新一代重组人粒细胞集落刺激因子的工业化发酵、复性和纯化  

Large-Scale Production,Refolding and Purification of a Novel Recombinant Human Granulocyte Colony Stimulating Factor

作　　者：宋爽[1] 蒋文宏 蒋永平[1,2] 

机构地区：[1]中国医学科学院北京协和医学院苏州方舟生物医药研发中心,苏州215126 [2]苏州方舟基因药业有限公司,苏州215126

出　　处：《中国生物医学工程学报》2012年第4期552-557,共6页Chinese Journal of Biomedical Engineering

基　　金：国家"重大新药创制"科技重大专项(2009ZX09102-250)

摘　　要：工业化大规模发酵、复性和纯化突变型人粒细胞集落刺激因子(rhG-CSFa),并测定其体外生物学活性。利用定点突变和DNA重组技术获得突变型G-CSF(rhG-CSFa)。通过40 L发酵罐发酵基因工程菌株获得rhG-CSFa包涵体,经过一系列透析后,通过离子交换柱和分子排阻层析进行纯化,并使用高效液相色谱技术对纯化后的rhG-CSFa进行纯度分析。利用G-CSF依赖细胞株(M-NFS-60)测定纯化后的rhG-CSFa体外活性效价。结果表明,rhG-CSFa表达量占全菌蛋白的31.2%,通过优化复性条件,rhG-CSFa复性率达到11.36%,纯化后rhG-CSFa的纯度达到99.11%。体外活性实验显示,与野生型G-CSF相比,rhG-CSFa在相同浓度下能诱导NFS-60细胞株获得更高的细胞增殖率。rhG-CSFa工程菌株能够高表达rhG-CSFa,可用于工业化大规模生产,生产的rhG-CSFa具有较高的生物活性、稳定性和纯度。rhG-CSFa大规模生产的成功为该药物未来的临床运用打下了良好的基础。The aim of this work is to produce, refold, and purify rhG-CSFa in large scale and determine its bioactivity. Using site-direct and additional mutagenesis, a structurally modified derivative of human G-CSF termed G-CSFa was constructed and fermented in a 40 liter bio-reactor. The rhG-CSFa inclusion bodies were extracted and refolded by a serial dialysis. The refolded G-CSFa was then purified by an ion-exchange column and size-exclusive chromatography, and the purity of rhG-CSFa was then analyzed by HPLC. Purified rhG-CSFa was tested for its biological activity in vitro using a G-CSF-dependent cell （M-NFS -60） assays. The 31.2% expressed G-CSFa protein yield was achieved and 11.36% refolding yield was obtained after optimizing the protein refolding conditions. The purity of rhG-CSFa was 99. 11% after purification. In vitro studies demonstrated that rhG-CSFa induced a much higher proliferation rate of M-NFS - 60 cell lines than wild-type G- CSF at the same concentrations. The large-scale of recombinant G-CSFa in M15 strain was expressed with high yield, purity, and its biological activity and stability was confirmed. The success of large-scale production of G- CSFa facilitates the clinical application of this potential drug.

关 键 词：重组人粒细胞集落刺激因子(rhG-CSF) 发酵 复性 纯化 生物活性 

分 类 号：R318[医药卫生—生物医学工程]