Title of Invention	AN IMPROVED PROCESS FOR PREPARING STABILIZED BIOLOGICAL PREPARATIONS SUCH AS TRIVALENT ORAL POLIO VACCINE (OPV)
Abstract	An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), comprising three serotypes PV1 [106 (TCID 50)], PV2 [105 (TCID 50)] and PV3 [105 8(TCID 50)] in 0.1 ml of OPV, minimum essential medium (Eagle) in Earle's salts(MEM) 0.95mg, tris buffer, and MgCl2 and heavy water, the process comprising i) dissolving MEM powder 9.5g in heavy water 1000ml; ii)i adding tris buffer to MEM powder solution obtained at the end of step (i), to adjust pH (as seen by pH meter) between 6.5 and 68; iii) adding anhydrous/deuterated MgCl2 to MEM powder solution obtained at the end of step (ii) sufficient to attain 1M MgGl2 strength in the MEM powder solution to form a medium; iv) sterilizing said medium by filtration through not more than 0.22 micron Millipore membrane filter; v) taking xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three serotypes PV1, PV2 and PV3 respectively individually at concentrations with said sterilised medium and mixing them together and making the final volume to 100 ml with said sterilised medium, wherein xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3 and 10yl, 10y2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively; vi) storing the vaccine prepared at the end of step (v) at not more than -20°C and subsequently mamtaining at different temperatures as per standard cold chain temperatures

Title of Invention

AN IMPROVED PROCESS FOR PREPARING STABILIZED BIOLOGICAL PREPARATIONS SUCH AS TRIVALENT ORAL POLIO VACCINE (OPV)

Abstract

An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), comprising three serotypes PV1 [106 (TCID 50)], PV2 [105 (TCID 50)] and PV3 [105 8(TCID 50)] in 0.1 ml of OPV, minimum essential medium (Eagle) in Earle's salts(MEM) 0.95mg, tris buffer, and MgCl2 and heavy water, the process comprising i) dissolving MEM powder 9.5g in heavy water 1000ml; ii)i adding tris buffer to MEM powder solution obtained at the end of step (i), to adjust pH (as seen by pH meter) between 6.5 and 68; iii) adding anhydrous/deuterated MgCl2 to MEM powder solution obtained at the end of step (ii) sufficient to attain 1M MgGl2 strength in the MEM powder solution to form a medium; iv) sterilizing said medium by filtration through not more than 0.22 micron Millipore membrane filter; v) taking xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three serotypes PV1, PV2 and PV3 respectively individually at concentrations with said sterilised medium and mixing them together and making the final volume to 100 ml with said sterilised medium, wherein xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3 and 10yl, 10y2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively; vi) storing the vaccine prepared at the end of step (v) at not more than -20°C and subsequently mamtaining at different temperatures as per standard cold chain temperatures

