基于MDCK悬浮细胞灌注培养的流感病毒生产过程开发

Perfusion Process Development of MDCK Suspension Cells for Influenza Virus Production

为了提升用MDCK悬浮细胞培养生产流感疫苗的效率,克服目前存在的产能低下问题,以期满足日益增长的流感疫苗市场需求和提升应对大流行爆发的能力。通过病毒驯化、稀释流加条件下的细胞生长和产毒能力分析、Semi-perfusion中的细胞高密度培养可行性分析以及ATF灌注反应器过程验证完成过程开发。驯化的病毒加速了其在细胞内的感染扩增,且在稀释流加培养中获得(3.57±0.17)log10(HAU/100μL)的病毒滴度。Semi-perfusion可实现细胞高密度培养至40×106 cells/mL以上,病毒产量达4 log10(HAU/100μL)以上,同时发现MOI和胰酶浓度对病毒的复制过程存在显著影响。在反应器中的ATF灌注培养过程得到了与Semi-perfusion相近的细胞密度及更高的病毒产量4.37 log10(HAU/100μL),并且通过降温措施维持了高密度下的单细胞病毒产量,克服了“细胞密度效应”。建立了基于MDCK悬浮细胞ATF灌注培养的流感病毒生产平台,通过同时提高细胞密度和单细胞病毒产率提升了流感病毒的生产效率,为基于细胞培养的流感疫苗产业化生产提供了新选择。

This work aims to increase the production efficiency of influenza vaccine,i.e.,overcome the current drawback of lowproduction,and to meet the growing market demand for influenza vaccines and be full preparedness to pandemic outbreaks.Processdevelopment was carried out using MDCK suspension cells by virus adaptation,extended batch cultivation to evaluate cell growth and virusproduction,semi-perfusion to verify the feasibility of MDCK high cell density cultivation,and ATF perfusion process verification in thebioreactor.Faster infection with adapted seed virus contributed to high virus titer of(3.57±0.17)log10(HAU/100μL)in extended batchcultivation.The high cell concentration over 40×106 cells/mL and high virus titer over 4 log10(HAU/100μL)in the semi-perfusion modelproved the feasibility of high cell density cultivation using MDCK suspension cells.MOI and trypsin concentration presented significant impactson the cell growth and virus propagation during the infection phase.Similar cell growth profile and higher virus titer of 4.37log10(HAU/100μL)in the ATF-based perfusion process were obtained in the bioreactor;and comparable cell-specific virus yield(CSVY)to the batchcultivation was maintained by implementing the temperature reduction strategy during infection phase,which overcame the“cell densityeffect”.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-basedindustrialization of influenza vaccine.