森林脑炎灭活疫苗(地鼠肾细胞)细胞工厂培养工艺的建立

Development of culture process for large-scale preparation of inactivated tick-borne encephalitis vaccine (hamster kidney cells) by cell factory

目的应用细胞工厂代替转瓶,培养原代地鼠肾细胞制备森林脑炎灭活疫苗,建立连续培养多次收获工艺,以提高疫苗产量。方法采用40层细胞工厂培养地鼠肾细胞,确定最佳细胞培养条件。用森林脑炎病毒森"张"株感染细胞,连续收获病毒液,经超滤浓缩、灭活、柱层析纯化后获得疫苗原液,按照《中国药典》三部(2015版)进行相关检测。结果确定了细胞工厂制备森林脑炎灭活疫苗的工艺参数:细胞工厂最适细胞接种量为3-4对地鼠肾/层;感染病毒96 h后第1次收获,之后每48 h收获1次,可连续收获3-5次。原液中抗原含量、蛋白质含量、地鼠肾细胞蛋白质残留量、牛血清白蛋白残余量均符合《中国药典》三部(2015版)规定。结论利用细胞工厂培养原代地鼠肾细胞制备森林脑炎灭活疫苗,获得了较高的细胞密度并收获了高毒力的病毒液,可替代转瓶培养工艺大规模制备森林脑炎灭活疫苗。

Objective To develop a procedure for continuous culture and multiple harvest of primary hamster kidney cells by cell factory instead of rolling bottles so as to improve the yield of vaccine.Methods Primary hamster kidney cells were cultured in a 40 layer cell factory,for which the condition was optimized.The cells were infected with tickborne encephalitis virus Sen“Zhang”strain,and the virus harvest liquid was concentrated,inactivated and purified by column chromatography.The obtained bulk of vaccine was subjected to control tests according to requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition).Results The technological parameters for preparation of inactivated tickborne encephalitis vaccine by cell factory were determined as follows:the optimal inoculation amount was 3 - 4 pairs of hamster kidneys/layer;the virus was harvested for the first time 96 h after infection,and harvested every 48 h afterwards,which could be harvested for 3 - 5 times.The antigen content,protein content,residual hamster kidney cell content and residual bovine serum albumin content met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition).Conclusion High cell density and virulence were obtained by preparation of inactivated tick-borne encephalitis vaccine by cell factory.The process may be used for large-scale production of inactivated tick-borne encephalitis vaccine instead of culture in rolling bottles.