Full Text	FORM 2 THE PATENTS ACT 1970 COMPLETE SPECIFICATION (See Section 10) TITLE APPLICANT Department of Atomic Energy, Government of India Anushakti Bhavan, Chatrapati Shivaji Maharaj Marg Mumbai 400001. The foUowing specifications particularly describes the nature of the invention and the manner in which it is to be performed :- 1. FIELD OF INVENTION ; This invention relates to the process of biological preparation such as trivalent poliovirus vaccine using heavy water as a stabilization medium. This invention particularly relates to the process of making biological preparation such as Oral Polio Vaccine (OPV), which improves the thermostability of such biological preparation by an order of magnitude. 2. BACKGROUND ON OPV : Tremendous efforts are ongoing in India for total eradication of poliomyelitis using mass administration of Oral Polio Vaccine (OPV). This has been generally successful and India is inching towards getting closer to complete immunisation. This will be followed up by continued administration of OPV under normal mode for next five to seven years. The main problem why the countries like India and other tropically hot climatic countries (South Asia and most of Afiica) are finding the problem of continued existence of poliomyelitis is due to the nature of the polio virus which exhibits high thermal inactivation. This calls for strict maintenance of a cold chain from the point of manufacture to the point of actual administration. The major problem so far has been the frequent break in such cold chains, especially in geographically remote areas in India. Therefore, there is an urgent need for developing a thermally stable carrier for OPV, which can maintain the potency of the vaccine at higher temperature levels. WHO specifies thermostability of any vaccine as the one that can maintain its TCID 50 value (Tissue Culture Infective Dose, a measure of the potency) at 37°C for seven days. At present, the MgCl2 stabilised OPV does not provide such thermostabilisation effect and hence needs additional support of cold chain. And this is where the problem of loss of viral potency comes in to being. In Indian scenario, the problem is further accentuated due to vary harsh climatic conditions and for the vaccine to be potent, one may like the extended stabilisation at 42°C for say, three days and even short term stabilisation for say, 12 hours at even elevated temperatures like 45 °C. This would cover almost major temperature variations across the country. During the early days of use of OPV, it became apparent that the attenuated strains has a tendency to mutate and that such mutations may occur not only in the course of virus replication in tissue culture but also within vaccine recipients. A few rare cases have been associated temporarily with the administration of the vaccine. The figures available to the WHO indicate that there is one recipient case and one contact case per 4 million doses of the vaccine distributed. According to Nkowane and others the reported overall frequency of vaccine associated poliomyelitis was 1 case per 2.6 million doses distributed. However, the ratio of cases associated with the first dose becomes more than 1 per million first doses and is proportionally less for subsequent doses Molecular biology has provided methods for analysing the genetic basis of virus attenuation. The molecular differences between the parent and vaccine viruses have been determined. For type 1, 56 mutations scattered through the 7441 nucleotides in the genome of the virus accounts for 21 amino acid differences. Reversion to neurovirulence requires several back mutations. This is an unlikely occurrence as the virus replicates in the vaccinated person and accounts for the greater safety of the type 1 component of OPV. For type 3, only 10 of the 7429 nucleotides in the genome are different in the vaccine from those of the parent strains and only two seem to be important for attenuation. For type 2, the situation in regard to frequency of base changes is intermediate, with 23 point mutations in the vaccine virus with the nucleotide at position 481 in the 5' non-coding region essential for achieving attenuation. For the three types, the most crucial mutations for attenuation seem to be at nucleotide 480 for type 1, at 481 for type 2 and at 472 for type 3. There has been a tremendous research interest in developing various ways and means by which the poliovirus can be made immune to the thermal degradations. Some of the attempts to improve the polio vaccine are discussed below. 2.1. Recombinant poliovirus strains as vaccine: The attenuation of poliovirus type 1 is very stable. Several laboratories have constructed recombinant viruses in which the capsid region of type 2 or type 3 vaccine virus has been exchanged with the corresponding segment of poliovirus type 1 genome. The recombinant viruses thus possess the stable genetic chaiacter of type 1 vaccine virus but include type 2 or type 3 neutralising antibodies. A vaccine consisting of poliovirus type 1 (Sabin), poliovirus type 1+2 (Sabin recombinant) and poliovirus type 1+3 (Sabin recombinant) has been proposed. In addition, hybrid viruses expressing antigenic epitopes of the three types have also been designed. 2.2. Genetically stabilised seed virus: The rate by which spontaneous mutations occur is especially high in single stranded RNA genome replication. That the single stranded RNA genome of poliovirus is not an exception to this high rate of mutation was demonstrated when plaque purified viruses from the same seeds of poliovirus were characterised after a few independent passages. The rapid accumulation of mutations has been attributed to the absence of proof reading and editing functions in RNA replication. As a result, even the original Sabin vaccine strains cannot be considered to be completely homogeneous. The high frequency of mutations that occurs during poliovirus genome replication makes it difficult to maintain the attenuation phenotype of poliovirus live vaccines. Racaniello and Baltimore assembled a full length clone of the genome of the Mahoney strain of poliovirus type 1 and showed that transfection of plasmid DN A from this clone would produce infectious poliovirus in cultured primate cells. Subsequently, an infectious cDNA clone of the genome Sabin 1 was prepared. Infectious cDNA clones may be used to preserve the vaccine quality of Sabin viruses and to provide a potentially unlimited supply of constant seed virus for polio vaccines. WHO has emphasized that trial with new candidates that involve humans should be conducted only after the strains have been proved in laboratory and animal tests to be as safe as or safer than the Sabin strains and have shown to meet applicable current WHO requirements. 2.3. Improving thermostability of opv: The oral poliovirus vaccine presently used requires storage under refrigeration temperature to maintain its thermostability. A considerable percentage of the total cost and effort of immunisation program relates to the creation and maintenance of a cold chain to ensure that vaccines are maintained under conditions that result in retention of potency to the point of administration. Cold chain conditions are defined by the requirements of the vaccine that is least stable, making it clear that efforts to improve the thermostability of vaccines in general must first be focused on OPV. EPI advisory committee on the technical aspects of polio eradication identified the need for significant improvements in the thermostability of OPV as a critical success factor for the WHO's initiative for global eradication of poliomyelitis by the year 2000. In 1991, the EPI set an ambitious target for an enhanced thermostable OPV; one that could survive 'a week in the pocket' that is 45°C for 7 days. However, in 1993, this target was relaxed to a vaccine that could withstand 37°C for 7 days (compared to 2 days with the current vaccine). Acceptable potency after thermal challenge was defined as 2.4. Freeze drying: Freeze drying & Lyophilization of poliovirus vaccine were the several different drying protocols examined. Trehalose (a.-D-glucophyranosyl-a.-D-gluophyronoside) was found to be of particular interest. However, the large initial loss in vaccine potency proved that these procedures were not suitable for OPV. 2.5. Pocket binding compounds as stabilisers: Oxazolinyl isoxazoles (WIN compounds) and pyridazinamines (Jensen compounds), known as enteroviral compounds interact with the viral capsid thus stabilising the capsid structures. It was postulated that high concentration of these compounds bind and stabilise poliovirus against thermal inactivarion. When vaccine is administered the bound antiviral compound will elute (reversible binding) from viral capsid against steep concentration gradient effected by dilution. The vims thus regains infectivity and would immunise the child. The pocket binding antiviral compounds had undergone safety and pharmacological studies and were cleared for human application. It was found that the stabilized poliovirus antigenicity with these compounds is measured by methods that detect intact viral antigens but the stabilisation of infectivity (vaccine potency) was not better than MgCl2. 2.6. Use of 1M MgCI2 as stabilizer: So far accepted stabilizing agent for poliovirus has been 1 M MgCl? which provides some additional stabilization but is not sufficient to meet WHO specification of seven days at 37°C. Therefore, use of MgCl2 is further reinforced through a cold chain where the vaccine is stored at refrigerated temperatures from the point of manufacture to the point of administration. 3. PRIOR ART: 3.1 Heavy water as a thermostabilising medium The stabilization of various microorganisms have been tried with heavy water since 1969 onwards. As early as 1969 by Y.B.Popov et al., have published the paper "Effect of deuterium oxide on the heat resistance of staphylophage". There have been various references available on use of heavy water for this purpose, which are listed below. 1. Y.B. Popov et al. "Effect of deuterium oxide on the heat resistance of staphylophage" - Chemical Abstracts : vol. 70, no.21, 26 Mai 1969, Columbus, Ohio, US; abstract no. 94238d voir abrege & BIOFIZIKA vol. 14, no. 1, 1969 pages 186-187. 2. P.R.M. Steele et al. "Factors affecting the viability of freeze thawed T4 bacteriophage II. Influence of certain electrolytes on the degree of inactivation"- Chemical Abstracts: vol.72, no. 15, 13 Avril 1970, Columbus, Ohio, US; abstract no. 76039b, voir abrege & J. HYG. vol. 67, no. 4, 1969 pages 679-690. 3. K. Jung "Effect of heavy water on the thermal resistance of microorganisms" -Chemical Abstracts : vol. 71, no. 23, 8 December 1969, Columbus, Ohio, US; abstract no. 110159g, voir abrege & NUCL. SCI. ABSTR, vol. 23, no. 12, 1969 page 22242. 4. C.S. Pittendrigh et al. "Very rapid enhancement by deuterium oxide of the temperature-tolerance of adult dropsophila" - Chemical Abstracts: vol.80, no.23, 10 Juin 1974, Columbus, Ohio, US; abstract no. 130872y, voir abrege & PROC. NAT.ACAD.SCI. USA, vol.71, no.2, 1974. 5. V.Ya. Aleksandrov et al. "Effect of heavy water (water-D2) on the resistance of procollagen to thermal denaturation and to the action of collagenase" - Chemical Abstracts: vol. 83, no. 1, 7 Juillet 1975, Columbus, Ohio, US; voir abrege & DOKL. AKAD.NAUK.SSSR vol.220, no. 6, 1975 pages 1445-1448. 6. U.Lemm et al. "Stabilisation of enzymes and antiserums by heavy water" -Chemical Abstracts: vol. 95, no.5, 3 Aout 1981, Columbus, Ohio, US; abstract no. 37552u, voir abrege & EUR.J.BIOCHEM. vol. 116, no.3, 1981 pages 441-445. 7. U.Lemm et al. "Stabilisation of biochemically active proteins by heavy water " -Chemical Abstracts: vol. 95, no. 19, 9 November 1981, Columbus, Ohio, US; abstract no. 164216n, voir abrege & STUD BIOPHYS. vol. 84, no. 1, 1981 pages 69-70. 8. V.Ya. Aleksandrov "Stabilising effect of heavy water (D20) on the cell" - Chemical Abstracts: vol. 105, nOo. 17, 27 October 1986, Columbus, Ohio, US; abstract no. 14827lw, voir abrege & TS1TOLOGIYA vol. 28, no. 8, 1986 pages 790 - 795. 9. C.Heyde et al. "Heavy water (D20) protective effect on proteins in pharmaceuticals. For example: Human cholinesterase" - Chemical Abstracts: vol. 116, no.4, 27 Janvier 1992, Columbus, Ohio, US; abstract no. 28099b, voir abrege & Z. NATURFORSCH. Vol. 46, no. 9-10, 1991 pages 789-793. 