Title of Invention

A PROCESS FOR LARGE SCALE PRODUCTION OF RUBELLA VACCINE

Abstract

A process for large scale production of Rubella Vaccine using RA-27/3 Rubella virus strain ( Plotkin) and human diploid cell strain MRC-5 comprising the steps of:- a) growing MRC-5 cells by i) essentially employing CellCube modules having predetermined surface area for seeding the said cells at predetermined density, after growing in tissue culture flask and subsequently tryspinising them after specified number of days; ii) allowing cell attachment to take place for specific time period, after seeding; and iii) circulating and thereby perfusing a cell medium of specific composition on the cells to maintain predetermined control conditions during growth followed by b) infecting the MRC-5 cells grown in said CellCube modules with Rubella working seed virus RA - 27/3 at specific controlled conditions.

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION [See section 10] A PROCESS FOR LARGE SCALE PRODUCTION OF RUBELLA VACCINE; SERUM INSTITUTE OF INDIA LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS "SAROSH BHAVAN" 16-B/1, DR. AMBEDKAR ROAD, PUNE - 411 001, MAHARASHTRA, INDIA; SPECIFICATION AND THE FOLLOWING DESCRIBES PARTICULARLY ASCERTAINS THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFOMED. GRANTED 14-5-2003 TITLE A PROCESS FOR LARGE SCALE PRODUCTION OF RUBELLA VACCINE. References: 1 Hyflic L, Moorehead P S ( 1961), The serial cultivation of human diploid cell strain Exp. Cell Res. 25, 585. 2 Hyflic I, (1965) The limited in-vitro lifetime human diploid cell strains Exp. Cell Res. 37,614. 3 Jacobs J P ( 1976) The status of human diploid cell strain MRC-5 as an approved substrate for the production of viral vaccines, 4,97. 4 Jacobs J P Perkins F T (1963) Cultivation and distribution of human diploid cell strains (HDCS) in proc. Symp. On the characterisation and use of human diploid cell strains Opatija, Yugoslavia 185-193. 5 Plotkins & Mortimer. ' Vaccines ' 2nd Edition, 1994, W. B. Sauders Company. Pages 303 to 335. 6 Meeting on preventing Congenial Rubella Syndrome (CRS) : Immunisation Strategies, Surveillance needs, WHO, 12-14 Jan 2000. FIELD OF THE INVENTION The present invention relates to a novel process for large scale production of Rubella vaccine and more particularly to a process for large scale production of Rubella Vaccine, using RA-27/3 Rubella Virus Strain (Plotkin) and MRC - 5 cells ( Human Diploid Cell). Rubella is a mild exanthematous viral infection of children and young adults. It assumes great importance when it occurs in a pregnant woman, from whom the causative agent can be transmitted to the foetus, with disastrous effects, such as deafness, blindness and heart disease in newborn child. ( Ref. 5 ) Rubella is a worldwide infection as may be inferred from serological surveys conducted in many different countries. The age of infection varies from area to area. Crowded places such as day-care centers, schools, colleges and military camps are places where Rubella is likely to become epidemic. Transmission of Rubella is primarily by respiratory route with implantation of the virus in nasopharynx (Ref. 5) Vaccination by commercially available vaccines of attenuated Rubella strains commenced in developed countries in 1970s, whereas in developing and underdeveloped countries it has still not reached it's fullest potential (Ref. 6). Hence a method for large scale production of Rubella Vaccine is of high significance to cater the need for large quantities of potent vaccine. The requirement of Rubella in mono and combined forms have started increasing since last three years as many of the developing countries have introduced Rubella vaccine in Universal Immunization programme. Considering the forecast of various international agencies, it is estimated that more than about 100 million doses of Rubella (mono & combination) would be required per year and therefore there is an urgent need to produce large quantities of vaccine. DESCRIPTION OF THE PRIOR ART A number of methods exist for large scale production of Rubella Vaccine, in the state of the art. One of such existing methods of manufacturing the Rubella vaccine is as described below: a) The lower passage cells ( MRC 5 ) stored in liquid nitrogen in the working cell bank are revived and grown initially in small Tissue-Culture Flask (75 sq.cm)(about 5 x 106 cells) using minimum essential medium (MEM) supplemented with Foetal Bovine Serum (FBS). b) The cells from this bottle after complete growth are trypsinised and added to 2 new flasks (Tissue Culture Flasks). c) By following similar procedure and doubling the number of Tissue Culture Flasks at every passage, the cells are grown to a desired doubling level. d) Thus starting from 1 Tissue Culture Flask over a period of about 45-50 days, the process reaches to about 1000 Tissue Culture Flasks, going through about 10 doubling cycles. e) The cells so grown are then used to prepare about 850 sq.cm. area Roller Bottles to continue the doubling for 2 more cycles. f) After growing the cells in Roller bottle, the cells are infected with Rubella (RA-27/3) working seed virus g) Multiple harvests are then collected from individual Rollers h) The harvest are then stored frozen and tested as per the prevailing pharmacopoeal guidelines. i) The bulk vaccine is prepared by pooling of the harvests, which is then