不同工艺制备CTN-1V株狂犬病病毒MOI的摸索及其在生物反应器规模化培养中的应用  

Exploration of rabies virus CTN-1V strain MOI prepared by different processes and its application in large-scale culture in bioreactor

作　　者：王月莉 曹烨 王玲 王玥 徐晓 胡文龙 陈杰杉 宋远涛 文聪 杨敏 陶涛 梁婧 Wang Yueli;Cao Ye;Wang Ling;Wang Yue;Xu Xiao;Hu Wenlong;Chen Jieshan;Song Yuantao;Wen Cong;Yang Min;Tao Tao;Liang Jing(Rabies Vaccine Department,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司狂犬病疫苗室,武汉430207

出　　处：《国际生物制品学杂志》2024年第3期133-138,共6页International Journal of Biologicals

基　　金：人畜共患烈性传染病临床救治创新技术与防护规范研究(2021YFC2600200)。

摘　　要：目的通过比较转瓶和反应器工艺制备的CTN-1V株狂犬病病毒在Vero细胞上适应性传代的最佳感染复数(multiplicity of infection, MOI), 评价应用反应器工艺规模化制备狂犬病病毒工作种子批的可行性。方法复苏Vero细胞后按照1∶4比例传代至生产代次, 接种不同MOI的CTN-1V株狂犬病病毒并培养。转瓶工艺毒种MOI为0.005、0.010、0.050、0.100、0.500;反应器工艺毒种MOI为0.010、0.030、0.050、0.080、0.100。在培养过程中分别采用ELISA和小鼠脑内滴定法进行狂犬病病毒G蛋白抗原含量和狂犬病病毒滴度检测, 确定最佳MOI, 并将最佳MOI分别应用于300 L反应器规模化病毒培养工艺。单次病毒收获液取样进行病毒G蛋白抗原含量和病毒滴度的检测, 并进行连续3批次确认试验。结果转瓶工艺毒种工作种子批(批号20181201)与反应器工艺毒种工作种子批(批号20201001), 在T225瓶中的最佳MOI均为0.050。将2批毒种分别用于300 L反应器灌流培养48 h后, 每批病毒收获液的G蛋白抗原含量连续9 d均达到1.0国际单位/ml以上, 病毒滴度均达到6.0 lg半数致死剂量/ml以上, 符合工艺标准。结论根据MOI比较结果, 反应器工艺可以替代原有的转瓶工艺制备狂犬病病毒工作种子批, 并应用到规模化生产中。ObjectiveTo evaluate the feasibility of large-scale preparation of rabies virus working seed lot by bioreactor process,by comparing the best multiplicity of infection(MOI)of adaptive passage of rabies virus CTN-1V strain on Vero cells with rolling bottle and bioreactor processes.MethodsAfter resuscitation,Vero cells were passaged to production generation at the ratio of 1∶4,and inoculated with CTN-1V rabies virus at different MOIs and cultured.The MOIs of rolling bottle process were 0.005,0.010,0.050,0.100 and 0.500,while the MOIs of bioreactor process were 0.010,0.030,0.050,0.080 and 0.100.The content of rabies virus G protein antigen and the virus titer were detected by ELISA and mouse brain titration,respectively during cultivation,and the best MOI was determined and applied to the large-scale virus culture process in 300 L bioreactor.Single virus harvest was sampled to detect the virus G protein antigen content and virus titer and three consecutive batches of confirmation tests were carried out.ResultsThe best MOIs in T225 bottle were both 0.050 for the working seed lot of rolling bottle(20181201)and bioreactor(20201001)processes.After 2 batches of working seed lot were cultured in 300 L bioreactor with perfusion culture process for 48 h,the G protein antigen content of every batch of single harvest for 9 consecutive days reached above 1.0 international unit/ml,and the virus titer reached above 6.0 lg median lethal dose/ml,which met the process standard.ConclusionThe bioreactor process can replace the original rolling bottle process to prepare rabies virus working seed lot and be applied to large-scale production,based on MOI comparison.

关 键 词：狂犬病病毒 VERO细胞 感染复数 G蛋白抗原含量 病毒滴度 

分 类 号：R392.33[医药卫生—免疫学]