virus of interest to collect the purified concentrated virus bulk;
(e) subjecting the purified concentrated virus bulk of step (d) to 0.45u PVDF
(polyvinylidene fluoride) membrane filtration to eliminate any contaminants in the process;
(f) simultaneous stabilization and inactivation of the purified virus of step (e) with formalin at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 5°C ±3°C;
(g) diafiltration and buffer exchange of the inactivated purified virus bulk of step (f) using 300kDa PESU membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
(h) 0.22u PVDF (polyvinylidene fluoride) membrane filtration of the purified inactivated virus bulk of step (g) to obtain the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.
The results of purification through celufine sulphate matrix in case of batch size production in 30 CF40s through multiple viral harvest is provided. The total virus bulk obtained for one batch of 30 CF40s with celufine sulphate in one month is mentioned below. Accordingly the number of doses of JE vaccine that may be made available with NLT (not less than) 5 (ig of inactivated JE per dose also increases considerably from at least 60,000 doses in case of 30 CF40s to at least 1 lakh doses to 3 lakh doses in disposable bioreactor-100 (DB-100) and disposable bioreactor-500 (DB-500) respectively.

The total virus bulk obtained for one batch of DB100 in one month is mentioned below. Accordingly the number of doses of JE vaccine that may be made available with 7 ug of inactivated JE per dose is also calculated and provided below:
The total virus bulk obtained for one batch of DB-500 in one month is mentioned below. Accordingly the number of doses of JE vaccine that may be made available with 7 ug of inactivated JE per dose is also calculated and provided below:

Hence, we can see that, the total JE bulk produced in one commercial batch in the disposable bioreactor DB-100 is equivalent to the amount of JE bulk that would have been produced in at least 48 CF40s, for the same period of time or even lesser (38 days). Similarly, in case of commercial batch with DB-500 the amount of JE bulk produced is equivalent to the amount of JE bulk that would have been produced in at least 147 CF40s for the same period of time (39 days). This establishes the significant technical advance accomplished by implementing commercial production of Japanese encephalitis virus in the novel disposable bioreactors of sizes DB-100 and DB-500.
Example 3: Purification of JE virus using agarose based matrix column and inactivation of the JE virus.
Purification of JE virus using an agarose based column Capto Core-700 is another novel aspect of this invention. The number of doses that may be manufactured in one batch size of DB-100 and DB-500 are even higher in comparison with celufine sulphate purification. Capto core 700 column consists of a highly cross-linked agarose matrix with a functional active ligand octylamine, responsible for both hydrophobic interactions and is also positively charged in order to interact strongly with most of the impurities over a wide range of pH and salt concentrations. In this method of purification of JE virus using a Capto Core-700 matrix, the advantage is concentration of viral harvest is not required. The principle involved in the Capto Core matrix is size exclusion together with affinity chromatographic techniques both acting simltaneously. The column purification is accomplished by passing the entire harvest volume through Capto Core matrix. The proteins having a molecular weight below 700 kDa and other genetic material present in the harvest will be bind to the Capto Core matrix. The flow through is collected comprising the live JE virus because viruses will not bind to the Capto Core matrix. This is followed by concentration through a 300kDa polyethersulfone (PESU) membranes cassette. The permeate is discarded, and the retentate comprises the live JE virus at this stage. Thereafter, the concentrated live JE virus present in the retentate is chemically inactivated through treatment with inactivating agent formalin or betapropiolactone (BPL) at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) for a period of 7 days at 22°C along with the stabilizer mixture containing 0.1-1% sorbitol, and 0.1-1% Glycine added during the inactivation process only.. Stabilizers at specific