handed over to filling, lyophilization & sealing section. For producing anywhere between 0.5 to 1.0 million doses, this system needs about 2400 sq.ft. of working area with class 10,000 as baseline and class 100 in working cubicles. Also personnel handling would be in the range of about 10 for scientific staff about 15 for skilled people and balance about 5 for doing menial work. Any increase desired in the capacity, multiple increase of above would be needed making the manufacture too complicated and labour intensive. Currently practiced Tissue culture Flask-Roller bottle technology for the production of Rubella vaccine is extensively labour oriented. Besides it requires large GMP facilities and highly trained manpower. OBJECTS OF THE INVENTION The process of large scale production of the Rubella Vaccine according to the present invention, has the following objectives and their related features. A foremost objective of the present invention is that Instead of handling large number of Tissue Culture flasks individually, a procedure has been invented to combine these operations using a compact system. The present invention achieves this by using a process of growing MRC-5 cells to a high density in a system called CellCube (Corning Costar) system. The process of growing MRC-5 cells to 3-4 population doubling in this compact system brings about cost saving. The space required to handle 400 Rollers (400 x 850 sq.cm.) per production cycle is already described. The alternative compact system has a surface area of 340,000 sq. cm. This reduces the need of the working area to almost 700 sq.ft. and also saves the cost by a reduction in the use of costly tissue culture media components. The unique advantage of the process according to this invention is total reduction of work burden because of unit operation of manufacture in less space and less manpower. The process according to this invention offers a great potential for a cost effective scale up which will help to reduce the cost of vaccine & make the vaccine available in very large quantities for the entire needy population. SUMMARY OF THE INVENTION According to the present invention, the process for large scale production of Rubella Vaccine comprises of using CellCube modules, which are essentially a stack of parallel plastic trays enclosed inside a compact cube. MRC-5 cells tryspsinized from the Tissue Culture Flask are seeded into the CellCube modules and are allowed for cell attachment. A cell medium of suitable composition is circulated and thereby perfused to maintain specified growth & controlled conditions. The cells grown in the CellCube modules are then surface infected with Rubella working seed virus in controlled manner. DISCLOSURE OF THE INVENTION The present invention discloses a novel process for large scale production of Rubella Vaccine, using human diploid cell strain MRC-5 and RA-27/3 Rubella Virus strain (Plotkin). The entire process is described herein as two phases. The first phase being growth of MRC-5 cells and the second phase being the growth of Rubella Virus RA-27/3. GROWTH OF MRC- 5 CELLS : According to the present invention, CellCube modules are used for seeding the MRC-5 cells, which were grown and trypsinized from the Tissue Culture Flask (TCF). The CellCube modules are configured as a stack of parallel plastic trays, which are fused and sealed inside a compact cube. The CellCube modules, along with other equipments and instruments manufactured by Corning Costar, U.S.A , are installed in a specially designed incubator facility. At the time of seeding the MRC- 5 Cells into the CellCube modules, the surface area of the CellCube module is in the range of 8500 sq cm to 85000 sq cm. or multiples thereof (1 x 85000, 2 x 85000, 4 x 85000). These cubes are seeded using cells pregrown in TC flasks / cell factory of suitable growth area. After completion of seeding, the cells are allowed for attachment over a period of time which is dependant on cell growth (the media used for supporting MRC-5 cell growth is the same used for Roller bottles). Subsequently, circulation and perfusion of a cell medium is started to control the growth conditions of the MRC-5 cells. A suitable glucose consumption rate which is within 0.008 g /1 / hr initially, and rises to as high as 0.08 g / I / hr is observed during peak cell densities. The control conditions which influence the cell growth are defined by a pH range of 7.1 to 7.6, dissolved Oxygen (D.O) Content of 80% and above and a temperature range of 32° C to 38° C at the time of circulation and perfusion of the cell medium. By following this procedure, it can be generally observed that a population doubling of nearly 3.9 could be achieved after 9 days of cell growth under controlled conditions. GROWTH OF RUBELLA RA-27/3 VIRUS : According to the present invention, in the phase of growing Rubella RA-27/3 virus on MRC-5 cells, the cells grown in the CellCube modules are surface infected with Rubella working seed virus at appropriate conditions. The conditions maintained during the surface infection are defined by ranges of Multiplicity Of Infection ( MOI ) and the prevailing temperature. The MOI is maintained in the range of 20 to 100 and the prevailing temperature is controlled to be within the range of 31° C to 38° C. The invention may be better understood by the reference to the following example which is intended