聚乙二醇干扰素α2b质控方法探讨

Quality Control Methods for PEG-IFNα2b

目的:采用分子量为30 KD的聚乙二醇(PEG)对干扰素α2b进行N端定点修饰获得聚乙二醇干扰素α2b(PEGIFNα2b),并建立PEG-IFNα2b的质控方法。方法:采用Wish细胞-VSV病毒系统,以细胞病变抑制法(CPE)测定PEG-IFNα2b的生物学活性,Lowry法测定蛋白含量,SDS-聚丙烯酰胺凝胶电泳法测定相对分子质量,高效液相凝胶过滤色谱法测定纯度,胰蛋白酶消化法测定肽图,紫外分光光度法测定紫外吸收光谱,地高辛(DIG)标记的核酸探针法测定外源性DNA残留量,ELISA法测定宿主菌菌体蛋白残留量。结果:聚乙二醇干扰素α2b的比活性为1.39×10~7 IU·mg^(-1)蛋白,蛋白含量为420μg·ml^(-1),相对分子质量约为65 000道尔顿,纯度超过99.0%,紫外最大吸收峰约在278 nm波长处,外源性DNA残留量小于100pg/剂量,宿主菌菌体蛋白残留量为0.010 27%。结论:该质控方法可用于聚乙二醇干扰素α2b的检定。

Objective： To establish the quality control methods for PEG-IFNα2b,30 KD PEG was used to modify the N-terminal site-specific amine of interferon ct2b based on optimized testing methods. Method： The anti-viral activity of PEG-IFNα2b was analyzed by evaluating the eytopathic effect using the WISH-VSV system,the Lowry method was used to measure the protein quantitation,the re- duced SDS-PAGE was used to determine the relative molecular weight,the protein purity was analyzed by SEC-HPLC,a peptide map- ping procedure utilizing trypsin digestion RP-HPLC analysis was developed to detect the subtle alteration, the ultraviolet absorption spectrum was recorded by ultraviolet spectroscopy, the high sensitivity hybridization assay for quantitation of residual E. colt DNA was carried out by using digoxin-labeled DNA and the residual host cell protein by ELISA. Result： The specific activity, concentration,relative molecular weight and SEC-HPLC purity of PEG-IFNα2b were 1.39 × 10^7 IU-mg^-1 protein,420 μg·ml -1,65 KD,and 99% respectively,its maximum ultraviolet absorption peak appeared at 278um,the quantitation of residual DNA was less than100 pg/dose and the amount of the residual host cell protein was 0. 010 27%. Conclusion： The developed quality control methods are suitable for evaluating the quality of PEG-IFNα2b product.