刘兴茂，刘红，叶玲玲，李世崇，吴本传，王启伟，陈昭烈

军事医学科学院生物工程研究所，北京 100071

中国仓鼠卵巢工程细胞无血清流加培养关键工艺参数的考察

刘兴茂*，刘红*，叶玲玲，李世崇，吴本传，王启伟，陈昭烈

军事医学科学院生物工程研究所，北京 100071

主要考察流加培养基中不同营养成分、流加起始时间及初始接种密度对 11G-S细胞无血清流加培养的影响。在研究中以悬浮适应的表达尿激酶原 (Pro-urokinase，Pro-UK) CHO工程细胞系11G-S为研究对象，在100 mL的摇瓶中无血清悬浮流加培养11G-S细胞，同时以活细胞密度、细胞活力及Pro-UK活性为评价依据。结果表明在培养基中氨基酸、无血清添加成分及无机盐对促进细胞生长、细胞活力维持及蛋白表达起着较为重要的作用；且流加起始时间为72 h及初始接种密度为3×105~4×105cells/mL的流加培养效果较好。在为期12 d的流加培养过程中，11G-S细胞的最大活细胞密度达到7.8×106cells/mL，Pro-UK的最大累积活性为8 570 IU/mL。在此基础上，分别考察了无血清悬浮流加培养及分批培养11G-S细胞的生长及代谢特征，流加培养中期11G-S细胞的比生长速率 (μ) 高于同期批次培养的11G-S细胞；流加培养中后期11G-S细胞的葡萄糖比消耗速率 (qglu) 和谷氨酰胺比消耗速率 (qgln) 均高于同期批次培养的11G-S细胞。

CHO工程细胞，无血清培养，流加培养，工艺参数，代谢特征

Abstract:Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at3×105~4×105cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8×106cells/mL with a peak Pro-UK activity at 8 570 IU/mL was achieved during 12 d fed-batch culture. Further, the μ of the 11G-S cells at the middle phase of the fed-batch culture, and both the qgluand qglnof the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.

Keywords:recombinant CHO cells, serum free culture, fed-batch culture, process parameter, metabolism character

动物细胞大规模培养过程中营养物的耗竭和大量代谢副产物的积累往往是限制细胞生长密度、影响培养过程的主要因素[1-2]。特别是在批次培养过程中，不断消耗的营养成分往往成为影响细胞密度、细胞活力和细胞表达产物生产的限制性因素。……