The Optimization Of Manufacturing Process And Verification Technology During The Preparation Of Vero Cells Of JE Inactivated Vaccine

Objective:In order to improve the quality of products, manufacture more economical, safer and more effective Japanese Encephalitis(JE) vaccine, we put it into practice from two aspects, production process and Verification technology. One is to simplify the production process, optimize all steps of the production process of the vaccine, choose the simplest, cheapest and the most effective ways, making the JE vaccine more safe, effective, and cheaper. The other aspect is to improve the verification technology, find the most suitable detection method, reduce interference factors, making the detection method simpler than before and the condition be controled easilier. We also formulate strict rules for operational procedures, all this make the test results close to the truth and reflect the real situation of the vaccine. Manufacturing a safer JE vaccine for human is our goal.Methods:1.Recovery the Vero cells, then cultivate and propagate in cell culture bottle, in the end establish the main cell bank(MCB) and working cells bank(WCB).2.Vero cells is infected by the P3 strain of JE virus of mouse brain, then prepare JE virus P3 strain of Vero cells (P3MV strain), and establish the Main Seed Lot(MSL) and Working Seed Lot(WSL).3.Vero cells is infected by the JE virus P3 strain of Vero cells and cultivated under optimum temperature. Harvest the virus solution, when the cytopathogenic effect(CPE) reached +++～++++. 4.The harvested virus solution was combined, inactivated, concentrated, purified, and finished product by diluent subpackaged and freeze-dried.5.All steps of the preparation of vaccines, whether semi-finished products or finished products should be verified in accordance with the requirements of "Chinese Pharmacopoeia three" 2005 Edition.Results:1.After cultivating and propagating Vero cells, establishing vero cells 131 as the MCB, an 135 as WCB.2.Cultivating JE virus P3 strain of Vero cells, after testing the virus has steady virulence and good immunogenicity. For all popular strains of the virus have a good protection. Establishing 5 as the generation of MSL, and 8 as WSL.3.Harvest virus solution, observe the cells every day after being infected virus, when the cytopathogenic effect(CPE) reached +++～1++++. Harvest virus solution once a day, for 3 to 5 days and add formaldehyde in combined virus solution for inactivating virus.4.Concentrate inactivated virus harvest fluids into 50 times by ultrafiltration, when the verification test of inactivation was qualified.5.Purify vaccine by gel chromatography, than remove unwanted proteins, and reduce the incidence of adverse reactions.6.The purified liquid was diluted and subpackaged, or lyophylized vaccine as final product.7.Finally, inspecting the quality of finished product in accordance with the requirements of Pharmacopoeia through comprehensive test.Conclusion:1.Use Vero cells as the production matrix of JE vaccine, the feature of cytological is stable. There is no risk of pollution and tumorigenicity by exogenous factors. The Vero cells was easy to cultivate, it could be used for a long time because of its high passages.2.Select inactivated process to prepare JE vaccines. Use formaldehyde as the classical inactivating agent inactivated JE virus, the concentration of formaldehyde is 1:2000 at 4℃for 28 days. The testing of inactivation validation is cell culture-animal. 3.Purify vaccine by gel filtration with sepharose 6FF. Its separation conditions are mild. It has a good reproducibility, and a high recovery.4.Use cells plaque method rather than mouse method for testing virus titer. Because the cells plaque method has several fortes, such as short test cycle, simple procedure and low cost.