10. EP, A, O, 332 826(TEVA) 20 September 1989 voir le document en entire. Use of heavy water for stabilizing polio vaccine has been described in a paper published by Wu, R., Crainic, R., et el. "Thermostabilisation of live virus vaccines by heavy water (D20)", Vaccine 1995 Vol 13 No 12 ppl058-1063, and subsequently by Milstein, J. B., et al. "{Development of a more Thennostable Polio Vaccine ", Journal of infectious Diseases 1997. The authors (Crainic, R. & Simpson, K) of the above paper have applied for a patent bearing the publication no. WO 94/21298 dated September 29, 1994. In the invention claimed by them, heavy water at concentration less than 95% D20 is tried with and without 1M MgCl2 for finding its suitability on specific serotype -3 of OPV. .The said prior art is restricted to stabilization for type-3 vaccine which is stated to be the most thermo labile and limits the overall concentration carrier medium to about 87% using 95% heavy water. It is well known that the polio vaccine is a mixture of three serotypes in a particular concentration, 106, 1050 & 1058 for the PV1, PV2 & PV3 per 0.1 ml respectively and as per the WHO recommendations, every dose of polio vaccine shall be administered in the above concentrations for immunization puiposes. This requires the stabilization experiments must be conducted when all the three species are present together in exactly the same compositions and not as single species i.e.,serotype 3, due to the interactive reactions. Importantly, the stabilization of WHO recommended vaccine comprising the three serotypes is complex as compared to the stabilization of single vaccine as that of the prior art due to obvious interactive reactions. Therefore the stability criteria as in the prior art for the individual serotype can neither be directly followed or extended to achieve the required stabilization for the combination vaccine of WHO recommendations. 4. OBJECT OF THE INVENTION : The basic object of the present invention is directed to provide a process whereby polio vaccine comprising mix of three serotypes as per the WHO recommendations can be produced in a stabilized form. Another object 6f the present invention is to provide the desired stability to stand accidental breaks in the standard cold chain temperature cycle which consists of -20°C at the point of vaccine manufacture to 8°C at the point of administration. Another object of the invention is to make the combination vaccine thermally stable upto 7 days at temperature of 37°C, upto 3 days at a temperature of 42°C and about a day at a temperature of 45°C. Yet another object is directed to provide a process for manufacture of trivalent polio vaccine as per WHO acceptable limits which would be adapted to maintain potency of trivalent polio vaccines without acceptable limits without the maintenance of the cold chain. 5. SUMMARY OF THE INVENTION : Accordingly, the present invention relates to an improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), comprising three serotypes PV1 [106 (TCID 50)], PV2 [105 (TCID 50)] and PV3 [1058(TCID 5o)] in 0.1 ml of OPV, minimum essential medium (Eagle) in Earle's salts(MEM) 0.95mg, tris buffer, and MgCl2 and heavy water, the process comprising i) dissolving MEM powder 9.5g in heavy water 1000 ml; ii) adding tris buffer to MEM powder solution obtained at the end of step (i), to adjust pH (as seen by pH meter) between 6.5 and 6.8; iii) adding anhydrous/deuterated MgCl2 to MEM powder solution obtained at the end of step (ii) sufficient to attain 1M MgCl2 strength in the MEM powder solution to form a medium; iv) stuilizing said medium by filtration through not more than 0.22 micron fMilhpore membrane filter; v) taking xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three serotypes PV1, PV2 and PV3 respectively individually at concentrations with said sterilised medium and mixing them together and making the final volume to 100 ml with said sterilised medium, wherein xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3 and 10yl, 10v2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively; vi) storing the vaccine prepared at the end of step (v) at not more than -20°C and subsequently maintaining at different temperatures as per standard cold chain temperatures thereby providing the desired stable trivalent oral polio vaccine under standard cold chain temperatures. 6. DETAILED DESCRIPTION : As mentioned in the summary of the invention, the present invention relates to an improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), wherein heavy water iised is from 90 % to 99.9 % D / (D +H) by weight, preferably 95.1 % to 99.9% D/(D +H) by weight and more preferably more than 99.8 % D/(D+H) by weight. In the present invention for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV) the deuterated MgCl2 is having 1-6 moles of D2O as water of hydration. This is in view of the fact that 1 M MgCl2 contains light water molecules as water of hydration (MgCi2.6H20) resulting in overall dilution of D20 from 95% to 87%. The process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV) described as herein in the text and in the examples is useful for other similar biological preparations also. 6.1 Example: The invention will now be illustrated with the help of an example. The example is by way of illustration only and in no way restrict the scope of the invention. Material and methods: 6.1.1. Cell culture: Hep-2, a continuous cell line of human carcinoma of larynx was originally obtained from National Institute of Biological Standards and Control (NIBSC) Potters Bar, England. The cells were preserved in the Cell bank. Minimum Essential Medium (Eagle) in Earle's salts (MEM) was used. Powdered medium was obtained from commercial sources (GIBCO/BRL) and prepared as per the manufacturer's instructions. The medium was supplemented with l-glutamine, antibiotics and foetal bovine serum (FBS). Cell growth medium contained 5% FBS whereas cell maintenance medium contained 2% FBS. Cultures were grown in 80cm and 175cm disposable tissue culture flasks as required. The cell culture procedure followed is briefly described below. Maintenance of cell line: 1. A cell culture flask (Hep-2 cells) with a confluent monolayer of cells was used to initiate new cultures. 2. The used culture medium was decanted (discarded) the from the flask and washed the monolayer with 5 ml of phosphate buffered saline (PBS). 3. Added 5 ml of pre-warmed (37°C) trypsin-EDTA solution and incubated the flask for 3 min. 4. Decanted (discarded) trypsin and continued the incubation until cells started to detach from the surface (usually 5min). 5. Tapped the flask to detach most of the cells and then added 5ml of growth medium. Gently resuspended the cells with the aid of a Pasteur pipette. 6. Using a haemocytometer obtained the cell count (cells per ml) of the cell suspension. 7. Labelled new sterile TC flasks with the details of the cells i.e., cells line, sub-culture number and date. 8. Added growth medium (80 mL for 80Cm2 flasks). . 9. Seeded at least 2.5 x 106 cells in each flask (80cm2) to initiate new cultures. Incubated the flasks at 37°C and observe daily under an inverted tissue culture microscope for cell growth. When the cell monolayer was formed (after 4 to 5 days), discarded the medium from the flasks and replenished it with maintenance medium. 10. Initiated fresh (new) cultures at every 7-8 days interval. Note: Cells from step No. 5 were used for various experiments. 6.1.2. Poliovirus: Live attenuated oral poliovirus vaccine strains (poliovirus type 1,2 and 3) were originally obtamed fromCommercial units at the S0+3 level (i.e.^ vaccine) ,A stock was maintained in the laboratory. The virus was passed in Hep-2 cells for use in all experiments relating to this project. All procedures requiring virus handling were performed in bio-safety cabinets. 6.1.3. Heavy water: Heavy water (D20 >99.8% D/(D+H) by weight%) was made available from the operating plants of Heavy Water Board of the Department of Atomic Energy. The heavy water used was virgin heavy water as it was extracted from the natural occurring substances like water. MEM powder was dissolved to make tissue culture medium. pH of the medium was adjusted by 1M Tris base prepared in D20. MgCl2 powder (desiccated) was dissolved in heavy water to obtain IM concentration in the final volume of the solution. The final volume was adjusted by adding milli Q purified heavy water after dissolving MEM and adjusting pH by IM Tris base. All solutions were sterilised by filtration through 0.22ji (pore size) Millipore membrane filters. 6.1.4. Preparation virus stock: 1. Confluent monolayer culture of Hep-2 cells in 175 cm2 TC flasks were used. One TC flask was used for each poliovirus type. 2. The cell culture medium was discarded. 3. 0.5ml of polidvirus suspension (108 0TCID5o/ml) was inoculated to the flask for initiating virus infection. The flasks were incubated at 37°C for 1 h for virus adsorption. 4. 40ml maintenance medium was added to the flasks. The culture flasks were further incubated at 37°C for virus growth. 5. The flasks were observed microscopically daily for virus induced cytopathogenic effect (CPE). When all cells showed CPE, the flasks were frozen (-20°C) and thawed to release all virus from the cells. The culture medium was collected (harvested) and centrifuged at 5000Xg for 15rnin to remove cell debris. The clear supernatant containing the virus was distributed in 2ml aliquots and stored at -20°C until used. 6.1.5. Virus titration: Live virus content of a virus suspension is expressed either as plaque forming units or tissue culture infective doseTCID50 per ml. The micro-titration method was used in all experiments. The following materials were used. 1. 96 well flat bottom tissue culture plates (Sterile) 2. Disposable screw capped tubes (5ml, polyethylene, sterile) 3. Disposable micro-tips (sterile, lOOjxl and lOOOfxl) , 4. Variable volume single channel pipettes (Eppendorf type) 5. Serological pipettes. 6. Dropping pipettes 50^,1 per drop 7. Hep-2 cell suspension 8. Tissue culture media For each test a worksheet was prepared for recordrngtbe results, Preparation of a 10-fold virus dilution series was done and proceeded as given below, 1. For each virus suspension to be titrated, labelled eight tubes (5ml) with the dilution step 10'1 to 10"8. 2. Dispensed 0.9ml maintenance medium in each tube using a sterile pipette/ pipette and disposable microtip. 3. Using a disposable micro-tip and a pipette transferred 100JJ.1 of virus suspension from the stock to the tube marked 10"1, discarded the tip and mixed the contents of the tube. 4. Using a fresh micro-tip transferred 100}j,l of virus suspension from 10" tube to the second tube marked 10"2. 5. Repeated the procedure till the 10~8 dilution was prepared taking the precaution to use a fresh tip for each step and thorough mixing of the dilutions prepared. 6. Labelled required number of microtitre plates with identification number and date of the experiment. 7. 10"3 to 10"8 dilutions were required for titration. 8. Using a disposable tip added lOOixl of the 10~3 dilution in the wells Al to A10 of the microtitration plate. 9. Using fresh tip continued to add lOOpl virus suspension of the 10-fold dilutions in 10 wells of each row of the plate until the 108 dilution is added. 10. Added lOOpil of medium to wells All to H12 (cell control wells). 11. Prepared Hep-2 cell suspension by trypsinization of a confluent monolayer culture. Adjusted the cell count to 105 cells per ml. 12. Added 2 drops (IOOJJI) of cell suspension in each well using the dropping pipettes. Also seeded cell control wells wherein lOOul of medium (no virus) was added. Replaced the plate cover. 13. Incubated the plates at 37°C in a CO2 incubator (5% C02 in air). 14. Observed the plates under an inverted TC microscope to record virus induced CPE on days 5 and 7 of incubation. 15. Calculated virus titre by using the Karber's formula. Expressed virus titre as TCID50 per ml. 6.1.6. Karber's formula: Virus titre is determined by determining the dilution of the virus suspension that produced CPE in 50% of the cultures. A reciprocal of this dilution is the virus titre in the stock suspension. Log TCID50 = L - D (S - 0.5) Where, L = lowest dilution used (log), D = difference between dilution steps (log), S = sum of proportion of CPE positive wells at each dilution. 6.1.7. Preparation of experimental vaccine: The heavy water concentration used for preparation of the vaccine was more than 99.8 %by weight D/(D+H). 1. Obtained the virus stocks of vaccine strains of poliovirus type 1,2 and 3 of known titre. 2. A trivalent ORV contains the three serotypes in different quantities. Poliovirus type 1 (lO^TCIDso), poliovirus type 2 (10?0TCID50) and poliovirus type 3 (1058 TCID50) per human dose of 0.1ml. 3. Calculated the volume of the stock virus suspension needed to give the required titre of the poliovirus serotype in the trivalent vaccine preparation. i) For example, the titre of the stock poliovirus type 1 was 108 5 TCID5o/ml and 100 ml vaccine was being prepared. 