Research On The Production Process Of Vero Cell Influenza Live Attenuated Vaccine

Currently,influenza vaccines in the market are mainly three kinds of inactivated virus vaccines,live attenuated virus vaccines and recombinant HA vaccines.Influenza virus attenuated live vaccine is generally immunized by intranasal inoculation to induce mucosal immune and cellular immunity,and achieve the same effect as natural infection process.In this study,Vero cell cold adapted strain A/Yunnan/1/2005 Vca was selected as the female parent strain.Through the exploration of various physical and chemical factors in the cultivation process,we optimized the screening conditions for the proliferation of influenza virus on Vero cells.The yield of virus was determined by measuring the TCID50 and coagulant titer of virus harvested liquid as the index of virus production.At home and abroad,cell factory culture technology has been a very mature and widespread technology of large number of cultured cells in vitro.Using cell factories to amplify the production scale of cells and viruses can provide a basis for mass production.This study used optimized conditions to amplify influenza virus into cell factories to improve production efficiency.The aim of this study is to reduce impurities,concentrate viruses,isolate viruses and impurities,through the purification process of centrifugal clarification,ultrafiltration,nuclease treatment and column chromatography through the culture of influenza virus cold adapted strain of Vero cells.In this study,through the comparison of two chromatography purification processes,the process of purification of influenza virus by Benzonase nuclease and Sepharose 4 Fast Flow was finally determined.The residual DNA,BSA content and protein content of Vero cells in the final virus purify solution are less than the prescribed value of the Pharmacopoeia,and meet the quality requirements for the preparation of the vaccine by Vero cells.To sum up,this study solved the problem of low yield of influenza virus in Vero cells,and determined the best culture condition of Vero cells as the substrate for the culture of influenza attenuated cold adaptable strain.In this study,a two step purification method was used to purify influenza virus from Vero cells.The virus has high recovery rate,and the inactivated influenza vaccine prepared is safe and effective.