concentration of 0.5% w/v glycine and 1% w/v sorbitol are added to the live JE virus along with inactivating agent either formalin or BPL. The stabilizers are added to prevent degradation of the virus during the inactivation incubation period of 7 days only, so mentioned above due to addition of the inactivating agent formalin or BPL. After inactivation, the JE bulk is diafiltered and concentrated by buffer exchange against phosphate buffered saline in a repeated manner for at least 5-6 times to remove formalin. In case of BPL inactivation diafiltration is not required. BPL will be neutralized by incubating the bulk at 37°C for 3 hours. The diafiltered JE bulk is sterile filtered by using 0.22)j, PVDF (polyvinylidene fluoride) membrane filtration. The inactivated Japanese encephalitis virus bulk is further stored at 5°C +3°C for at least 24 months to 36 months. The purification and inactivation steps for Kolar strain of Japanese encephalitis bulk according to one of the methods disclosed in this invention can be summarized below:
(a) clarification of the viral harvest with 0.45 u membrane to remove cell debris from the viral harvest:
(b) column purification through combined size-exclusion and affinity chromatography with a Capto Core-700 matrix, wherein the said matrix consists of consists of a highly cross-linked agarose matrix with a functional active ligand octylamine group, to collect the flowthrough containing purified virus;

(c) concentration and diafiltration of the flowthrough of step (b) using 300kDa polyethersulfone (PESU) membranes cassette to obtain the purified concentrated virus bulk;
(d) subjecting the purified concentrated virus bulk of step (c) to 0.45u. PVDF (polyvinylidene fluoride) membrane filtration to eliminate any contaminants in the process;
(e) simultaneous stabilization and inactivation of the purified virus of step (d) with formalin
at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of
formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine
and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between
1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated)
along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a
period of 7 days at 5°C ±3°C;

(f) diafiltration and buffer exchange of the inactivated purified virus bulk of step (e) using 300kDa PESU membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
(g) 0.22 ji PVDF (polyvinylidene fluoride) membrane filtration of the purified inactivated virus bulk of step (f) to obtain the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.
The results of purification through Captocore in case of batch size production in 30 CF40s, DB-100, DB-500 through multiple viral harvests are provided in the below tables. The total virus bulk obtained for one batch of 30 CF40s with Captocore purification in one month is mentioned below. Accordingly the number of doses of inactivated JE vaccine that may be made available with NLT 5 p.g per dose is also increased considerably from approximately 1 lakh doses in case of 30 CF40s to at least 9 lakh doses to 20 lakh doses in case of DB-100 to DB-500 respectively.

Further a comparison of increase in yield of purified inactivated JEV bulk through using a Capto Core-700 column in case of batch size of 30 CF40, DB-100 and DB-500 respectively is presented in the below table (Table 3.4). We can see that the total JEV bulk in case of 30

CF40 through capto core purification is 642857 mg, and in case of DB-100 is 964285 mg which amounts to an equivalent of production of 45 CF40s within the same amount of time required, and 2206428 mg of inactivated JEV bulk which amounts to an equivalent of production of approximately 140 CF40s within the same amount of time required. Summary of comparison between Celufine sulfate and through Capto Core 700 purification of JEV bulk is provided below:
Example 4: Stability studies of inactivated JEV bulk.
The dialyzed inactivated JEV bulk was stored with suitable proportion of with or without chemical stabilizer(s) at 5±3°C. The antigen was drawn at different interval and tested by plaque reduction neutralization (PRNT50). The stability of antigen was performed with a viral neutralization titer of mouse antiserum as per IP.2007; WHO TRS No.771. The virus neutralization of the antiserum was determined by the 50% plaque reduction method. The neutralization titer against the virus was expressed as the reciprocal of the serum dilution that showed 50% plaqu^ reduction compared to the plaque number of the control wells without live viral antigen.

