生物反应器微载体Vero细胞罐外消化放大培养对狂犬病病毒CTN-1Ⅴ株产毒能力的影响  

Effect of bioreactor scale⁃up culture of micro⁃carrier Vero cells after extra⁃tank trypsinization on virus⁃producing ability of rabies virus CTN⁃1V strain

作　　者：杨敏 宋远涛 王玲 徐晓 文聪 陈杰杉 王月莉 梁婧 YANG Min;SONG Yuantao;WANG Ling;XU Xiao;WEN Cong;CHEN Jieshan;WANG Yueli;LIANG Jing(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207

出　　处：《中国生物制品学杂志》2024年第5期532-536,共5页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2021YFC2600200).

摘　　要：目的探讨微载体Vero细胞从30 L生物反应器经罐外细胞消化放大培养至300 L生物反应器对狂犬病病毒(rabies virus,RABV)CTN-1Ⅴ株产毒能力的影响。方法将第140代Vero细胞于37℃培养72~120 h后,按1∶4的细胞密度比传代扩增至10层细胞工厂,继续培养72~120 h;将单层致密细胞消化后接种至30 L生物反应器,微载体7~10 g/L,培养温度37℃,pH(7.0~7.4),溶氧30%~80%,搅拌速度10~50 r/min,连续灌流培养72~120 h,共培养3批;微载体Vero细胞经罐外细胞消化后放大培养至300 L生物反应器,微载体5~8 g/L,培养温度37℃,pH(7.0~7.4),溶氧30%~80%,搅拌速度30~80 r/min,灌流培养72~120 h;按MOI=0.05接种RABV CTN-1Ⅴ株,每24 h收获病毒液1次,检测病毒滴度和抗原含量。结果30 L生物反应器微载体Vero细胞培养96 h后密度约为1×10^(7)个/mL;300 L生物反应器微载体Vero细胞培养96 h后密度约为7.4×10^(6)个/mL。病毒接种96 h达峰值,病毒平均滴度为6.8 lgLD50/mL,抗原平均含量为2.58 IU/mL。结论微载体Vero细胞从30 L生物反应器经罐外细胞消化放大培养至300 L生物反应器的工艺稳定可行,对RABV CTN-1Ⅴ株的产毒能力影响较小,可为RABV灭活疫苗大规模生产提供参考资料。Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1Ⅴstrain.Methods The 140-passage of Vero cells were cultured at 37℃for 72-120 h,then amplified by passaging at a cell density ratio of 1∶4 into the 10×cell factory.After incubation at 37℃for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37℃,pH 7.0-7.4,dissolved oxygen 30%-80%,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h.Total three batches of microcarrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier 5-8 g/L,culture temperature 37℃,pH 7.0-7.4,dissolved oxygen 30%-80%,stirring speed 30-80 r/min,and perfusion culture 72-120 h.RABV CTN-1Ⅴwas inoculated at the MOI of 0.05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content.Results The cell density was about 1×10^(7) cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7.4×10^(6 )cells/mL after culture for 96 h in the 300 L bioreactor.At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6.8 lgLD50/mL and the average antigen content of 2.58 IU/mL.Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsinization from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴstrain,which provides a reference for the large-scale production of inactivated RABV vaccine.

关 键 词：狂犬病病毒 VERO细胞 生物反应器 微载体 罐外消化 放大培养 

分 类 号：R392-33[医药卫生—免疫学]