重组人粒细胞集落刺激因子宿主蛋白质含量的测定

目的根据重组人粒细胞集落刺激因子 (rhG CSF)的生产工艺 ,建立宿主蛋白质 (HCP)含量的测定方法。方法用不含rhG CSF基因的空质粒转染E .coli宿主 (BL2 1) ,按rhG CSF的生产工艺进行发酵、纯化、制备HCP。常规法免疫家兔 ,制备抗HCP多抗血清 ,经纯化后 ,过碘酸钠法标记辣根过氧化物酶 (HRP) ,建立双抗夹心酶标记免疫吸附测定 (ELISA)法测定HCP在rhG CSF原液中的含量。结果所建HCP测定方法能够测定 7.8～ 2 5 0ng/ml范围内的HCP。结论重组蛋白质药物宿主蛋白质含量测定应依据不同的工艺 ,制定不同的方法。

Determining the host cell protein impurities in recombinant human granulocyte colony stimulating factor preparation

PurposeThe method to measure host cell protein(HCP) content in recombinant human granulocyte colony stimulating factor(rhG-CSF) was established based on according to its manufacture process.MethodsE.coil BL21 strain was trans-fected with the exact plasmid same as rhG-CSF producer except uncontained hG-CSF gene,and then was fermented and purified in accordance with rhG-CSF manufacture process and the preparation thus generated was HCP.Rabbit was immunized with regular method to prepare polyclonal anti-HCP antibody.After purification,the polyclonal anti-HCP antibody was labeled with horseradish peroxidase using sodium periodic acid method,then the polyclonal anti-HCP antibidy and the labeled polyclonal anti-HCP antibidy was used in sandwich ELISA method to determine HCP content in rhG-CSF preparation.ResultsThe ELISA method described in this paper could determine HCP in the range of 7.8～250 ng/ml.ConclusionThe assay to determine HCP content in recombinant protein pharmaceuticals should be established based on the respective manufacture process of each product.