苑志刚,刘岩,陈立新,韩慧丽,吴洋,李春艳,杨屹
YUAN Zhi-gang,LIU Yan,CHEN Li-xin,HAN Hui-li,WU Yang,LI Chun-yan,YANG Yi

• 1:长春生物制品研究所有限责任公司

摘要(Abstract)：

目的利用NBS 14 L篮式生物反应器大规模培养乙型脑炎病毒(Japanese encephalitis virus,JEV),并对病毒收获液进行灭活和纯化,为乙型脑炎灭活疫苗(Vero细胞)的大批量生产提供参考。方法用NBS 14 L篮式生物反应器,以片状载体FibralCel DISKⅠ为载体,进行Vero细胞高密度培养,分别按0.01、0.1和0.3 MOI接种JEV(P3V2)毒种,灌流培养,收获3批病毒液,检测病毒滴度;分别使用不同浓度的甲醛(100、200、400 ng/ml)于4、25℃和不同浓度的β-丙内酯(β-丙内酯︰病毒液分别为1︰2 000、1︰4 000、1︰8 000)于4℃对病毒收获液进行灭活,取灭活后的病毒液,接种昆明小鼠,验证病毒灭活效果;用截留相对分子质量为10万的超滤器对病毒收获液进行超滤浓缩,用Sepharose 4FF层析柱对病毒浓缩液进行层析纯化。结果共培养3批Vero细胞,平台期密度约为1.6×107个/ml。3批病毒收获液的滴度分别为8.4、8.1和7.9 lgLD50/ml。以甲醛为灭活剂,作用浓度为200 ng/ml,温度为4℃时,作用7 d可完全灭活病毒,且免疫原性合格;以β-丙内酯为灭活剂,作用浓度为1︰4 000,温度为4℃时,作用12 h可完全灭活病毒,且免疫原性合格。3批病毒浓缩液病毒液经Sepharose 4FF层析柱层析纯化后,蛋白去除率达99%以上,抗原回收率在95%以上,Vero细胞蛋白质残留量和DNA残留量均符合《中国药典》三部(2010版)的相关规定。结论成功实现了JEV的大规模生物反应器培养,层析纯化后病毒的抗原回收率较高,为乙型脑炎灭活疫苗(Vero细胞)的大批量生产提供了参考。
Objective To culture Japanese encephalitis virus(JEV) in a large scale in NBS 14 L bioreactors,inactivate and purify the harvest and provide a reference for large-scale production of inactivated JEV vaccine(Vero cells). Methods Vero cells were cultured at a high density in NBS 14 L bioreactor using FibralCel DISKⅠ carrier,into which JEV(P3V2)seeds were inoculated at MOIs of 0. 01,0. 1 and 0. 3 and subjected to perfused culture. Three batches of virus liquids were harvested and determined for titer,then inactivated with formaldehyde at various concentrations(100,200 and 400 ng / ml)at 4 and 25 ℃ and with β-propionolactone(BPL) at various ratios(BPL ∶ virus liquid were 1 ∶ 2 000,1 ∶ 4 000 and 1 ∶ 8 000)at 4 ℃ respectively. The inactivated virus liquid was inoculated to Kunming mice to verify the inactivation efficacy. The virus harvest was concentrated by ultrafiltration using an ultrafilter with entrapped relative molecular mass of 100 000,and purified by Sepharose 4FF chromatography. Results Three batches of Vero cells were cultured,at a platform density of about 1. 6 × 107 cells / ml. The titers of three batches of virus harvest were 8. 4,8. 1 and 7. 9 lgLD50/ ml respectively.The virus was completed inactivated with 200 ng / ml formaldehyde at 4 ℃ for 12 h,while the immunogenicity was qualified. After purification by Sepharose 4FF chromatography,more than 99% of proteins in the three batches of concentrated virus liquids were removed,while the recovery rate of antigen was more than 95%. Both the residual Vero cell protein and DNA met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). Conclusion JEV was cultured in a large scale in bio-reactor,and the recovery rate of antigen in the virus after purification by chromatography was high,which provided an experimental basis for large-scale production of inactivated JEV vaccine(Vero cells).

关键词(KeyWords)： 乙型脑炎病毒;灭活疫苗;Vero细胞;生物反应器;柱层析
Japanese encephalitis virus(JEV);Inactivated vaccine;Vero cells;Bioreactor;Column chromatography

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 苑志刚,刘岩,陈立新,韩慧丽,吴洋,李春艳,杨屹
YUAN Zhi-gang,LIU Yan,CHEN Li-xin,HAN Hui-li,WU Yang,LI Chun-yan,YANG Yi

DOI: 10.13200/j.cnki.cjb.000618

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