应用篮式生物反应器制备森林脑炎灭活疫苗(Vero细胞)  

Preparation of inactivated tick-borne encephalitis vaccine(Vero cells)by using basket bioreactor

作　　者：刘岩松[1] 王华[1] 孙宏亮[1] 安继新 纪权 邢坤鹏 张朔[1] 刘梦妮 邹勇[1] 

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062 [2]长春祁健生物制品有限公司,吉林长春130012

出　　处：《中国生物制品学杂志》2015年第12期1324-1326,1331,共4页Chinese Journal of Biologicals

基　　金：国家科技部重大新药创制项目(2013ZX09402302-205)

摘　　要：目的探讨篮式生物反应器制备森林脑炎灭活疫苗（Vero细胞）。方法应用14 L篮式生物反应器,以Vero细胞作为森林脑炎病毒增殖的细胞基质,采用片状载体对Vero细胞进行高密度培养,探讨不同细胞接种浓度对生物反应器内细胞密度及病毒产量的影响。连续收获的森林脑炎病毒液经超滤浓缩、1∶4 000（v/v）β-丙内酯灭活、柱层析纯化后,获得疫苗原液,按照《中国药典》三部（2010版）进行相关检测。结果用（5-7.5）×105个/ml细胞浓度接种Vero细胞培养森林脑炎病毒可连续收获30 d以上,平均病毒滴度为8.4 lg LD50/ml;1∶4 000（v/v）β-丙内酯灭活24 h可完全灭活病毒,3批病毒浓缩液灭活验证试验均未检出残余活病毒;柱层析纯化后杂蛋白去除率可达89.56%;疫苗原液经无菌检查合格,其他各项质量指标均符合《中国药典》三部（2010版）规定。结论应用篮式生物反应器培养Vero细胞制备了合格的森林脑炎灭活疫苗,该工艺适用于森林脑炎灭活疫苗的规模化生产。Objective To prepare inactivated tick-borne encephalitis（TBE） vaccine（Vero cells） by using a basket bioreactor. Methods Vero cells was cultured at a high density in a 14 L basket bioreactor with a commercial fibracel,and used as the substrate for propagation of TBE virus. The effect of various cell concentration for inoculation on cell den-sity and virus yield in bioreactor were investigated. The continuously harvested TBE virus was concentrated by ultrafiltra-tion, inactivated with 1 ∶ 4 000（v / v）β-propiolactone and purified by gel filtration chromatography. The obtained bulk of vaccine was subjected to control tests according to the requirements in Chinese Pharmacopeia（Volume Ⅲ, 2010 edition）.Results The TBE virus inoculated to Vero cells at a concentration of（5 - 7. 5） × 10^5 cells / ml could be harvested for more than 30 d, of which the mean titer was 8. 4 lg LD50/ ml. The virus was inactivated completely within 24 h with1 ∶ 4 000（v / v）β-propiolactone. No residual live viruses were detected in three batches of concentrated virus liquids in inactivation confirmation test. The removal rate of foreign protein after purification by gel filtration chromatography was89. 56%. The bulk of vaccine was qualified in sterility test, of which the other quality indexes met the requirements in Chinese Pharmacopeia（Volume Ⅲ, 2010 edition）. Conclusion Qualified inactivated TBE vaccine was prepared by culture of Vero cells using basket bioreactor. The procedure was suitable for the large-scale production of inactivated TBE vaccine.

关 键 词：生物反应器 森林脑炎灭活疫苗 VERO细胞 

分 类 号：R373.31[医药卫生—病原生物学] R392-33[医药卫生—基础医学]