Development Of The Fed-batch Process For The Efficient Production Of Influenza Vaccine By MDCK Cells

Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. The majority of vaccine doses are being produced in embryonated chicken eggs. While egg-based influenza vaccines have a proven safety and efficacy record, their manufacturing is associated with severe limitations. These limitations include complex logistics for the supply of millions of embryonated eggs, constrains in scale-up, and low yields for some strains. Cell culture-based processes with highly susceptible mammalian cell lines have become an important alternative to embryonated chicken eggs. Nowadays MDCK cells microcarrier culture system has been successfully industrialized. But this system involves high cost and complicated operation, which makes it difficult to achieve real large-scale production. Meanwhile, it takes such a long term for MDCK cells to adapt suspension culture by using the existing commercial serum-free medium, which also ultimately affect the final virus yield. Thus, it is urgent to develop a serum-free medium for MDCK suspension cells and an economic and efficient fed-batch culture process.Therefore this research takes MDCK cells for influenza virus cultivation as the research object. Firstly, the five backup medium and additives were mixed according to the mixture design, which is one of the technologies of DOE. The experiment results showed that the new medium XP gained 82.40% higher the maximum viable cell density value and IVCC increased by 74.40% compared to the original serum-free medium SFM. At the same time, simple methods were used to make MDCK cells adapt suspension culture and stable growth for only 3 weeks. The maxium viable cell denity of new suspension cell line MDCK-S2 cells reached (14.05±0.07)×106 cells/mL during batch culture while the average specific growth rate during exponential growth phase was 0.63 d-1.Secondly, based on the experimental results in last chapter, further research was carried out on the MOI and trypsin concentration effects on cell growth and influenza virus production. The results showed that as MOI equaled 0.01 and trypsin concentration was 5.0 μg/mL, it could both guarantee strong infectious ability and high yield of influenza virus.Finally, in order to solve the problem of batch culture nutrition limitations, two kinds of serum-free medium were used as nutrition additives:the basic medium XP and precisely designed medium FM. The results showed that using 1.0×106 cells/mL as seeding cell density, then adding 100% the volume of medium XP at 72 h obtained the highest HA titer 21300±0.00 HAU/50 μL and the best single virus yield (47224±35) virions/cell. Compared with batch progress, HA titer increased by 3 HA units while svy improved by 168%.Through the research work, an economic and efficient fed-batch culture process was rapidly established and laid the foundation for the efficient industrial production of influenza vaccines. In addition, the established fast and efficient development method will also provide beneficial reference to other animal cell-based influenza vaccine development.