重组人促红细胞生成素纯化工艺的优化

采用堆积床生物反应器,用无血清培养基培养分泌重组人促红细胞生成素（rhEPO）的工程细胞株ZK9703.所收集的上清,采用阴离子交换层析－反相层析－分子筛层析三步纯化工艺路线,分别用Q－Sepharose XL-C4-S-200（方法Ⅰ）和DEAE Sepharose FF-Source-S-200(方法Ⅱ）纯化3批产品,所得EPO纯度达98％以上,体外比活性大于1.3^10^5IU/mg。方法Ⅰ、方法Ⅱ纯化过程的EPO体外活性回收率分别为23.56%和28.57%。本纯化方法Ⅱ工艺纯化日程短,分离效果好,EPO体内、体外活性回收率较高,更适合于大规模生产重组人促红细胞生成素。

Optimized PurificationProcess of Human Recombinant Erythropoietin

Gene-engineered CHO cell line ZK9703 with the capacity to express huma n erythropoietin were cultured in packed bed bioreactor in a perfusion culture m ode. Harvest medium was collected and purified with a three-step purificati on pr ocession-exchanger, reverse phase and molecular sieve chromatogra phy. Three lot s of harvest medium was purified with a purification processQ-Sep harose XL, C4 reverse phase and S-200( the first method), three lots of harvest medium wa s pu rified with a purification processDEAE FF, source reverse phase an d S-200(the second method),the final products were analyzed for its purity and specific in vitro biological activity. Its purity is over 98%, and its specific in vit ro bio logical activity is over 1.3×105 IU/mg.The total recovery of EPO activity wa s 23.56% (by the fist method) and 28.57%.( by the second method), respectively. Th e results show that the second method of purification process is rapid and effic ient with high recovery, and could be more useful in large-scale production of rhEPO.