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

Evaluation of the critical process parameters for the cultivation of recombinant Chinese hamster ovary cells in serum-free fed-batch mode

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3×105~4×105 cells/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7.8×106 cells/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。

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 at 3×105~4×105 cells/mL and the start time of feeding set at 72 h,a maximum viable cells density of 7.8×106 cells/mL with a peak Pro-UK activity at 8 570 IU/mL was achieved during 12 d fed-batch culture.Further,the of the 11G-S cells at the middle phase of the fed-batch culture,and both the qglu and qgln of 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.