3.16ml of the stock diluted to 100 ml will have poliovirus type 1 titre of 106 TCID50 per 0.1 ml. ii) The three viruses were mixed in the required proportion to obtain correct blend of the vaccine. The dilution of the stock solution and its blending with the sterilized medium was done as mentioned below. xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three serotypes PV1, PV2 and PV3 respectively were taken individually at the concentrations and mixed them together. The same was made up to the final volume of 100 ml with sterilized,pH adjusted and MgCl2 added MEMmedium, heavy water, wherein xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3 and 10yl, 10y2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively; iii) Storing the vaccine prepared at the end of step (v) at not more than -20°C and >'■' '■...•'.-,. ■' subsequently mamtaining at different temperatures as per standard cold chain temperatures 6.1.8. Determination of concentration of individual virus component in the tnvalent polio vaccine: Principle: Determination of individual serotype concentration in trivalent polio vaccine was based on specific neutralisation of two of the three virus using serotype specific anti-poliovirus antibodies. For example, neutralisation of poliovirus type 2 and 3 by a combination of anti-poliovirus type 2 and anti-poliovirus type 3 antisera permitted the titration of poliovirus type 1. Antisera: Good quality monospecific antisera of known titre against the three types of polioviruses were used for analysis of trivalent polio vaccine. The dilutions of these antisera used in preparation of combination sera for neutralisation of poliovirus serotypes was determined by experiments in advance. Procedure: 1. Antisera combinations were prepared as indicated below. The aliquots were made,frozen and stored for ready use. Anti-PVI (1:2000) + Anti-PV2 (1:4000); Anti-PVl (1:2000) + Anti-PV3 (1:2000); Anti-PV2 (1:4000) + AntiPV3 (1:2000). 1 "7 2. Diluted bivalent vaccine sample 10" to 10". 3. Two microtitre plates were required for one sample and labelled the plates. 4. Added 50[xl of antiserum combination (anti-P2+P3) in first set of 6 rows of 8 wells. 5. Added 50μ1 of antiserum combination (an.ti-Pl+P3) in the second set of 6 rows of 8 wells. 6. Added 50μ1 of antiserum combination (anti-Pl+P2) in me third set of 6 rows of 8 wells. 7. Added 50μl of tissue culture medium (no antiserum) in the fourth set of 6 rows of 8 wells. 8. Added 50μ1 of diluted trivalent vaccine 10" to 10" dilution to the sets of 6 rows. Mix and incubate at 37°C in a C02 incubator for 90 min. 9. Prepared sufficient quantity of Hep-2 cell suspension (105 cells per ml). At the end of the incubation added 100Μ.1 Hep-2 cells in each well, mixed and returned to the incubator. 10. Observed the plates after 7 days under an inverted microscope and recorded CPE reading. 11. Calculated virus titre in each of the four sets of titration using Karber formula. The 1st set gave value of poliovirus type 1, the second for type 2, the third type 3 and the fourth set indicated a combined titre for the trivalent preparation (total virus content). 12. Virus titre was expressed as titre per human dose (0.1ml). 6.1.9. Estimation of magnesium chloride concentration: Concentration of magnesium chloride in MEM was determined by titration of a small aliquot of the medium against a standard solution of EDTA. i."'5WAmmonia: Dissolve 37.5ml ammonia in 100 ml heavy water. r 2. Ammonia-ammonium chloride buffer, NH4C1 5.4g 5N Ammonia 70.0 ml Milli Q purified heavy water To 100 ml 3. EDTA(0.05M). EDTA disodium 18.6g Milli Q purified heavy water To 1000 ml 4. Indicator: solochrome black. Procedure-Diluted 2 ml of the test medium with 10 ml distilled water. Added 5ml ammonia-NR4Cl buffer. Added a small quantity (a pinch) of solochrome black powder as the indicator. Titrated the solution against 0.05 M EDTA solution (in burette) to obtain an end point which is from wine red to blue colour. Molarity of MgCl2= Volume of EDTA X Molarity of EDTA r! Volume of test medium The vaccine samples prepared as described above were exposed to temperature at 37 ° C. Samples of vaccine samples were withdrawn at different time intervals of exposure. The amount of live virus was determined by titration in cell cultures. The difference between the titre of virus suspension before and after exposure to heat was a measure of virus inactivation. The results were compared with heat inactivation of poliovirus suspensions;, in medium containing 1M MgCl2 prepared in H20, similar to the OPV being currently used. 6.1.10 Estimating mean titre and variation: The samples of OPV prepared in heavy water of the above said purity and that prepared in normal water were taken for analysis. The poliovirus suspensions were distributed in 1ml aliquots in 2ml screw-capped vials and stored at -20°C until used. To determine the virus concentration in these suspensions, samples were titrated in Hep-2 cells. To determine inter-experiment variation each suspension was titrated ten times. Briefly, the virus suspension was serially diluted ( 10"1 to 10~8) in tissue culture medium. IOOJLXI of each dilution ( 10"2 to 10"8) were inoculated in 8 wells of a microtitre plate. 100(il of Hep-2 cell suspension (105cells/ml) were then added. The plates were incubated at 37°C in a C02 incubator. The plates were observed under an inverted TC microscope 22 to record CPE reading on the 7 day of incubation. Virus titre was calculated according to Karber's formula. Ten experiments were carried out on poliovirus types 1, 2 and 3 and the titres were used to calculate mean number of infectious unit (titre) and also the 95% confidence limits (2s.d.). The maximum variation in these assays were about 0.2 Hog, well within the fiducial limits of assays of 0.5 log allowed by the WHO for vaccine titration. In each of the experiments carried out using the above mentioned virus suspensions the above mean titres were used for validation. Vials stored at -20°C were used as internal control in all the experiments. An experiment was considered, as valid if the control vial titre was within the predetermined limits. 6.2 Results and discussions: The results are as shown in figure below. It can be clearly inferred that the product of example 1 is more stable at 37 ° C than that of the conventional vaccine. It can be noted that the loss in potency is upto 0.5 loglO at 37 °G for a period of 7 days for the vaccine made with heavy water of 99.8 % concentration while the loss of potency for the conventional vaccine prepared in normal water over the above value after two days itself. Thus, the potency loss for the vaccine prepared in example 1 can be seen to have a lojfs of potency within the acceptable limits while that of the normal vaccine is far away from the same after the storage for one day itself indicating the advantages of the new process. It may be noted that the above said vaccine samples prepared in heavy water and that prepared in conventional normal water were exposed to 42°C and 45°C temperatures also and the loss of potency after exposure to different time periods were found. While the loss of potency of the trial vaccine at 42°C was within acceptable limits over 3 days and 45°C was over a day. The same for normal vaccine was much less than this. The trivalent vaccine prepared as the above said process indicated a marked improvement over the case illustrated for the monovalent virus strain PV3 in the patent publication mentioned in prior art of the present invention. The marked improvement in the thermostabilisation observed in the vaccine prepared by the present invention is due to the new process used in the present invention for preparing the trivalent OPV using heavy water of more than 99.8% D/(D+H) by weight. As would be evident from the above disclosure the object of the present invention of providing stability to trivalent oral polio vaccine is surprisingly achieved by selective reduction of light water content of the vaccine to maximum extent which is not taught in any of the available prior art. 7. ADVANTAGES ; The principle advantage of the present invention is to obtain a thermostable triple polio vaccine which is stored at -20°C and subsequently maintaining at different temperatures as per standard cold chain temperatures ;and giving stability by retaining the potency of the vaccine against accidental breaks intiie cold chain. The vaccine prepared by the new invention makes it thermally stable upto ? days at temprature of 37ifc» upto 3 days at a temperature of 42°C and about a day at a temperature of 45°C, The vaccine manufactured using the invention maintains the potency of trivalent polio vaccine as per WHO acceptable limits without the maintenance of cold chain. Other advantages of the vaccine prepared by the new process is given below. 1. The vaccine prepared by the process of present invention is having more stability with respect to higher temperature as compared to the presently available vaccine prepared in normal water. 2. The process helps in retaining the potency of the vaccine to the acceptable levels despite breakage in the cold chain. The existing vaccine is not stable at 45 °C beyond few hours while the thermostabilised vaccine can allow breakage in the cold chain for over a day, even at 45 °C which is highly advantageous to tropically located developing countries like India. 3. The thermostabilized vaccine meets with the WHO recommendations for the OPV for 7 days storage at 37 °C. 4. The vaccine prepared by the present invention having improved thermostability will be very helpful in the set goal of complete polio eradication by 2005 particularly in India. 5. The improvement brought in by usage of heavy water of higher purity in the therrno labile nature of biological preparations such as polio vaccine can be gainfully utilized in other fields also. 8. CLAIMS: We Claim 1. An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), comprising three serotypes PV1 [106 (TCID 50)], PV2 [105 (TCID 50)] and PV3 [105 8(TCID 50)] in 0.1 ml of OPV, minimum essential medium (Eagle) in Earle's salts(MEM) 0.95mg, tris buffer, and MgCl2 and heavy water, the process comprising i) dissolving MEM powder 9.5g in heavy water 1000ml; ii)i adding tris buffer to MEM powder solution obtained at the end of step (i), to adjust pH (as seen by pH meter) between 6.5 and 68; iii) adding anhydrous/deuterated MgCl2 to MEM powder solution obtained at the end of step (ii) sufficient to attain 1M MgGl2 strength in the MEM powder solution to form a medium; iv) sterilizing said medium by filtration through not more than 0.22 micron Millipore membrane filter; v) taking xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three serotypes PV1, PV2 and PV3 respectively individually at concentrations with said sterilised medium and mixing them together and making the final volume to 100 ml with said sterilised medium, wherein xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3 and 10yl, 10y2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively; vi) storing the vaccine prepared at the end of step (v) at not more than -20°C and subsequently mamtaining at different temperatures as per standard cold chain temperatures 2. Afi! improved process for manufacturing stabilized biological^preparations such as trivalent oral polio vaccine (OPV),as claimed in claim 1, wherein heavy water used is from 90 % to 99.9 % 0 / (0 +H) by wt. 3. An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV),as claimed in claim 1 or 2, wherein heavy water used is from 95.1 % to 99.9% D/(D +H) by wt. 4. An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV),as claimed in claim 1-3, wherein heavy water used is from more than 99.8 % D/(D+H) by wt. 5. A process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV),as claimed in any claim 1-4, wherein deuterated MgCl2 is having 1-6 moles of D20 as water of hydration. 6. A process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV) substantially as herein described in the text and in the example^. *************** Date : 8.2.2001 APPLCANT'S NAME : Dept. of Atomc Energy Govt, of India APPLICATION NO. : 158/MUM/2001 TOPV H-Mg AT 37 C TOPV 100 D-Mg AT 37 C 06 , D.V i- 1.2 v -^3— PV1 ; : - a PV2 '■! ~*~- PV3 i rs —-n- /" W 0.2 t— ^—■ ot J , :-&>L 0 > &-- ■■■££?—"S n 0 2 4 —»- -PV1 ..«. PV2 - A-- PV3 TOPV 100 D-Mg AT 42C TOPV 10OD-Mg AT 45C —e— PVI -S- PV2 Dated this 12th day of February 2001 K >THA SIDDHARTHA NAG Of S.MAJUMDAR&CO Applicant's Agent