We claim:
1. A viral vaccine composition for prophylaxis against infections caused by Indian Kolar strain of Japanese encephalitis virus comprising a vaccine antigen, wherein the vaccine antigen is inactivated Kolar strain of the Japanese encephalitis virus adapted in Vero Cells grown in a disposable bioreactor to harvest live Japanese encephalitis virus bulk.
2. The vaccine composition according to claim 1, wherein the said vaccine antigen is grown in the disposable bioreactor in Vero Cells to collect live Japanese encephalitis virus bulk, and the said virus bulk is harvested multiple times (multiple viral harvest) from at least more than once upto five times or at least three times from a single culture of Vero Cells and single infection of the Kolar strain of the Japanese encephalitis virus.
3. The vaccine composition according to claim 1, wherein the disposable bioreactor is maintained at a dissolved oxygen in the range of 50 ppm-70 ppm, temperature range between 35.5°C to 37.5 °C at a maintained pH of 7.2 to 7.6 with an agitation of 260 rpm to 280 rpm at an in-flow rate in 80-150 ml/min and an outflow rate of 84-156 ml/min.
4. The vaccine composition according to claim 2, wherein the multiple viral harvests obtained are further purified and inactivated comprising the steps:

(a) clarification of the viral harvest with 0.45 {i membrane to remove cell debris from the viral harvest:
(b) concentration of the harvest volume to at least 4 times with 300kDa membrane cassette to obtain the concentrated retentate;
(c) purification of the concentrated harvest retentate of step (b) through celufine sulphate matrix, wherein the said matrix consists of a cellulose support matrix with the presence of an activated group sulphate ester to collect the elute containing the desired virus, high amount of salts cellular proteins and nucleic acid material;
(d) diafiltration and buffer exchange using 300kDa membrane cassette against phosphate buffered saline of the elute of step (c) for at least 5-6 times to collect the retentate to remove the undesirable high amount of salts cellular proteins and

nucleic acid material from the virus of interest to collect the purified concentrated virus bulk;
(e) subjecting the purified concentrated virus bulk of step (d) to 0.45u membrane filtration to eliminate any contaminants in the process;
(f) simultaneous stabilization and inactivation of the purified virus of step (e) with formalin at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 5°C ±3°C;
(g) diafiltration and buffer exchange of the inactivated purified virus bulk of step (f) using 300kDa membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
(h) 0.22p membrane filtration of the purified inactivated virus bulk of step (g) to obtain the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.
5. The vaccine composition according to claim 2, wherein the multiple viral harvests obtained are further purified and inactivated comprising the steps:
(a) clarification of the viral harvest with 0.45 JJ. membrane to remove cell debris from the viral harvest;
(b) purification through combined size-exclusion and affinity chromatography with a Capto Core-700 matrix, wherein the said matrix consists of a highly cross-linked agarose matrix with a functional active ligand octylamine group, to obtain the flowthrough containing purified virus;
(c) concentration and diafiltration of the flowthrough of step (b) using 300kDa membranes cassette to obtain the purified concentrated virus bulk;

(d) subjecting the purified concentrated virus bulk of step (c) to 0.45fa membrane filtration to eliminate any contaminants in the process;
(e) simultaneous stabilization and inactivation of the purified virus of step (d) with formalin at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 5°C ±3°C;
(f) diafiltration and buffer exchange of the inactivated purified virus bulk of step (e) using 300kDa membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
(g) 0.22u membrane filtration of the purified inactivated virus bulk of step (f) to obtain the final and stabilized inactivated JEV bulk at 5°C± 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.

6. A method of adaptation of the Kolar strain of the Japanese encephalitis virus in Vero cells capable of industrial production in a disposable bioreactor to collect live Japanese encephalitis virus bulk, and the said virus bulk is harvested multiple times (multiple viral harvest) from at least more than once upto five times or at least three times from a single culture of Vero Cells and single infection of the Kolar strain of the Japanese encephalitis virus.
7. The method according to claim 6, wherein the multiple viral harvest of the Japanese encephalitis virus bulk is scaled up from laboratory scale batches in a disposable bioreactor of size equivalent to 4 cell factories to production size batches in a disposable bioreactor of size equivalent to approximately 200 cell factories.
8. The method according to claim 6 and 7, wherein the said disposable bioreactor is maintained at a dissolved oxygen in the range of 50 ppm-70 ppm, temperature range

between 35.5°C to 37.5 °C at a maintained pH of 7.2 to 7.6 with an agitation of 260 rpm to 280 rpm at an in-flow rate in 80-150 ml/min and an outflow rate of 84-156 ml/min.
9. The method according to claim 7, wherein the multiple viral harvests of Japanese encephalitis virus bulk are obtained for at least thrice in a disposable bioreactor of size equivalent to approximately 200 cell factories, within a period of at least 35-40 days.
10. A method of simultaneous purification and inactivation of the viral harvest of Japanese encephalitis virus bulk of claim 7, comprising the steps:

(a) clarification of the viral harvest with 0.45 u membrane to remove cell debris from the viral harvest:
(b) concentration of the harvest volume to at least 4 times with 300kDa membrane cassette to obtain the concentrated retentate;
(c) purification of the concentrated harvest retentate of step (b) through celufine sulphate matrix column, wherein the said matrix consists of a cellulose support matrix with the presence of an activated group sulphate ester to collect the elute containing the desired virus, high amount of salts cellular proteins and nucleic acid material;
(d) diafiltration and buffer exchange using 300kDa membrane cassette against phosphate buffered saline of the elute of step (c) for at least 5-6 times to collect the retentate to remove the undesirable high amount of salts cellular proteins and nucleic acid material from the virus of interest to collect the purified concentrated virus bulk;
(e) subjecting the purified concentrated virus bulk of step (d) to 0.45|u membrane filtration to eliminate any contaminants in the process;
(f) simultaneous stabilization and inactivation of the purified virus of step (e) with formalin at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein

the said stabilizers consists of glycine and sorbitol for a period of 7 days at 5°C
±3°C; (g) diafiltration and buffer exchange of the inactivated purified virus bulk of step (f)
using 300kDa membrane cassette against phosphate buffered saline for at least 5-6
times to remove formalin only in case of formalin inactivation; (h) 0.22|i membrane filtration of the purified inactivated virus bulk of step (g) to obtain
the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to
36 months free from any contaminants suitable for formulation of the Japanese
encephalitis vaccine.
11. A method of simultaneous purification and inactivation of the viral harvest of Japanese encephalitis virus bulk of claim 7, comprising the steps:
(a) clarification of the viral harvest with 0.45 u membrane to remove cell debris from the viral harvest:
(b) purification through combined size-exclusion and affinity chromatography with a Capto Core-700 matrix, wherein the said matrix consists of consists of a highly cross-linked agarose matrix with a functional active ligand octylamine group, to collect the flowthrough containing purified virus;
(c) concentration and diafiltration of the flowthrough of step (b) using 300kDa membranes cassette to obtain the purified concentrated virus bulk;
(d) subjecting the purified concentrated virus bulk of step (c) to 0.45ju membrane filtration to eliminate any contaminants in the process;
(e) simultaneous stabilization and inactivation of the purified virus of step (d) with formalin at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 22°C or with beta-propio lactone at a ratio between 1:1500 v/v to 1: 2500 v/v (i.e. to 2500 volume of live JE, 1 volume of formalin is treated) along with stabilizers, wherein the said stabilizers consists of glycine and sorbitol for a period of 7 days at 5°C ±3°C;

(f) diafiltration and buffer exchange of the inactivated purified virus bulk of step (e) using 300kDa membrane cassette against phosphate buffered saline for at least 5-6 times to remove formalin only in case of formalin inactivation;
(g) 0.22(i membrane filtration of the purified inactivated virus bulk of step (f) to obtain the final and stabilized inactivated JEV bulk at 5°C+ 3°C for at least 24 months to 36 months free from any contaminants suitable for formulation of the Japanese encephalitis vaccine.
12. A method of increase of a single batch-size commercial production of purified inactivated Japanese encephalitis virus (JEV) bulk in a disposable bioreactor upto at least 8 folds to 10 folds of purified inactivated JEV bulk by using a combined size-exclusion and affinity chromatography with a Capto Core-700 matrix, wherein the said matrix consists of consists of a highly cross-linked agarose matrix with a functional active ligand octylamine group within a period of 35-40 days, as compared to purified inactivated JEV bulk obtained in a single batch-size commercial production of JEV in 30 cell factories within the same 35-40 days of time period.