THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
Section 10
"A NOVEL PROCESS FOR LARGE SCALE PRODUCTION OF VIRAL
ANTIGEN"
Serum Institute of India Ltd., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

Title: A NOVEL PROCESS FOR LARGE SCALE PRODUCTION OF VIRAL ANTIGEN
Objective: To Provide a process for large scale production of viral vaccines.
BACKGROUND OF THE INVENTION:
Considerable progress has been made in the field of production of vaccines against viruses such as rabies, poliomyelitis, where the host cells are grown on microcarriers in suspension at large volume. The large scale production of rabies vaccine using Vero cells in a liquid culture containing serum, medium and micro carriers were reported by Wiktor et al(US 4,664,912).
Cory J et al discusses high titre of poliovirus grown on Vero cells with serum free medium in a static system as compared to microcarrier or roller bottle. However, in a culture method using a serum-free medium and a microcarrier, as a support for culturing anchorage dependent cells, there has been a problem that the attachment rate to the microcarrier is reduced, making it difficult to efficiently culture large amount of cells.
According to Ronaldo et al J. Bras. Patol. Med. Lab. 2004 cells grown in Roux bottles presented 1.3 to 6.7 times more virus particles than those in the microcarrier systems. Ronaldo et al also suggested that the physical characteristic of the support as well as the conformation can be responsible for variations in viral titres.The titres were found to be higher when the rabies virus was grown on BHK or Vero and adhered to a flat support than in microcarriers.Similar results were reported by Ronaldo et al for Measles and Polio viruses grown on Vero.
Optimization of rabies virus yield using a bioreactor with medium, serum and micro carriers was discussed earlier by Khaled Trabelsi et al, Journal of Biotechnology 121 (2006) 261-271.
A major contributor to the low rabies virus antigen yield from the microcarrier cultures was attributed to the fact that the cells gradually fell off the beads during the infection period. Based on these results, microcarrier technology was reported to be suboptimal for commercial scale production of rabies virus antigen.
The above cited prior art methods produce less number of harvests as the cells get detached from the micro carriers before producing the antigen; resulting in low yield of viral antigen.

SUMMARY OP THE INVENTION
The present invention is based on a surprising finding that if microcarriers along with virus infected ceils are transferred to a flat surface ,instead of continuing the harvesting in a bioreactor,then the infected cells are found to live longer resulting in higher unitage of viral antigen.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention higher unitage of viral antigen can be obtained when microcarriers along with virus infected cells are transferred to a flat surface ,instead of continuing the harvesting in a bioreactor.
As part the present invention the simultaneous addition of cell pool and virus can be carried on microcarrier in a stirred tank bioreactor, and the said microccariers along with infected cells are then transferred to a flat surface under static environment.Alternatively the cell pool can be added initially followed by addition of Virus seed to microcarrier beads.
The present invention relates to a process of large scale production of rabies vaccine, said process including the steps of expansion of MRC-5 cells obtained from MLTCF to CYTODEX-1 microcarriers in stirred tank bioreactors; simultaneously adding rabies virus seed /growing infected cells until 50-90% confluency is reached; washing cells with virus maintenance serum free medium; transferring the infected MRC-5 cells along with microcarriers to a flat surface under static condition; at a temperature between 33 degree C to 37 degree C at a pH of about 7.2 to 7.8; multiple harvesting from supernatant; optionally purifying the harvests or using the harvests as it is ; inactivation by beta-propio lactone and adsorbed on aluminium phosphate gel.
The present invention also relates to a process of large scale production of rabies vaccine, said process including the steps of growing MRC-5 cells on CYTODEX-1 microcarriers in stirred tank bioreactors until 20-90% confluency is reached; infecting - said MRC-5 cells with rabies virus; washing cells with virus maintenance serum free medium; transferring the infected MRC-5 cells along with microcarriers to a flat surface under static condition; at a temperature between 33 degree C to 37