Full Text

FORM 2
THE PATENTS ACT 1970
COMPLETE SPECIFICATION (See Section 10)
TITLE

APPLICANT
Department of Atomic Energy, Government of India Anushakti Bhavan, Chatrapati Shivaji Maharaj Marg
Mumbai 400001.
The foUowing specifications particularly describes the nature of the invention and the
manner in which it is to be performed :-

1. FIELD OF INVENTION ;
This invention relates to the process of biological preparation such as trivalent poliovirus vaccine using heavy water as a stabilization medium. This invention particularly relates to the process of making biological preparation such as Oral Polio Vaccine (OPV), which improves the thermostability of such biological preparation by an order of magnitude.
2. BACKGROUND ON OPV :
Tremendous efforts are ongoing in India for total eradication of poliomyelitis using mass administration of Oral Polio Vaccine (OPV). This has been generally successful and India is inching towards getting closer to complete immunisation. This will be followed up by continued administration of OPV under normal mode for next five to seven years. The main problem why the countries like India and other tropically hot climatic countries (South Asia and most of Afiica) are finding the problem of continued existence of poliomyelitis is due to the nature of the polio virus which exhibits high thermal inactivation. This calls for strict maintenance of a cold chain from the point of manufacture to the point of actual administration. The major problem so far has been the frequent break in such cold chains, especially in geographically remote areas in India. Therefore, there is an urgent need for developing a thermally stable carrier for OPV, which can maintain the potency of the vaccine at higher temperature levels. WHO specifies thermostability of any vaccine as the one that can maintain its TCID 50 value (Tissue Culture Infective Dose, a measure of the potency) at 37°C for seven days. At present, the MgCl2 stabilised OPV does not provide such thermostabilisation effect and hence needs additional support of cold chain. And this is where the problem of loss of viral potency comes in to being. In Indian scenario, the problem is further accentuated due to vary harsh climatic conditions and for the vaccine to be potent, one may like the extended stabilisation at 42°C for say, three days and even short term stabilisation for say,

12 hours at even elevated temperatures like 45 °C. This would cover almost major temperature variations across the country.
During the early days of use of OPV, it became apparent that the attenuated strains has a tendency to mutate and that such mutations may occur not only in the course of virus replication in tissue culture but also within vaccine recipients. A few rare cases have been associated temporarily with the administration of the vaccine. The figures available to the WHO indicate that there is one recipient case and one contact case per 4 million doses of the vaccine distributed. According to Nkowane and others the reported overall frequency of vaccine associated poliomyelitis was 1 case per 2.6 million doses distributed. However, the ratio of cases associated with the first dose becomes more than 1 per million first doses and is proportionally less for subsequent doses
Molecular biology has provided methods for analysing the genetic basis of virus attenuation. The molecular differences between the parent and vaccine viruses have been determined. For type 1, 56 mutations scattered through the 7441 nucleotides in the genome of the virus accounts for 21 amino acid differences. Reversion to neurovirulence requires several back mutations. This is an unlikely occurrence as the virus replicates in the vaccinated person and accounts for the greater safety of the type 1 component of OPV.
For type 3, only 10 of the 7429 nucleotides in the genome are different in the vaccine from those of the parent strains and only two seem to be important for attenuation. For type 2, the situation in regard to frequency of base changes is intermediate, with 23 point mutations in the vaccine virus with the nucleotide at position 481 in the 5' non-coding region essential for achieving attenuation. For the three types, the most crucial mutations for attenuation seem to be at nucleotide 480 for type 1, at 481 for type 2 and at 472 for type 3.

There has been a tremendous research interest in developing various ways and means by which the poliovirus can be made immune to the thermal degradations.
Some of the attempts to improve the polio vaccine are discussed below.
2.1. Recombinant poliovirus strains as vaccine:
The attenuation of poliovirus type 1 is very stable. Several laboratories have constructed recombinant viruses in which the capsid region of type 2 or type 3 vaccine virus has been exchanged with the corresponding segment of poliovirus type 1 genome. The recombinant viruses thus possess the stable genetic chaiacter of type 1 vaccine virus but include type 2 or type 3 neutralising antibodies. A vaccine consisting of poliovirus type 1 (Sabin), poliovirus type 1+2 (Sabin recombinant) and poliovirus type 1+3 (Sabin recombinant) has been proposed. In addition, hybrid viruses expressing antigenic epitopes of the three types have also been designed.
2.2. Genetically stabilised seed virus:
The rate by which spontaneous mutations occur is especially high in single stranded RNA genome replication. That the single stranded RNA genome of poliovirus is not an exception to this high rate of mutation was demonstrated when plaque purified viruses from the same seeds of poliovirus were characterised after a few independent passages. The rapid accumulation of mutations has been attributed to the absence of proof reading and editing functions in RNA replication. As a result, even the original Sabin vaccine strains cannot be considered to be completely homogeneous. The high frequency of mutations that occurs during poliovirus genome replication makes it difficult to maintain the attenuation phenotype of poliovirus live vaccines.

Racaniello and Baltimore assembled a full length clone of the genome of the Mahoney strain of poliovirus type 1 and showed that transfection of plasmid DN A from this clone would produce infectious poliovirus in cultured primate cells. Subsequently, an infectious cDNA clone of the genome Sabin 1 was prepared. Infectious cDNA clones may be used to preserve the vaccine quality of Sabin viruses and to provide a potentially unlimited supply of constant seed virus for polio vaccines.
WHO has emphasized that trial with new candidates that involve humans should be conducted only after the strains have been proved in laboratory and animal tests to be as safe as or safer than the Sabin strains and have shown to meet applicable current WHO requirements.
2.3. Improving thermostability of opv:
The oral poliovirus vaccine presently used requires storage under refrigeration temperature to maintain its thermostability. A considerable percentage of the total cost and effort of immunisation program relates to the creation and maintenance of a cold chain to ensure that vaccines are maintained under conditions that result in retention of potency to the point of administration. Cold chain conditions are defined by the requirements of the vaccine that is least stable, making it clear that efforts to improve the thermostability of vaccines in general must first be focused on OPV.
EPI advisory committee on the technical aspects of polio eradication identified the need for significant improvements in the thermostability of OPV as a critical success factor for the WHO's initiative for global eradication of poliomyelitis by the year 2000.
In 1991, the EPI set an ambitious target for an enhanced thermostable OPV; one that could survive 'a week in the pocket' that is 45°C for 7 days. However, in 1993, this target was relaxed to a vaccine that could withstand 37°C for 7 days (compared to 2 days with

the current vaccine). Acceptable potency after thermal challenge was defined as 2.4. Freeze drying:
Freeze drying & Lyophilization of poliovirus vaccine were the several different drying protocols examined. Trehalose (a.-D-glucophyranosyl-a.-D-gluophyronoside) was found to be of particular interest. However, the large initial loss in vaccine potency proved that these procedures were not suitable for OPV.
2.5. Pocket binding compounds as stabilisers:
Oxazolinyl isoxazoles (WIN compounds) and pyridazinamines (Jensen compounds), known as enteroviral compounds interact with the viral capsid thus stabilising the capsid structures. It was postulated that high concentration of these compounds bind and stabilise poliovirus against thermal inactivarion. When vaccine is administered the bound antiviral compound will elute (reversible binding) from viral capsid against steep concentration gradient effected by dilution. The vims thus regains infectivity and would immunise the child. The pocket binding antiviral compounds had undergone safety and pharmacological studies and were cleared for human application. It was found that the stabilized poliovirus antigenicity with these compounds is measured by methods that

detect intact viral antigens but the stabilisation of infectivity (vaccine potency) was not better than MgCl2.
2.6. Use of 1M MgCI2 as stabilizer:
So far accepted stabilizing agent for poliovirus has been 1 M MgCl? which provides some additional stabilization but is not sufficient to meet WHO specification of seven days at 37°C. Therefore, use of MgCl2 is further reinforced through a cold chain where the vaccine is stored at refrigerated temperatures from the point of manufacture to the point of administration.
3. PRIOR ART:
3.1 Heavy water as a thermostabilising medium
The stabilization of various microorganisms have been tried with heavy water since 1969
onwards. As early as 1969 by Y.B.Popov et al., have published the
paper "Effect of deuterium oxide on the heat resistance of staphylophage". There have
been various references available on use of heavy water for this purpose, which are listed
below.
1. Y.B. Popov et al. "Effect of deuterium oxide on the heat resistance of staphylophage" - Chemical Abstracts : vol. 70, no.21, 26 Mai 1969, Columbus, Ohio, US; abstract no. 94238d voir abrege & BIOFIZIKA vol. 14, no. 1, 1969 pages 186-187.
2. P.R.M. Steele et al. "Factors affecting the viability of freeze thawed T4 bacteriophage II. Influence of certain electrolytes on the degree of inactivation"- Chemical Abstracts: vol.72, no. 15, 13 Avril 1970, Columbus, Ohio, US; abstract no. 76039b, voir abrege & J. HYG. vol. 67, no. 4, 1969 pages 679-690.

