Abstract:

This work aims to increase the production efficiency of influenza vaccine,i.e.,overcome the current drawback of low production,and to meet the growing market demand for influenza vaccines and be full preparedness to pandemic outbreaks. Process development was carried out using MDCK suspension cells by virus adaptation,extended batch cultivation to evaluate cell growth and virus production,semi-perfusion to verify the feasibility of MDCK high cell density cultivation,and ATF perfusion process verification in the bioreactor. Faster infection with adapted seed virus contributed to high virus titer of（3.57±0.17）log₁₀（HAU/100 μL）in extended batch cultivation. The high cell concentration over 40×10⁶ cells/mL and high virus titer over 4 log₁₀（HAU/100 μL）in the semi-perfusion model proved the feasibility of high cell density cultivation using MDCK suspension cells. MOI and trypsin concentration presented significant impacts on the cell growth and virus propagation during the infection phase. Similar cell growth profile and higher virus titer of 4.37log₁₀（HAU/100 μL）in the ATF-based perfusion process were obtained in the bioreactor;and comparable cell-specific virus yield（CSVY）to the batch cultivation was maintained by implementing the temperature reduction strategy during infection phase,which overcame the “cell density effect”. In conclusion,the platform of producing influenza virus based on the MDCK suspension cell ATF perfusion process is established,and it enhanced the influenza virus productivity by increasing the cell density and CSVY,which provides a new option for the cell-based industrialization of influenza vaccine.

Key words: MDCK cells, influenza virus, perfusion process, semi-perfusion