degree C at a pH of about 7.2 to 7.8; multiple harvesting from supernatant;concentrating the harvest;inactivation by beta-propio lactone and adsorbed on aluminium phosphate gel.
Also according to the instant invention washing step for removal of FBS can be effected either in bioreactor or on flat surface.
The rabies virus strain used in the present invention is preferably Pitman Moore 3218.
Further as per the present invention there is a significant increase in viral antigen when the microcarriers along with cells are transferred from stirred bioreactor to a flat surface as compared to the conventional method wherein the entire process is carried on microcarriers in a stirred bioreactor.
According to the instant invention higher yields of rabies virus can be obtained. Also the process of instant invention affords higher number of harvests as compared to the conventional processes. The present invention is directed to an improved and cost effective process for production of rabies vaccine. Further the instant process is easily scaleable.
Yet another aspect of the instant invention is that the said process can be utilized for viruses selected from a group of but not limited to Polio, Small Pox, Measles, Mumps, Rubella and rabies.
The process acoording to the present invention enables the large scale production of the vaccine using MRC-5, Vero, WI-38, BHK-21 or Chicken embryo fibroblast cells.
The microcarrier can be selected from the group of but not limited to microcarriers based on dextran, collagen, plastic, gelatine and cellulose. Preferably, the microcarrier is selected from the group of microcarriers such as CYTODEX I microcarrier, CYTODEX II microcarrier , CYTODEX III microcarrier, CYTOPORE microcarrier and CYTOLINE microcarrier.
Further according to an aspect of the present invention the flat surface is made of any medical grade material more preferably of glass,stainless steel or plastic.The vessel having flat surface can be selected from but not limited to a tissue culture flask,multilayer tissue culture flask.

The instant invention utilizes stepwise combination of microcarriers under stirred and microcarriers under static culture conditions for virus production that can be considered as a potential alternative to conventional virus production systems like roller bottle, microcarriers, stirred bioreactor or static environment.
EXAMPLES;
EXAMPLE 1
Bioreactor (Biostat C 10 - 2, Make - B. Braun, Working capacity - 10 L) Preparation:
Bioreactor vessel was cleaned, conductivity was checked, pH probe was calibrated, sterilize - 121°C, 30 min. 35 g Cytodex 1 was added in empty vessel followed by addition of 10 L PBS. The beads were allowed to soak for 3 hrs. PBS was withdrawn and fresh 6 L PBS was added. Sterilize -121°C, 30 min. DO probe was calibrated to zero% at 121°C
EXAMPLE 2
Conditioning of beads (Cytodex 1)
PBS was withdrawn from bioreactor vessel through sieve tube. Sterile conditioning medium (10L) was added. The beads were stirred for 40 min. The conditioning medium was withdrawn. Fresh sterile conditioning medium (10L) was then added.
EXAMPLE 3
Cell seeding in to bioreactor
Cell seeding was done 18-24 hrs post conditioning the beads. Agitation was stopped for 40 min. After 40 min conditioning medium was withdrawn through harvest port. Approximately 3 L conditioning medium & beads were present in the vessel. Approximately 3 L of cell pool was added. The cells were allowed to get attached for 3 to 3.5 hrs. After 3 to 3.5 hrs, cell growth medium was added up to 10 L volume. Sample were withdrawn to check cell attachment. Growth parameters maintained were mainly

Temperature of about 36.5±0.5°C, RPM 30, DO 30 - 40% & pH 7.2
Routine sampling from bioreactor was carried to check -1] cells under inverted microscope, 2] glucose content in medium using YSI biochemistry analyzer, 3] cell count using nuclei counting method
EXAMPLE 4
COUNTING OF MRC -5 CELLS GROWN ON CYTODEX 1
Citric acid in counting stain plays dual role - It detaches the cells from microcarriers & secondly it dissolves the cell membrane. The released nuclei get stained with crystal violet and look purple & ovoid when observed under inverted microscope. Each nucleus corresponds to single cell. Therefore; number of nuclei are directly equivalent to number of cells. The procedure employed is as given below:
1. 10 ml culture sample was removed aseptically from bioreactor while stirring.
2. After withdrawal from bioreactor, the sample was kept stirring on the magnetic stirrer.
3. 1 ml of culture was removed while stirring on the magnetic stirrer with a 1 ml plastic pipette.
4. The pipette was held straight while releasing the sample in to a Eppendorf tube(2 or 1.5 ml).
5. Centrifugation was carried for 1 min. to 4 min. at 2000 rpm
6. With the help of Pasteur pipette supernatant was removed carefully without disturbing the pellet.
7. Counting stain was added to this pellet so as to make up the volume to 1 ml.
8. The contents of the tube were mixed well with vibro-mixer.
9. The tube was incubated at 37 °C for 1 hr. It was ensured that evaporation does not occur during incubation by properly sealing the tube.