3. K. Jung "Effect of heavy water on the thermal resistance of microorganisms" -Chemical Abstracts : vol. 71, no. 23, 8 December 1969, Columbus, Ohio, US; abstract no. 110159g, voir abrege & NUCL. SCI. ABSTR, vol. 23, no. 12, 1969 page 22242.
4. C.S. Pittendrigh et al. "Very rapid enhancement by deuterium oxide of the temperature-tolerance of adult dropsophila" - Chemical Abstracts: vol.80, no.23, 10 Juin 1974, Columbus, Ohio, US; abstract no. 130872y, voir abrege & PROC. NAT.ACAD.SCI. USA, vol.71, no.2, 1974.
5. V.Ya. Aleksandrov et al. "Effect of heavy water (water-D2) on the resistance of procollagen to thermal denaturation and to the action of collagenase" - Chemical Abstracts: vol. 83, no. 1, 7 Juillet 1975, Columbus, Ohio, US; voir abrege & DOKL. AKAD.NAUK.SSSR vol.220, no. 6, 1975 pages 1445-1448.
6. U.Lemm et al. "Stabilisation of enzymes and antiserums by heavy water" -Chemical Abstracts: vol. 95, no.5, 3 Aout 1981, Columbus, Ohio, US; abstract no. 37552u, voir abrege & EUR.J.BIOCHEM. vol. 116, no.3, 1981 pages 441-445.
7. U.Lemm et al. "Stabilisation of biochemically active proteins by heavy water " -Chemical Abstracts: vol. 95, no. 19, 9 November 1981, Columbus, Ohio, US; abstract no. 164216n, voir abrege & STUD BIOPHYS. vol. 84, no. 1, 1981 pages 69-70.
8. V.Ya. Aleksandrov "Stabilising effect of heavy water (D20) on the cell" - Chemical Abstracts: vol. 105, nOo. 17, 27 October 1986, Columbus, Ohio, US; abstract no. 14827lw, voir abrege & TS1TOLOGIYA vol. 28, no. 8, 1986 pages 790 - 795.

9. C.Heyde et al. "Heavy water (D20) protective effect on proteins in pharmaceuticals. For example: Human cholinesterase" - Chemical Abstracts: vol. 116, no.4, 27 Janvier 1992, Columbus, Ohio, US; abstract no. 28099b, voir abrege & Z. NATURFORSCH. Vol. 46, no. 9-10, 1991 pages 789-793.
10. EP, A, O, 332 826(TEVA) 20 September 1989 voir le document en entire.
Use of heavy water for stabilizing polio vaccine has been described in a paper published by Wu, R., Crainic, R., et el. "Thermostabilisation of live virus vaccines by heavy water (D20)", Vaccine 1995 Vol 13 No 12 ppl058-1063, and subsequently by Milstein, J. B., et al. "{Development of a more Thennostable Polio Vaccine ", Journal of infectious Diseases 1997. The authors (Crainic, R. & Simpson, K) of the above paper have applied for a patent bearing the publication no. WO 94/21298 dated September 29, 1994. In the invention claimed by them, heavy water at concentration less than 95% D20 is tried with and without 1M MgCl2 for finding its suitability on specific serotype -3 of OPV. .The said prior art is restricted to stabilization for type-3 vaccine which is stated to be the most thermo labile and limits the overall concentration carrier medium to about 87% using 95% heavy water.
It is well known that the polio vaccine is a mixture of three serotypes in a particular concentration, 106, 1050 & 1058 for the PV1, PV2 & PV3 per 0.1 ml respectively and as per the WHO recommendations, every dose of polio vaccine shall be administered in the above concentrations for immunization puiposes. This requires the stabilization experiments must be conducted when all the three species are present together in exactly the same compositions and not as single species i.e.,serotype 3, due to the interactive reactions. Importantly, the stabilization of WHO recommended vaccine comprising the three serotypes is complex as compared to the stabilization of single vaccine as that of the prior art due to obvious interactive reactions. Therefore the stability criteria as in the prior art for the individual serotype can neither be directly followed or extended to

achieve the required stabilization for the combination vaccine of WHO recommendations.
4. OBJECT OF THE INVENTION :
The basic object of the present invention is directed to provide a process whereby polio vaccine comprising mix of three serotypes as per the WHO recommendations can be produced in a stabilized form.
Another object 6f the present invention is to provide the desired stability to stand accidental breaks in the standard cold chain temperature cycle which consists of -20°C at the point of vaccine manufacture to 8°C at the point of administration.
Another object of the invention is to make the combination vaccine thermally stable upto 7 days at temperature of 37°C, upto 3 days at a temperature of 42°C and about a day at a temperature of 45°C.
Yet another object is directed to provide a process for manufacture of trivalent polio vaccine as per WHO acceptable limits which would be adapted to maintain potency of trivalent polio vaccines without acceptable limits without the maintenance of the cold chain.
5. SUMMARY OF THE INVENTION :
Accordingly, the present invention relates to an improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), comprising three serotypes PV1 [106 (TCID 50)], PV2 [105 (TCID 50)] and PV3 [1058(TCID 5o)] in

0.1 ml of OPV, minimum essential medium (Eagle) in Earle's salts(MEM) 0.95mg, tris buffer, and MgCl2 and heavy water, the process comprising
i) dissolving MEM powder 9.5g in heavy water 1000 ml;
ii) adding tris buffer to MEM powder solution obtained at the end of step (i), to
adjust pH (as seen by pH meter) between 6.5 and 6.8; iii) adding anhydrous/deuterated MgCl2 to MEM powder solution obtained at the
end of step (ii) sufficient to attain 1M MgCl2 strength in the MEM powder
solution to form a medium; iv) stuilizing said medium by filtration through not more than 0.22 micron
fMilhpore membrane filter;
v) taking xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three
serotypes PV1, PV2 and PV3 respectively individually at concentrations with
said sterilised medium and mixing them together and making the final volume
to 100 ml with said sterilised medium, wherein xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3 and 10yl, 10v2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively; vi) storing the vaccine prepared at the end of step (v) at not more than -20°C and
subsequently maintaining at different temperatures as per standard cold chain
temperatures thereby providing the desired stable trivalent oral polio vaccine
under standard cold chain temperatures.

6. DETAILED DESCRIPTION :
As mentioned in the summary of the invention, the present invention relates to an improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), wherein heavy water iised is from 90 % to 99.9 % D / (D +H) by weight, preferably 95.1 % to 99.9% D/(D +H) by weight and more preferably more than 99.8 % D/(D+H) by weight. In the present invention for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV) the deuterated MgCl2 is having 1-6 moles of D2O as water of hydration. This is in view of the fact that 1 M MgCl2 contains light water molecules as water of hydration (MgCi2.6H20) resulting in overall dilution of D20 from 95% to 87%.
The process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV) described as herein in the text and in the examples is useful for other similar biological preparations also.
6.1 Example:
The invention will now be illustrated with the help of an example. The example is by way of illustration only and in no way restrict the scope of the invention.
Material and methods:
6.1.1. Cell culture:
Hep-2, a continuous cell line of human carcinoma of larynx was originally obtained from National Institute of Biological Standards and Control (NIBSC) Potters Bar, England. The cells were preserved in the Cell bank.

Minimum Essential Medium (Eagle) in Earle's salts (MEM) was used. Powdered medium was obtained from commercial sources (GIBCO/BRL) and prepared as per the manufacturer's instructions. The medium was supplemented with l-glutamine, antibiotics and foetal bovine serum (FBS). Cell growth medium contained 5% FBS whereas cell maintenance medium contained 2% FBS. Cultures were grown in 80cm and 175cm disposable tissue culture flasks as required. The cell culture procedure followed is briefly described below.
Maintenance of cell line:
1. A cell culture flask (Hep-2 cells) with a confluent monolayer of cells was used to
initiate new cultures.
2. The used culture medium was decanted (discarded) the from the flask and washed the
monolayer with 5 ml of phosphate buffered saline (PBS).
3. Added 5 ml of pre-warmed (37°C) trypsin-EDTA solution and incubated the flask for 3 min.
4. Decanted (discarded) trypsin and continued the incubation until cells started to detach from the surface (usually 5min).
5. Tapped the flask to detach most of the cells and then added 5ml of growth medium. Gently resuspended the cells with the aid of a Pasteur pipette.
6. Using a haemocytometer obtained the cell count (cells per ml) of the cell suspension.
7. Labelled new sterile TC flasks with the details of the cells i.e., cells line, sub-culture number and date.
8. Added growth medium (80 mL for 80Cm2 flasks). .

9. Seeded at least 2.5 x 106 cells in each flask (80cm2) to initiate new cultures. Incubated the flasks at 37°C and observe daily under an inverted tissue culture microscope for cell growth. When the cell monolayer was formed (after 4 to 5 days), discarded the medium from the flasks and replenished it with maintenance medium.
10. Initiated fresh (new) cultures at every 7-8 days interval.
Note: Cells from step No. 5 were used for various experiments.
6.1.2. Poliovirus:
Live attenuated oral poliovirus vaccine strains (poliovirus type 1,2 and 3) were originally obtamed fromCommercial units at the S0+3 level (i.e.^ vaccine) ,A stock was maintained in the laboratory. The virus was passed in Hep-2 cells for use in all experiments relating to this project.
All procedures requiring virus handling were performed in bio-safety cabinets.
6.1.3. Heavy water:
Heavy water (D20 >99.8% D/(D+H) by weight%) was made available from the operating plants of Heavy Water Board of the Department of Atomic Energy. The heavy water used was virgin heavy water as it was extracted from the natural occurring substances like water.
MEM powder was dissolved to make tissue culture medium. pH of the medium was adjusted by 1M Tris base prepared in D20.

