营养补偿和代谢控制提高细胞蛋白表达的研究

Study on Increasing the Protein Expression of Cells by Specific Nutrient Compensation and Metabolism Control Strategy

为解决连续灌注培养产物因营养物质不能有效利用而导致产率偏低的问题,降低生产成本,通过在发酵过程中测定表达重组肿瘤坏死因子受体p75:Fc(TNFRp75:Fc)蛋白的CHO工程细胞株对氨基酸成分的不同消耗速率,定量添加特定氨基酸作为营养补偿,提高了培养基中氨基酸的综合利用效率。同时,在连续灌注过程中通过对葡萄糖补加的限量控制,使培养体系中葡萄糖始终低于0.5 g／L,减少了乳酸蓄积对细胞的毒性作用,从而有效降低了灌注速率。结果显示, 在30L工作体积发酵规模上经过氨基酸补偿和葡萄糖控制的连续灌注培养工艺使最终重组蛋白产率(mg／L)和最终产量较工艺改进前提高2．1倍和3．7倍,分别达到388mg／L和244．4g,生产周期延长了一周。工艺改进前后重组蛋白的唾液酸含量和体外生物比活没有改变。通过营养补偿和代谢控制工艺策略可以有效提高连续灌注培养工艺重组肿瘤坏死因子受体p75:Fc蛋白的产率和产能,从而降低产业化成本。

To increase the productivity and yield Every amino acid consumption rate in continual perfusi of recombinant protein in on culture of engineering continual perfusion processing, CHO cell line which expressed recombinant TNFRp75：Fc fusion protein were analyzed. Then rational amino acids were accordingly added to improve its comprehensive utilizing. At the same time, glucose supply was controlled to make the concentration of glucose below 0.5 g/L for ameliorating the toxicity of lactate accumulation in order to decrease the perfusion rate. The result showed that the productivity of recombinant protein was 3.1 times （388mg/L） and the total yield was 4.7 times （244.4g） that of control cultures after nutrient compensation and metabolism control in 30 liter working volume, and the fermentation period was prolonged one week longer. The sialic acid content and bioactivity in vitro of recombinant TNFRp75 ： Fc were not changed after nutrient compensation and glucose control supply. Nutrient compensating and metabolic control in continual perfusion fermentation could significantly increase the productivity and yield of recombinant TNFRp75： Fc, and thus reduced relative industrialization costs.