10. After incubation, the contents were mixed well.
11. The released nuclei were counted using Neubauer chamber under inverted microscope.

EXAMPLE 5 Infection
After reaching cell growth to 6000 to 8000 million cells (day 2 or 3 after cell seeding) infection was carried out by addition of rabies working seed virus (PM 3218) Pitman Moor.
After settling of the beads 7 L of spent medium was removed from harvest port and rabies working seed virus with MOI 1:10 to 1:80 (MOI - Multiplicity Of Infection, 1:10 - One virus particle for 10 cells) was added. After 50 min. cell growth medium was added up to 10 L volume & growth parameters were set as Temperature range. 36.5+0.5°C, RPM 30, DO 30 - 40% and pH 7.2
EXAMPLE 6 Washing
Partial removal of FBS was done 24 hr to 60 hrs post infection. After settling of the beads, 7 L of spent medium was removed from harvest port. MEM (Earle's) without FBS was then added. Stirring was done for 20 min. washing was carried in similar manner for 4 times. After washing MEM {Earle's} without FBS was added up to 10 L volume & growth parameters were set of about Temp. 33.5+0.5°C, RPM 30, DO 20 - 30% and pH 7.6
EXAMPLE 7
Multiple harvesting
Harvesting interval was 24 to 48 hrs after washing. Period between two harvests was 24 to 48 hrs. After settling of- the beads, 7 L of medium (harvest) was removed from harvest port. Clarification was done through 6 urn filter. The harvest was stored at +4 to +8C. 7 L of fresh MEM (Earle's) without FBS was added in to bioreactor & growth parameters were set at temperature of. 33.5±0.5°C, RPM 30, DO 20 - 30% and pH 7.6.Harvests were then checked for antigen content (IU/ml) by ELISA .Harvesting was terminated when there were no cells observed on beads.
EXAMPLE 8
Flat surface / static culture
100 ml of bead suspension was withdrawn {beads with
cells; cells infected with rabies seed virus) from
bioreactor after washing step and transferred to a flat
surface {TCF - Tissue culture flask - 175 cm2)

Multiple Harvesting was carried by removal of supernatant
(without beads) 70 ml to 90 ml & fresh MEM (Earle's)
without FBS -100 ml (Incubation condition - 33.5 ±0.5C)
was added.
EXAMPLE 9
A) Estimation of rabies glycoprotein in rabies virus harvest & rabies antigen using Enzyme Linked Immunosorbent Assay (ELISA)
Step I; Coating ELISA. strips (F8 Maxisorp with monoclonal antibody (Rab-50) (Abeam)
100 μL of the coating solution was dispensed into each micro well of the strip.
ELISA strips were incubated at 37+1 °C for In.
Step II- Blocking of ELISA strips.
250 μL of blocking buffer was dispensed in each micro well of ELISA strip.
Strips were incubated at 37+_l °C for lh.
After completion of incubation, strips were washed thrice with washing buffer using ELISA plate washer.
Step III: Incubation with standard and test samples
1:10; 1:20; 1:40 and 1:80 dilutions of NIBSC standard (NIBSC, London) (5 IU/mL) were prepared in the dilution buffer.
100 uL of each dilution of standard and test samples were added, in the prelabelled wells of ELISA strip respectively (each sample in triplicates). For blank wells, 100μL/ well dilution buffer was added in Duplicate.
ELISA strips were incubated at 37°C for 30 min.
After completion of incubation, strips were washed thrice with washing buffer in ELISA plate washer.