MgCl2 powder (desiccated) was dissolved in heavy water to obtain IM concentration in the final volume of the solution. The final volume was adjusted by adding milli Q purified heavy water after dissolving MEM and adjusting pH by IM Tris base. All solutions were sterilised by filtration through 0.22ji (pore size) Millipore membrane filters.
6.1.4. Preparation virus stock:
1. Confluent monolayer culture of Hep-2 cells in 175 cm2 TC flasks were used. One TC flask was used for each poliovirus type.
2. The cell culture medium was discarded.
3. 0.5ml of polidvirus suspension (108 0TCID5o/ml) was inoculated to the flask for initiating virus infection. The flasks were incubated at 37°C for 1 h for virus adsorption.
4. 40ml maintenance medium was added to the flasks. The culture flasks were further incubated at 37°C for virus growth.
5. The flasks were observed microscopically daily for virus induced cytopathogenic effect (CPE). When all cells showed CPE, the flasks were frozen (-20°C) and thawed to release all virus from the cells. The culture medium was collected (harvested) and centrifuged at 5000Xg for 15rnin to remove cell debris. The clear supernatant containing the virus was distributed in 2ml aliquots and stored at -20°C until used.
6.1.5. Virus titration:
Live virus content of a virus suspension is expressed either as plaque forming units or tissue culture infective doseTCID50 per ml. The micro-titration method was used in all experiments.

The following materials were used.
1. 96 well flat bottom tissue culture plates (Sterile)
2. Disposable screw capped tubes (5ml, polyethylene, sterile)
3. Disposable micro-tips (sterile, lOOjxl and lOOOfxl) ,
4. Variable volume single channel pipettes (Eppendorf type)
5. Serological pipettes.
6. Dropping pipettes 50^,1 per drop
7. Hep-2 cell suspension
8. Tissue culture media
For each test a worksheet was prepared for recordrngtbe results, Preparation of a 10-fold virus dilution series was done and proceeded as given below,
1. For each virus suspension to be titrated, labelled eight tubes (5ml) with the dilution step 10'1 to 10"8.
2. Dispensed 0.9ml maintenance medium in each tube using a sterile pipette/ pipette and disposable microtip.
3. Using a disposable micro-tip and a pipette transferred 100JJ.1 of virus suspension from the stock to the tube marked 10"1, discarded the tip and mixed the contents of the tube.
4. Using a fresh micro-tip transferred 100}j,l of virus suspension from 10" tube to the second tube marked 10"2.
5. Repeated the procedure till the 10~8 dilution was prepared taking the precaution to use a fresh tip for each step and thorough mixing of the dilutions prepared.
6. Labelled required number of microtitre plates with identification number and date of the experiment.
7. 10"3 to 10"8 dilutions were required for titration.

8. Using a disposable tip added lOOixl of the 10~3 dilution in the wells Al to A10 of the microtitration plate.
9. Using fresh tip continued to add lOOpl virus suspension of the 10-fold dilutions in 10 wells of each row of the plate until the 10*8 dilution is added.
10. Added lOOpil of medium to wells All to H12 (cell control wells).
11. Prepared Hep-2 cell suspension by trypsinization of a confluent monolayer culture. Adjusted the cell count to 105 cells per ml.
12. Added 2 drops (IOOJJI) of cell suspension in each well using the dropping pipettes. Also seeded cell control wells wherein lOOul of medium (no virus) was added. Replaced the plate cover.
13. Incubated the plates at 37°C in a CO2 incubator (5% C02 in air).
14. Observed the plates under an inverted TC microscope to record virus induced CPE on days 5 and 7 of incubation.
15. Calculated virus titre by using the Karber's formula. Expressed virus titre as TCID50 per ml.
6.1.6. Karber's formula:
Virus titre is determined by determining the dilution of the virus suspension that produced CPE in 50% of the cultures. A reciprocal of this dilution is the virus titre in the stock suspension.
Log TCID50 = L - D (S - 0.5)
Where,
L = lowest dilution used (log),
D = difference between dilution steps (log),
S = sum of proportion of CPE positive wells at each dilution.

6.1.7. Preparation of experimental vaccine:
The heavy water concentration used for preparation of the vaccine was more than 99.8 %by weight D/(D+H).
1. Obtained the virus stocks of vaccine strains of poliovirus type 1,2 and 3 of known titre.
2. A trivalent ORV contains the three serotypes in different quantities. Poliovirus type 1 (lO^TCIDso), poliovirus type 2 (10?0TCID50) and poliovirus type 3 (1058 TCID50) per human dose of 0.1ml.
3. Calculated the volume of the stock virus suspension needed to give the required titre of the poliovirus serotype in the trivalent vaccine preparation.
i) For example, the titre of the stock poliovirus type 1 was 108 5 TCID5o/ml and 100 ml vaccine was being prepared. 3.16ml of the stock diluted to 100 ml will have poliovirus type 1 titre of 106 TCID50 per 0.1 ml.
ii) The three viruses were mixed in the required proportion to obtain correct blend of the vaccine.
The dilution of the stock solution and its blending with the sterilized medium was done as mentioned below.
xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three serotypes PV1, PV2 and PV3 respectively were taken individually at the concentrations and mixed them together. The same was made up to the final volume of 100 ml with sterilized,pH adjusted and MgCl2 added MEMmedium, heavy water, wherein

xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3
and 10yl, 10y2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively;
iii) Storing the vaccine prepared at the end of step (v) at not more than -20°C and
>'■' '■...•'.-,. ■'
subsequently mamtaining at different temperatures as per standard cold chain
temperatures
6.1.8. Determination of concentration of individual virus component in the tnvalent polio vaccine:
Principle: Determination of individual serotype concentration in trivalent polio vaccine was based on specific neutralisation of two of the three virus using serotype specific anti-poliovirus antibodies. For example, neutralisation of poliovirus type 2 and 3 by a combination of anti-poliovirus type 2 and anti-poliovirus type 3 antisera permitted the titration of poliovirus type 1.
Antisera: Good quality monospecific antisera of known titre against the three types of polioviruses were used for analysis of trivalent polio vaccine. The dilutions of these antisera used in preparation of combination sera for neutralisation of poliovirus serotypes was determined by experiments in advance.

Procedure:
1. Antisera combinations were prepared as indicated below. The aliquots were
made,frozen and stored for ready use.
Anti-PVI (1:2000) + Anti-PV2 (1:4000); Anti-PVl (1:2000) + Anti-PV3 (1:2000); Anti-PV2 (1:4000) + AntiPV3 (1:2000).
1 "7
2. Diluted bivalent vaccine sample 10" to 10".
3. Two microtitre plates were required for one sample and labelled the plates.
4. Added 50[xl of antiserum combination (anti-P2+P3) in first set of 6 rows of 8 wells.
5. Added 50μ1 of antiserum combination (an.ti-Pl+P3) in the second set of 6 rows of 8 wells.
6. Added 50μ1 of antiserum combination (anti-Pl+P2) in me third set of 6 rows of 8 wells.
7. Added 50μl of tissue culture medium (no antiserum) in the fourth set of 6 rows of 8 wells.
8. Added 50μ1 of diluted trivalent vaccine 10" to 10" dilution to the sets of 6 rows. Mix and incubate at 37°C in a C02 incubator for 90 min.
9. Prepared sufficient quantity of Hep-2 cell suspension (105 cells per ml). At the end of the incubation added 100Μ.1 Hep-2 cells in each well, mixed and returned to the incubator.
10. Observed the plates after 7 days under an inverted microscope and recorded CPE reading.

11. Calculated virus titre in each of the four sets of titration using Karber formula. The 1st set gave value of poliovirus type 1, the second for type 2, the third type 3 and the fourth set indicated a combined titre for the trivalent preparation (total virus content).
12. Virus titre was expressed as titre per human dose (0.1ml).
6.1.9. Estimation of magnesium chloride concentration:
Concentration of magnesium chloride in MEM was determined by titration of a small aliquot of the medium against a standard solution of EDTA.
i."'5WAmmonia: Dissolve 37.5ml ammonia in 100 ml heavy water. r
2. Ammonia-ammonium chloride buffer,

NH4C1 5.4g
5N Ammonia 70.0 ml
Milli Q purified heavy water To 100 ml
3. EDTA(0.05M).

EDTA disodium 18.6g
Milli Q purified heavy water To 1000 ml
4. Indicator: solochrome black. Procedure-Diluted 2 ml of the test medium with 10 ml distilled water. Added 5ml ammonia-NR4Cl buffer.

