长效重组人凝血因子Ⅷ细胞培养条件的优化  

Optimization of Cell Culture Conditions of Long-acting Recombinant Human Coagulation FactorⅧ

作　　者：向雨秘 费保进 聂艳桃 胡珍霞 侯仰帅 梁洪 XIANG Yu-mi;FEI Bao-jin;NIE Yan-tao;HU Zhen-xia;HOU Yang-shuai;LIANG Hong(Chengdu Rongsheng Pharmaceuticals Co.Ltd.,Chengdu 610041,China;Bejing Tiantan Biological Products Co.Ltd.,Beijing 100024,China)

机构地区：[1]成都蓉生药业有限责任公司,四川成都610041 [2]北京天坛生物制品有限公司,北京100024

出　　处：《药物生物技术》2023年第1期25-31,共7页Pharmaceutical Biotechnology

摘　　要：优化国产长效重组人凝血因子Ⅷ的细胞培养条件,为后续研究提供实验依据。采用分批补料培养方式,将长效重组人凝血因子Ⅷ细胞接种至1 L发酵罐中进行培养,分析在不同补料培养基浓度(2%Cell Boost 2,3%Cell Boost 2,5%Cell Boost 2,补料培养基为Cell Boost 2和细胞培养基按照W/V比配制的培养基)、不同细胞培养pH(pH=6.9、pH=7.0、pH=7.1)、不同培养基(CDM4 PERMAb、CD11V、Veros),培养基补加Cu^(2+)的情况下,细胞密度、细胞活率、细胞代谢以及发酵液中蛋白活性的变化,从而确定最优培养条件。最佳培养条件:补料浓度2%Cell Boost 2,细胞培养pH 7.0,培养基为添加40μmol/L Cu^(2+)的CD11V,整个细胞培养周期17 d,细胞培养液收获体积是初始培养体积的2.6倍;细胞密度峰值达2375×10^(4)cells/mL,在收获时活率高于90%;在蛋白表达最高时,乳酸仅为0.3 mmol/L,蛋白活性高达2443.94 IU/mL,是同等条件下CDM4 PERMAb培养基(1182.17 IU/mL)的2.1倍。该研究优化了国产长效重组人凝血因子Ⅷ的细胞培养条件,提高了目的蛋白的活性,延长了整个培养周期,提升了目的蛋白的产量,对国产长效重组人凝血因子Ⅷ的产业化具有促进意义。To provide experimental basis for subsequent research of domestic long-acting recombinant human coagulation factor VIII which was used for treating Haemophilia A,this study optimized the cell culture conditions of the long-acting recombinant human coagulation factor VIII cell line.The long-acting recombinant human coagulation factor VIII cells were inoculated into 1 L fermenter for culture by fed-batch culture method.The optimum condition was determined by analyzing the different conditions:different feeding concentrations(2%Cell Boost 2,3%Cell Boost 2,5%Cell Boost 2,it is prepared from Cell Boost 2 and cell culture medium according to the mass/volume ratio),different cell culture pH(pH=6.9,pH=7.0,pH=7.1),different cell culture medium(CDM4 PERMAb,CD11V,Veros)and cell culture medium supplemented with Cu^(2+),how effected the changes of the cell in cell density,cell viability,cell metabolites and protein activity in fermentation broth.The most suitable conditions:the supplementary medium concentration was 2%Cell Boost 2 media,the cell culture pH was 7.0,the cell culture medium was domestic media which was CD11V supplemented with 40μmol/L Cu^(2+).In this case,the entire culture period was extended to 17 days,and the harvested volume of cell culture medium was 2.6 times of the initial volume.The peak cell density reached 2375×10^(4)cells/mL,and the viability was higher than 90%at harvest.At the highest protein expression,the lactic acid was only 0.3 mmol/L,and the protein activity was as high as 2443.94 IU/mL,which was 2.1 times that of CDM4 PERMAb medium(1182.17 IU/mL)under the same conditions.This study optimized the cell culture conditions of domestic long-acting recombinant human coagulation factor VIII,improved the activity of the target protein,prolonged the entire culture cycle,increased the output of the target protein,and successfully realized the localization of the imported medium,which was of great significance for the industrialization of domestic long-acting recombinant human coagulation factor VIII.

关 键 词：血友病A 长效重组人凝血因子Ⅷ 中国仓鼠卵巢细胞 分批补料培养 接种培养 细胞培养 蛋白活性 

分 类 号：TQ464[化学工程—制药化工]