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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 obtain pegylated interferon α2b (PEGIFNα2b) by N-terminal modification of interferon α2b using polyethylene glycol (PEG) with a molecular weight of 30 KD and to establish a method for the quality control of PEG-IFNα2b. Methods: The biological activity of PEG-IFNα2b was determined by using the Cell-VVSV system. The protein content of PEG-IFNα2b was determined by Lowry’s method. The relative molecular mass, high performance liquid phase Gel filtration chromatography was used to determine the purity. Peptide mapping was determined by trypsin digestion. UV-Vis absorption spectra were determined by ultraviolet spectrophotometry. Exogenous DNA residues were detected by digoxigenin (DIG) Cell protein residue. Results: The specific activity of peginterferon α2b was 1.39 × 10 ~ 7 IU · mg -1, the protein content was 420 μg · ml -1, the relative molecular mass was about 65 000 dalton , The purity is more than 99.0%, the maximum ultraviolet absorption peak is at the wavelength of 278 nm, the amount of exogenous DNA is less than 100 pg / dose, and the residual amount of the host bacterial protein is 0.010 27%. Conclusion: The quality control method can be used for the determination of peginterferon α2b.