摘要目的 优化基于中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达的Fc融合蛋白制备工艺.方法 对于上游细胞培养工艺,先在摇瓶中分别对培养基配比和流加培养方案进行优化,再将优化工艺放大到生物反应器规模并确认其可行性.对于下游蛋白纯化工艺,分别对A蛋白亲和层析的洗脱条件和阴离子交换层析的平衡条件进行优化.结果 对于上游细胞培养工艺,以种子培养基∶基础培养基(CD OptiCHO)∶基础培养基(CDM4Mab)为2∶1∶1的配比配制培养基,并以3、6和9d补料的流加培养方案进行细胞培养,可获得理想的细胞密度和活率,且成本较低.对于下游蛋白纯化工艺,以pH3.3的洗脱液进行A蛋白亲和层析纯化时,Fc融合蛋白的回收率为94.7％,纯度为98.64％;以pH5.0～pH5.5的平衡液进行阴离子交换层析纯化时,Fc融合蛋白的回收率达到98％以上、纯度达到99％.结论 基于CHO细胞表达的Fc融合蛋白制备工艺的上游细胞培养和下游蛋白纯化工艺得到成功优化,这为此类抗体药物制备工艺的优化提供了思路.

abstractsObjective To optimize Chinese hamster ovary (CHO) cell expression-based Fc fusion protein preparation process.Methods For the upstream cell culture process,the medium proportion and feeding strategy were optimized first in the shake flask,and then the optimized process was scaled up to the bioreactor and its feasibility was confirmed.For the the downstream protein purification process,the elution condition of protein A affinity chromatography and the balance condition of anion exchange chromatography were optimized.Results For the upstream cell culture process,the seed culture medium,basic medium (CD OptiCHO) and basic medium (CDM4Mab) were mixed at 2 ∶ 1 ∶ 1 ratio.Cell density and viability of fed-batch culture by feeding at 3,6 and 9 d were good,and this feeding design cost was low.For the downstream protein purification process,when pH3.3 eluant was used for protein A affinity chromatography,the recovery and purity of Fc fusion protein were 94.7％ and 98.64％,respectively;when pH5.0-pH5.5 balance liquid was used for anion exchange chromatography,the recovery and purity of Fc fusion protein were more than 98 ％ and 99 ％,respectively.Conclusion CHO cell expression-based Fc fusion protein preparation process is optimized successfully,and provides ideas for optimization of the therapeutic antibody preparation processes.