Step IV:Incubation with anti-rabies sheep serum (PM strain, Center for Biologies Evaluation and Research, USFDA or ERA strain)
1:1000 dilution of anti-rabies sheep antibodies was prepared in the dilution buffer.
100 uL of the diluted anti-rabies sheep antibodies was added in each micro well of ELISA strip.
ELISA strips were incubated at 37°C for 30 min.
After completion of incubation, strips were washed thrice with washing buffer in ELISA plate washer.
Step V: Incubation with anti-sheep HRP conjugated antibodies (Sigma)
1.4:10000 dilution of HRP conjugated anti-sheep antibodies was prepared in the dilution buffer.
IOOuL of the diluted antibodies was added in each micro well of ELISA strips.
ELISA strips were incubated at 37 + 1 °C for 30 min.
After completion of incubation wash the strips thrice with washing buffer using ELISA plate washer.
Step VI: Incubation with substrate-chromogen buffer.
IOOμL of the freshly prepared substrate was added in each micro well of ELISA strips.
ELISA strips were kept at room temperature for 15 min in dark for colour development.
Step VII: Stopping the reaction and reading of absorbance
After 15 min of incubation the reaction was stopped by adding 50μL /well of stop solution.
Absorbance was immediately measured (not later than 10 min) at 450 nm using ELISA reader.

ACCEPTANCE LIMIT
• For calculations, the absorbance (at 450 nm) for all standard as well as test samples should fall in the range of 0.1-1.00.
• Range of absorbance (at 450 run) for blank,
• Absorbance of the blank (at 450 nm) < 0.2.
• For calculations, blank values should be subtracted from all the sample values.
• Range of absorbance (at 450 nm) for international standards (NIBSC) after subtracting blank values: 1:10 dilution: 0.8 + 0.2; 1:20 dilution: 0.4 + 0.1; 1:40 dilution: 0.2 + 0.05; 1:80 dilution: 0.1 + 0.025.
CALCULATIONS
Mean of all three absorbance values of all standard and test samples was taken. Rabies glycoprotein content was calculated in the harvest samples by plotting the standard curve [Absorbance@4 50 Vs antigen content (IU/mL) ] .
QUANTITATIVE RANGE
Quantitative range of this ELISA falls in the range of 0.5 to 0.05 IU/mL.

B) Comparison table for rabies antigen obtained in stirred culture (bioreactor) & static culture (flat surface/ tissue culture flask)
The antigen present in all harvests is expressed in units obtained by ELISA

Batch 2 (Bioreactor) B 2 Tissue Culture Flask / flat surface Batch 3 (Bioreactor) B 3 Tissue Culture Flask / flat surface
ELISA units (lU/ml) ELISA units (Ill/ml) ELISA units (lU/ml) ELISA units (lU/ml)
Harvest 1 0.09 0.24 0.13 0.18
Harvest 2 0.15 0.25 0.15 0.20
Harvest 3 0.1 0.37 0.18 0.24
Harvest 4 0.3 0.34 0.17 0.19
Harvest 5 0.345 0.44 0.24 0.43
Harvest 6 0.367 0.29 0.24 0.5
Harvest 7 0.18 0.30 0.20 0.51
Harvest 8 0.16 0.33 0.26 0.52
Harvest 9 0.172 0.38 0.25 0.43
Harvest 10 , 0.17 0.45 0.30 0.50
Harvest 11 - 0.52 0.29 0.42
Harvest 12 - 0.45 0.28 0.31
Harvest 13 - 0.43 0.26 0.20
Harvest 14 - 0.20 0.26 0.19
Harvest 15 - 0.17 0.22 -
Harvest 16 - 0.15 0.25 -
Harvest 17 - - 0.28 -
Harvest 18 - - 0.3 -
The results indicate that a higher unitage of rabies virus antigen can be obtained by the instant method.Also the yield per harvest is found to be 1.5 to 2 times higher when microcarriers along with infected cells are transferred to a flat surface as compared to the yield per harvest obtained from bioreactor.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.