for the purpose of illustration and is not to be construed as, in any way limiting the scope of the present invention, which is defined in the claims appended hereto. EXAMPLE Experiment: Growth of MRC-5 cells : First few experiments were done using smaller CellCube modules having a surface area of 8500 sq.cm. Cells trypsinized from TCFs were used for seeding the CellCube module at a density of 1 x 104 cells per sq.cm. Six hours time was allowed for the attachment of cells seeded on side 1 whereas an overnight time duration was given for attachment of cells seeded on side 2. Circulation and perfusion was then started . The cells were trypsinized after 9 days. It was observed that side 1 was less confluent than side 2. Hence in the next experiment 24 hours time was given for cell attachment after seeding of each side, without changing other controlled conditions. Better viable counts were obtained in this case. Based on this experience, cell seeding and cell growth was carried out in bigger CellCube modules (85000 sq.cm). The cell counts obtained in repeated consecutive experiments are shown in Table 1. Table 1 Parameter description CellCube Single container surface area : 85000 cm2 Cells seeded : Example 01 Example 02 Example 03 PDL-30, 925x10® PDL-30, 875x10® PDL-28, 925X10® Final Cell Count: Example 01 Example 02 Example 03 10000x10® 13000x10® 10000x10® PDL achieved : Example 01 Example 02 Example 03 3.44 3.90 3.44 It was generally observed that a population doubling of about 3.9 could be achieved after 9 days of cell growth under controlled conditions. EFFECT OF AVAILABILITY OF LIMITING NUTRIENTS DURING CELL GROWTH : In order to avoid starvation of growing cells, fresh cell medium was used for circulation and perfusion at a schedule shown in table 2 which helped to maintain the limiting nutrients above starvation level. Approximate perfusion schedule of cell medium during cell growth phase in the CellCube system is shown in Table 2. Table 2 Day after seeding Volume perfused Fourth day One tenth the system volume Fifth day One fifth the system volume Sixth day One third the system volume Seventh day One half the system volume Eighth day One full system volume Growth of rubella RA-27/3 virus : The conventional system showed invariably lower and inconsistent virus yields. It was found out that there are 2 main reasons for poor virus yield. 1. The pH of the medium, if drops below 6.3, virus yield drops considerably. 2. Temperature of the medium if exceeds 33°C. virus yield drops accordingly. Also it is not possible to maintain the appropriate dissolved oxygen concentration in the conventional system which may have it's effect on the poor virus yield. Considering these factors, the output of Rubella RA-27/3 virus growing on MRC-5 cells in the CellCube system was assessed. The cells grown in the CellCube system were surface infected by the Rubella working seed virus. Periodic multiple harvests of virus were then collected from the system. The harvest titers obtained in the CellCube system are given in Table 3. Table-3 Harvest titers (log TCID 50 per 0.5 ml) obtained in CellCube system. Run No. HI H2 H3 H4 H5 H6 H7 H8 R-CC3 5.2 5.2 4.84 4.77 4.2 3.77 3.64 3.64 R-CC5 4.85 5.04 5.06 5.0 4.6 4.26 4.04 3.2 R-CC6 5.3 5.15 5.13 4.94 4.93 4.59 4.65 4.45 R-CC8 5.49 5.4 4.94 4.75 4.49 4.37 3.77 3.35 R-CC9 5.28 5.34 5.24 5.13 4.83 4.75 4.51 4.22 H - indicates Harvest - ( Multiple harvests ) R-CC - indicates the Run number. Values expressed as virus titre, log CCID 50 / 0.5 ml. ( Cell Culture Infective Dose 50 ), when titrated on RK- 13 cells. CONCLUSION : It can be observed from the results that the process of growing MRC-5 cells to 3.9 population doublings in a very compact space and cost saving manner can be practiced. The space required to handle 400 Roller (400 X 850 sq.cm.) per production cycle is already discussed in the prior art. If this is compared with the present invention where the space required (3,40,000 sq. cm cell cube area equivalent to 400 roller surface area) is much less and can be handled by only three trained qualified people and two semi¬skilled workmen. The reduced cGMP area requirement will be approximately as follows: 300 sq.ft. of class 100000 area. 300 sq.ft. of class 10000 area & 100 sq.ft. of class 100 area & The foremost advantage of this system is the reduced work burden because of unit operation of manufacture in less space and less manpower. The process therefore offers a great potential for a cost effective scale up which will help to reduce the cost of vaccine & make the vaccine available in very large quantities for the entire needy population. The present invention can be easily carried out by an ordinary skilled person in the art. Any modifications and changes may be deemed to be within the spirit and scope of the present invention as defined in the following claims

Documents:

485-mum-2003-claims(granted)-(14-05-2003).doc

485-mum-2003-claims(granted)-(14-05-2003).pdf

485-mum-2003-correspondence(05-01-2005).pdf

485-mum-2003-correspondence(ipo)-(14-06-2007).pdf

485-mum-2003-form 1(14-05-2003).pdf

485-mum-2003-form 19(15-09-2003).pdf

485-mum-2003-form 2(granted)-(14-05-2003).pdf

485-mum-2003-form 3(06-05-2004).pdf

485-mum-2003-form 3(14-05-2003).pdf

485-mum-2003-form 5(14-05-2003).pdf

485-mum-2003-form-2-(granted)-(14-05-2003).doc

485-mum-2003-power of authority(08-05-2003).pdf