Added a small quantity (a pinch) of solochrome black powder as the indicator.
Titrated the solution against 0.05 M EDTA solution (in burette) to obtain an end point
which is from wine red to blue colour.
Molarity of MgCl2= Volume of EDTA X Molarity of EDTA r!
Volume of test medium
The vaccine samples prepared as described above were exposed to temperature at 37 ° C. Samples of vaccine samples were withdrawn at different time intervals of exposure. The amount of live virus was determined by titration in cell cultures. The difference between the titre of virus suspension before and after exposure to heat was a measure of virus inactivation. The results were compared with heat inactivation of poliovirus suspensions;, in medium containing 1M MgCl2 prepared in H20, similar to the OPV being currently used.
6.1.10 Estimating mean titre and variation:
The samples of OPV prepared in heavy water of the above said purity and that prepared in normal water were taken for analysis.
The poliovirus suspensions were distributed in 1ml aliquots in 2ml screw-capped vials and stored at -20°C until used. To determine the virus concentration in these suspensions, samples were titrated in Hep-2 cells. To determine inter-experiment variation each suspension was titrated ten times.
Briefly, the virus suspension was serially diluted ( 10"1 to 10~8) in tissue culture medium. IOOJLXI of each dilution ( 10"2 to 10"8) were inoculated in 8 wells of a microtitre plate. 100(il of Hep-2 cell suspension (105cells/ml) were then added. The plates were incubated at 37°C in a C02 incubator. The plates were observed under an inverted TC microscope
22

to record CPE reading on the 7 day of incubation. Virus titre was calculated according to Karber's formula.
Ten experiments were carried out on poliovirus types 1, 2 and 3 and the titres were used to calculate mean number of infectious unit (titre) and also the 95% confidence limits (2s.d.). The maximum variation in these assays were about 0.2 Hog, well within the fiducial limits of assays of 0.5 log allowed by the WHO for vaccine titration. In each of the experiments carried out using the above mentioned virus suspensions the above mean titres were used for validation. Vials stored at -20°C were used as internal control in all the experiments. An experiment was considered, as valid if the control vial titre was within the predetermined limits.
6.2 Results and discussions:
The results are as shown in figure below. It can be clearly inferred that the product of example 1 is more stable at 37 ° C than that of the conventional vaccine. It can be noted that the loss in potency is upto 0.5 loglO at 37 °G for a period of 7 days for the vaccine made with heavy water of 99.8 % concentration while the loss of potency for the conventional vaccine prepared in normal water over the above value after two days itself. Thus, the potency loss for the vaccine prepared in example 1 can be seen to have a lojfs of potency within the acceptable limits while that of the normal vaccine is far away from the same after the storage for one day itself indicating the advantages of the new process.

It may be noted that the above said vaccine samples prepared in heavy water and that prepared in conventional normal water were exposed to 42°C and 45°C temperatures also and the loss of potency after exposure to different time periods were found. While the loss of potency of the trial vaccine at 42°C was within acceptable limits over 3 days and 45°C was over a day. The same for normal vaccine was much less than this.
The trivalent vaccine prepared as the above said process indicated a marked improvement over the case illustrated for the monovalent virus strain PV3 in the patent publication mentioned in prior art of the present invention. The marked improvement in the thermostabilisation observed in the vaccine prepared by the present invention is due to the new process used in the present invention for preparing the trivalent OPV using heavy water of more than 99.8% D/(D+H) by weight.

As would be evident from the above disclosure the object of the present invention of providing stability to trivalent oral polio vaccine is surprisingly achieved by selective reduction of light water content of the vaccine to maximum extent which is not taught in any of the available prior art.
7. ADVANTAGES ;
The principle advantage of the present invention is to obtain a thermostable triple polio vaccine which is stored at -20°C and subsequently maintaining at different temperatures as per standard cold chain temperatures ;and giving stability by retaining the potency of the vaccine against accidental breaks intiie cold chain. The vaccine prepared by the new invention makes it thermally stable upto ? days at temprature of 37ifc» upto 3 days at a temperature of 42°C and about a day at a temperature of 45°C, The vaccine manufactured using the invention maintains the potency of trivalent polio vaccine as per WHO acceptable limits without the maintenance of cold chain. Other advantages of the vaccine prepared by the new process is given below.
1. The vaccine prepared by the process of present invention is having more stability with respect to higher temperature as compared to the presently available vaccine prepared in normal water.
2. The process helps in retaining the potency of the vaccine to the acceptable levels despite breakage in the cold chain. The existing vaccine is not stable at 45 °C beyond few hours while the thermostabilised vaccine can allow breakage in the cold chain for over a day, even at 45 °C which is highly advantageous to tropically located developing countries like India.
3. The thermostabilized vaccine meets with the WHO recommendations for the OPV for 7 days storage at 37 °C.

4. The vaccine prepared by the present invention having improved thermostability will be very helpful in the set goal of complete polio eradication by 2005 particularly in India.
5. The improvement brought in by usage of heavy water of higher purity in the therrno labile nature of biological preparations such as polio vaccine can be gainfully utilized in other fields also.

8. CLAIMS:
We Claim
1. An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV), comprising three serotypes PV1 [106 (TCID 50)], PV2 [105 (TCID 50)] and PV3 [105 8(TCID 50)] in 0.1 ml of OPV, minimum essential medium (Eagle) in Earle's salts(MEM) 0.95mg, tris buffer, and MgCl2 and heavy water, the process comprising
i) dissolving MEM powder 9.5g in heavy water 1000ml;
ii)i adding tris buffer to MEM powder solution obtained at the end of step (i), to
adjust pH (as seen by pH meter) between 6.5 and 68; iii) adding anhydrous/deuterated MgCl2 to MEM powder solution obtained at the
end of step (ii) sufficient to attain 1M MgGl2 strength in the MEM powder
solution to form a medium; iv) sterilizing said medium by filtration through not more than 0.22 micron
Millipore membrane filter; v) taking xl ml, x2 ml and x3 ml of polio virus stock suspensions of said three
serotypes PV1, PV2 and PV3 respectively individually at concentrations with
said sterilised medium and mixing them together and making the final volume
to 100 ml with said sterilised medium, wherein
xl=10 E08/10yl x2=10 E08/10y2l x3 =10 E08/10y3
and 10yl, 10y2 and 10y3 are strength of said polio virus stock suspensions of three serotypes PV1, PV2 and PV3 respectively;

vi) storing the vaccine prepared at the end of step (v) at not more than -20°C and subsequently mamtaining at different temperatures as per standard cold chain temperatures
2. Afi! improved process for manufacturing stabilized biological^preparations such as trivalent oral polio vaccine (OPV),as claimed in claim 1, wherein heavy water used is from 90 % to 99.9 % 0 / (0 +H) by wt.
3. An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV),as claimed in claim 1 or 2, wherein heavy water used is from 95.1 % to 99.9% D/(D +H) by wt.
4. An improved process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV),as claimed in claim 1-3, wherein heavy water used is from more than 99.8 % D/(D+H) by wt.
5. A process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV),as claimed in any claim 1-4, wherein deuterated MgCl2 is having 1-6 moles of D20 as water of hydration.
6. A process for manufacturing stabilized biological preparations such as trivalent oral polio vaccine (OPV) substantially as herein described in the text and in the example^.
****************

Date : 8.2.2001

APPLCANT'S NAME : Dept. of Atomc Energy Govt, of India APPLICATION NO. : 158/MUM/2001

TOPV H-Mg AT 37 C

TOPV 100 D-Mg AT 37 C

06 , D.V i-
1.2 v

-^3— PV1 ; :
- a PV2 '■! ~*~- PV3 i

rs —-n-
/"
W
0.2 t— ^—■
ot J , :-&>L
0 > &-- ■■■££?—"S n
0 2 4

—»- -PV1
..«. PV2
- A-- PV3

TOPV 100 D-Mg AT 42C

TOPV 10OD-Mg AT 45C

—e— PVI
-S- PV2

Dated this 12th day of February 2001

K

>THA
SIDDHARTHA NAG Of S.MAJUMDAR&CO Applicant's Agent

Documents:

158-mum-2001-cancelled page(12-2-2001).pdf

158-mum-2001-claim(granted)-(12-2-2001).pdf

158-mum-2001-claims(granted)-(12-2-2001).doc

158-mum-2001-correspondence(1-6-2005).pdf

158-mum-2001-correspondence(ipo)-(12-1-2004).pdf

158-mum-2001-form 1(12-2-2001).pdf

158-mum-2001-form 19(30-10-2003).pdf

158-mum-2001-form 2(granted)-(12-2-2001).doc

158-mum-2001-form 2(granted)-(12-2-2001).pdf

158-mum-2001-form 3(12-2-2001).pdf

158-mum-2001-power of attorney(12-2-2001).pdf

158-mum-2001-power of attorney(28-7-2003).pdf

« Previous Patent

Next Patent »

Patent Number

197836

Indian Patent Application Number

158/MUM/2001

PG Journal Number

40/2008

Publication Date

03-Oct-2008

Grant Date

16-Jan-2006

Date of Filing

12-Feb-2001

Name of Patentee

DEPARTMENT OF ATOMIC ENERGY

Applicant Address

GOVERNMENT OF INDIA, ANUSHAKTI BHAVAN, CHHATRAPATI SHIVAJI MAHARAJ MARG, MUMBAI

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. SONDE RAMAKRISHNA RAMANATH	CHAIRMAN & CHIEF EXECUTIVE, HEAVY WATER BOARD, V-FLOOR, VIKRAM SARABHAI BHAVAN, DEPARTMENT OF ATOMIC ENERGY, GOVERFNMENT OF INDIA, ANUSHAKTINAGAR, MUMBAI 400094,
2	DR. DESHPANDE JAGADISH MOHANRAO	DIRECTOR, ENTEROVIRUS RESEARCH CENTRE, ICMR, HAFFKIN INSTITUTE COMPOUND, PAREL, MUMBAI 400014
3	MR. KAMATH HIREBETTU SADANANDA	CHAIRMAN & CHIEF EXECUTIVE, HEAVY WATER BOARD, V-FLOOR, VIKRAM SARABHAI BHAVAN, DEPARTMENT OF ATOMIC ENERGY, GOVERFNMENT OF INDIA, ANUSHAKTINAGAR, MUMBAI 400094
4	MR. MUNDAYUR BHASKARAN	SCIENTIFIC OFFICER-G, HEAVY WATER BOARD, V-FLOOR, VIKRAM SARABHAI BHAVAN, DEPARTMENT OF ATOMIC ENERGY, GOVERFNMENT OF INDIA, ANUSHAKTINAGAR, MUMBAI 400094

PCT International Classification Number

N/A

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA