固化床反应器大规模灌注培养CHO-C28细胞生产乙型肝炎病毒表面抗原  

Large scale perfusion culture of CHO-C28 cells in fixed bed reactor for production of hepaqtitis B surface antigen

作　　者：刘伯宁[1] 王英 周兴军[1] 赵宏远[1] 侯丽媛 彭瑞珍[1] 耿欣[1] 李幼勃 容艳勇 刘欢欢 高健[1] 

机构地区：[1]华北制药集团新药研究开发有限责任公司抗体药物研制国家重点实验室,河北石家庄050015 [2]华北制药金坦生物技术有限责任公司,河北石家庄050015

出　　处：《中国生物制品学杂志》2014年第7期954-958,共5页Chinese Journal of Biologicals

摘　　要：目的开发一种应用固定化反应器大规模培养CHO-C28细胞的灌注培养工艺,用于制备重组乙型肝炎病毒表面抗原(HBsAg)。方法利用自行研发的微型反应器以轴向旋转模拟固化床内培养基流经载体表面,分析固化床反应器灌注培养CHO-C28细胞表达HBsAg的可行性;使用AP20小型固化床反应器进行小试工艺;将AP20反应器消化后收集的2.3×1010个细胞接种至AP200固化床反应器,对灌注工艺进行中试放大,收获培养液,测定溶氧、pH、葡萄糖、乳酸及HBsAg蛋白浓度,并与转瓶培养工艺进行比较。结果预装无纺纸片的微型反应器接种CHO-C28细胞后,可连续灌注培养60 d,HBsAg表达水平为1 100 ng/ml。AP20反应器接种CHO-C28细胞后,HBsAg表达水平在维持期约1 100 ng/ml;将AP20反应器培养至第10天的CHO-C28细胞消化后,收集2.3×1010个细胞,接种AP200反应器,可连续灌注培养60 d,生长期中,葡萄糖消耗速率最高可达450 g/d,维持期葡萄糖消耗速率降至250 g/d,整个维持期内葡萄糖浓度维持在约1.5 g/L,收液中乳酸积累量为22 mmol/L,HBsAg表达水平约1 200 ng/ml。固化床灌注培养工艺的工艺条件控制精度高,培养环境更适合CHO-C28细胞生长,培养周期与转瓶工艺相当。结论固化床反应器灌注培养CHO-C28细胞工艺与转瓶工艺HBsAg表达水平相当,具备较高的产业化应用价值。Objective To develop a large-scale perfusion culture process of CHO-C28 cells in fixed bed reactor for preparation of recombinant hepatitis B surface antigen（HBsAg）. Methods The feasibility of perfusion culture of CHOC28 cells in fixed bed reactor was investigated by mock culture in prepared mini-bioreactor in which medium flew through the surface of carriers in an axial rotation mode, based on which a bench-scale suspension culture process of CHO-C28 cells was developed by using AP20 fixed bed reactor. The 2. 3 × 1010cells after digestion in AP20 reactor were collected and inoculated to AP200 fixed bed reactor to develop a pilot process The culture liquids were harvested and determined for dissolved oxygen,pH value as well as glucose,lactic acid and HBsAg concentrations,and the results were compared with those of culture in rolling bottle. Results CHO-C28 cells inoculated into mini-bioreactor with nowoven paper could be cultured continuously by perfusion for 60 d,in which the expression level of HBsAg was 1 100 ng / ml.However,the expression level in CHO-C28 cells in AP20 reactor was about 1 100 ng / ml in maintain period. A portion of2. 3 × 1010CHO-C28 cells were harvested on day 10 after culture in AP20 reactor,and was cultured continuously by perfusion for 60 d,during which the glucose consumption rate reached 450 g / d at most in growth period and decreased to 250 g / d in maintain period. In the whole maintain period,the glucose concentration was about 1. 5 g / L,while the lactic acid accumulation in harvest was 22 mmol / L,and the expression level of HBsAg was about 1 200 ng / ml. The technological condition of perfusion culture process in fixed bed reactor was controlled precisely,while the culture environment was more suitable for the growth of CHO-C28 cells,and the culture cycle was equivalent to that of culture in rolling bottle. Conclusion The expression level of HBsAg of in CHO-C28 cells cultured by perfusion culture process in fixed bed was equivalent to that by rolling bottle culture,which wa

关 键 词：乙型肝炎表面抗原 反应器 灌注培养 

分 类 号：R373.21[医药卫生—病原生物学] R392-33[医药卫生—基础医学]