150 L生物反应器规模化扩增狂犬病病毒工艺的建立  

Development of large-scale amplification process of rabies virus in 150 L bioreactor

作　　者：原国强 郭宏庄 崔晓峰 崔艳霞 孙志华 安文珏 李津 潘若文 YUAN Guoqiang;GUO Hongzhuang;CUI Xiaofeng;CUI Yanxia;SUN Zhihua;AN Wenjue;LI Jin;PAN Ruowen(Hualan Biological Bacterin Inc.,Xinxiang 453003,Henan Province,China)

机构地区：[1]华兰生物疫苗股份有限公司,河南新乡453003 [2]河南省生物医药产业研究院,河南新乡453003 [3]河南省预防型疫苗工程技术研究中心,河南新乡453003 [4]河南省生物医药(制剂)工程技术研究中心,河南新乡453003

出　　处：《中国生物制品学杂志》2024年第6期718-722,730,共6页Chinese Journal of Biologicals

基　　金：河南省先进制造业发展专项(新财预[2018]75号);郑洛新自创区产业集群专项(181200211800)。

摘　　要：目的建立150 L生物反应器大规模培养狂犬病病毒(rabies virus,RABV)的工艺,为后期开发更大规模高密度微载体反应器工艺奠定基础。方法应用30 L(型号:C30-2)和150 L(型号:VESSEL FERMENTER 300L)生物反应器,以灌流方式培养Vero细胞和CTN-1V株RABV,Cytodex-1微载体浓度20 g/L,培养温度36~38℃,DO 20%~60%,pH 7.0~7.4,连续13 d收获病毒液,培养过程中取样检测细胞密度、病毒滴度,并对病毒收获液进行无菌和支原体检查及抗原、宿主细胞蛋白(host cell protein,HCP)、牛血清白蛋白(bovine serum albumin,BSA)、DNA残留量检测。结果30和150 L生物反应器的细胞培养密度均可达1.2×10^(7)个/mL以上,在培养过程中各时间点的细胞密度差异均无统计学意义(t=0.225~2.173,P=0.096~0.833)。2种规模生物反应器病毒收获液均在感染后6 d达最高病毒滴度(8.5 lgLD_(50)/mL),且差异无统计学意义(t=1.000,P=0.374)。2种规模生物反应器病毒收获液的抗原、HCP、BSA和DNA残留量基本一致。结论可用150 L生物反应器大规模培养RABV,病毒收获液符合《中国药典》三部(2020版)相关标准。Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foundation for the further development of a larger-scale and high-density microcarrier reactor process.Methods Vero cells and RABV strain CTN-1V were cultured in 30 L(model:C30-2)and 150 L(model:VESSEL FERMENTER 300L)bioreactors by perfused culture with 20 g/L Cytodex-1 microcarrier and DO 20%-60%,at culture temperature 36-38℃and pH 7.0-7.4.During the culture process,the cell density and virus titer were measured.The virus culture media was harvested for consecutive 13 d and detected for the sterility,mycoplasma,and the residues of antigen,host cell protein(HCP),bovine serum albumin(BSA)and DNA.Results The density of cultured cells in 30 L and 150 L bioreactors all reached above 1.2×10^(7)cell/mL.There was no significant difference in cell density at different time points during the culture(t=0.225-2.173,P=0.096-0.833).The highest virus titer(8.5 lgLD_(50)/mL)was found in the both bioreactors 6 d after infection with no significant difference(t=1.000,P=0.374).The residues of antigen,HCP,BSA and DNA in the virus suspension from the two bioreactors were basically the same.Conclusion 150 L bioreactor can be used for the large-scale culture of RABV,and the harvested virus conformed to the relevant standards in Chinese Pharmacopoeia(VolumeⅢ,2020 edition).

关 键 词：狂犬病病毒 VERO细胞 生物反应器 微载体 

分 类 号：Q815[生物学—生物工程]