Study On The Key Control Points Of H9 Avian Influenza Vaccine Production By Serum-free Suspension Culture Of MDCK

Avian influenza not only endangers poultry raising,causing enormous economic losses,but also seriously threatens human health.At present time,vaccination is the most effective preventive means.Avian influenza vaccine has been produced by chicken embryo for a long time,and a lot of defects existed in the process.However,animal cell culture is gradually recognized worldwide for its significant superiority.Especially the suspension cell cultured in serum-free medium is easy to scale-up and could sustain high cell density culture with lower cost and higher stability,becoming the main direction of influenza vaccine production.Currently,there is no H9 subtype influenza vaccine product manufactured by serum-free suspension cell culture in China.Therefore,it is necessary to study this process and establish high virus yield process to fit industrial production.The thesis analyzed the problem from two extreme processes:batch process and centrifugation process.There were three main conditions that might result in huge yield difference,which were nutrition,metabolic byproducts,and environment.Although centrifugation process led to high yield,it was hard to operate in industrial production.In this research,three main different conditions between two processes were considered as guides to improve batch process and promote virus yield.For the lack of nutrition in batch process,various nutrients were added to batch process to explore the effects on promoting virus titer.Results showed that adding glucose or glutamine alone could not promote virus titer;adding feeding medium could reach 2^10.3 HAU/25?l with complex operation;adding original culture medium could reach 29.0 HAU/25?l with simple operation,and higher titer might be realized through parameters adjustment.For the accumulation of metabolic byproducts in batch process,lactate or ammonium were added to centrifugation process on the base of abundant nutrition to explore the influence of byproducts on virus production.Results showed that neither the lactate below 40 mM nor the ammonium below 8 mM at infection time could affect maximum virms titer.For the different environment of virus production phase between batch and centrifugation processes,pH value and temperature were optimized.Results showed that pH?6.0 would lead to the drop of HA titer,and suitable pH in virus production phase might be controlled as 7.0±0.2.Furthermore,properly lowering temperature?33-35??was beneficial to virus production.Summarizing the research of conditions above,the process with dilution and temperature-down was established.In the bioreactor tests,the process could reach HA titer as 2^10.6±0.2 HAU/25?l,and its cell-specific virus yield achieved to?16060±786?virions/cell,which exceeded the centrifugation processes.This process was efficient and easy to operate,providing basal data support for industrial production of influenza vaccine.