We Claim,,
1. A method for production of virus comprising viral antigen, comprising the steps of a) simultaneous addition of a culture of adherent cells and virus to a microcarrier in a stirred bioreactor system; (b) growing the infected cells to confluence; (c)washing of cells; (d) transferring said microcarriers along with infected cells to a flat surface under static environment.
2. A method for production of virus comprising viral antigen, comprising the steps of a) addition of a culture of adherent cells to a microcarrier in a stirred bioreactor system; (b) growing cells to confluence; (c)infecting the cells with virus (d)washing of cells and transferring said
microcarriers along with infected cells to a flat surface under static environment.
3. The method according to claim 1 or claim 2 , further comprising the step (e) harvesting virus propagated from flat surface.
4. The method according to claim 1 or claim 2, further comprising the step (f) purifying the harvested virus to obtain a purified virus or viral antigen.
5. The method according to claim 1 or claim 2, further comprising the step (g) inactivating virus using beta-propiolactone.
6. The method according to claim 1 or claim 2, further comprising the step (h) adsorbing the inactivated virus with aluminium phosphate.

7. The method according to claim 3 or 4, wherein the virus produced on flat surface is harvested from supernatant.
8. The method according to step (a) of claim 1 or claim 2, wherein said cells are selected from the group of adherent cells of VERO, BHK, CHO, RK, RK4 4, RK13, MRC-5, MDCK, CEF,WI-38 or diploid monolayer cells.
9. The method according to to step (a) of claim 1 or claim 2, wherein said microcarrier is selected from the group of microcarriers made of dextran, collagen, polystyrene, polyacrylamide, gelatine, glass, cellulose, polyethylene and plastic.

10. The method according to to step (a) of claim 1 or (c) of claim 2, wherein said virus is selected from a group of Rabies, polio, small pox, measles, rubella, mumps and influenza.
11. The method according to step (d) of claim 1 or claim 2, wherein the flat surface is made of any suitable material,more particularly of a medical grade material.
12. A method for production of rabies virus comprising viral antigen, comprising steps of expansion of MRC-5 cells obtained from Multilayer tissue culture flask to CYTODEX-1 microcarriers in stirred tank bioreactors; simultaneously infecting said MRC-5 cells with rabies virus; growing infected cells until 50-90% confluency is reached; washing cells with virus maintenance serum free medium;

transferring the infected MRC-5 cells along with microcarriers to a flat surface under static condition; at a temperature between 33 degree C to 37 degree C at a pH of about 7.2 to 7.8; multiple harvesting from supernatant; optionally purifying the harvests or using the harvests as it is; inactivation by beta-propio lactone and adsorbed on aluminium phosphate gel.
13. A method for production of rabies virus comprising viral antigen, comprising steps of growing MRC-5 cells on CYTODEX-1 microcarriers in stirred tank bioreactors until 20-90% confluency is reached; infecting said MRC-5 cells with rabies virus/ washing cells with virus maintenance serum free medium; transferring the infected MRC-5 cells along with microcarriers to a flat surface under static condition; at a temperature between 33 degree C to 37 degree C at a pH of about 7.2 to 7.8; multiple harvesting from supernatant; optionally purifying the harvests or using the harvests as it is; inactivation by beta-propio lactone and adsorbed on aluminium phosphate gel.
14. The method according to step (a) of claim 1 and 9, wherein said microcarrier is Cytodex 1.
15. The method according to any of the claims 1 to 14, wherein the virus or viral antigen produced is Rabies virus or Rabies virus antigen.
16. The method according to step (a) of claim 1 and 8, wherein said cells are MRC-5 tells.

17. The method according to step (c) of claim 1, wherein the washing is effected in bioreactor or on flat surface.
18. The method according to step (d) of claim 1 and 11, wherein the flat surface is made of stainless steel.
19. The method according to step (d) of claim 1 and 11, wherein the flat surface is inade of plastic.
20. The method according to claim 2, wherein harvests are single to multiple.