用填充床生物反应器连续灌流培养CHO细胞生产HBsAg  

Continuously Perfused Cultivation of Genetically-engineered CHO Cells Producing HBsAg in a Packed Bed Bioreactor

作　　者：龚伟[1] 方学平[1] 刘燕[1] 王晓燕[1] 范昌发[1] 梅兴国[1] 章扬培[2] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850 [2]军事医学科学院输血研究所,北京100850

出　　处：《中国生物工程杂志》2004年第7期82-84,共3页China Biotechnology

基　　金：国家"973"计划资助项目 (2 0 0 2CB713 80 4)

摘　　要：为了获得重组CHO细胞持续高效表达HBsAg的最佳条件 ,应用填充床生物反应器和聚酯片 ,连续灌流培养分泌HBsAg的重组CHO细胞 ,考察灌流培养基中葡萄糖浓度对细胞代谢和HBsAg生产的影响。连续培养 60天 ,细胞密度可达 5 0× 1 0 6 ml。灌流培养液中葡萄糖浓度从5 0g L增加到 7 6g L时 ,葡萄糖消耗速率和乳酸浓度均随之上升。当葡萄糖浓度继续增加至9 3g L时 ,葡萄糖消耗速率和乳酸浓度呈下降趋势 ;当将葡萄糖浓度降回至 7 6g L后又开始回升。培养过程中最高抗原滴度达 1∶5 1 2 ,出现在以含 5 0g L葡萄糖的培养液灌流阶段的末期 ,即第 2 6天 ,维持 1 0天左右即迅速下降至 1∶1 2 8水平。共收液 335L ,纯化后获得抗原蛋白2 1 3 0 4mg ,平均产率为 635 94μg L ,较传统转瓶工艺 ( 4 1 4μg L)提高 5 3 6%。表明CHO细胞较长时间处于高乳酸水平下 ( >30mmol L)会严重影响产物的表达 ,应控制灌流培养液中的葡萄糖浓度在较低水平 ,或通过适当提高灌流速率使培养基中乳酸水平维持在较低水平 。Genetically-engineered CHO cells producing HBsAg were cultured in a 7.5L packed bed bioreactor employing 100g polyester fiber disk. The glucose consumption rate, the lactate concentration in bioreactor and the production of HBsAg under different glucose concentration in perfusion medium were investigated. The highest cell density over 5 0×10 6/ml was achieved during 60 days' culture period. Glucose consumption rate and the lactate concentration increased when glucose concentration in perfusion medium was increased from 5 0g/L to 7 6g/L, but decreased when glucose concentration was increased to 9 3g/L. But Glucose consumption rate and the lactate concentration increased again with the decrease of glucose concentration from 9 3 to 7 6g/L. The highest RPHA titre of HBsAg was achieved 1:512 at the end of culture stage with 5 0g/L glucose in perfusion medium and maintained for 10 days and then fell to 1:128. Total amount of 213 04 mg HBsAg protein was obtained from about 335L collected medium. Average yield rate of HBsAg was 635 94μg/L, which was 53 6 percent higher than that obtained from traditional rolling bottle(414μg/L). It is suggested that the expression of HBsAg is seriously effected as CHO cells exposed in medium with high concentration lactate(>30mmol/L) for long time. ;

关 键 词：填充床生物反应器 HBSAG 细胞培养 聚酯片 培养基 

分 类 号：Q813.11[生物学—生物工程]