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目的建立测定重组人干扰素α2a成品中干扰素α2a蛋白含量的体积排阻高效液相色谱法(size exclusion-high performance liquid chromatography,SE-HPLC),并进行验证。方法应用SE-HPLC法测定重组人干扰素α2a对照品及供试品蛋白质含量,并对方法的线性、精密度、稳定性、重复性及准确性进行验证。采用建立的SE-HPLC法检测5家企业生产的24批样品中干扰素α2a蛋白含量,同时每批样品按《中国药典》三部(2010版)方法测定生物学活性,并计算比活性(IU/mg)。结果重组人干扰素α2a在0.364 8～11.67μg范围内,与峰面积呈良好的线性关系(r2=0.999 9)。用建立的方法连续进样6次,检测对照品溶液的干扰素α2a蛋白含量,RSD为2.4%;同一批样品进样12次,检测供试品溶液的干扰素α2a蛋白含量,RSD为2.8%;同一批样品取6份进样,检测供试品溶液的干扰素α2a蛋白含量,RSD为2.1%;5家企业生产的24批样品,平均加标回收率为103.5%,平均比活性为1.65×108IU/mg。结论建立了测定重组人干扰素α2a蛋白含量的SE-HPLC法,该方法简便,精密度、稳定性、重复性及准确性好,可用于重组人干扰素α2a成品中干扰素α2a蛋白含量的测定及比活性评价。
Objective To establish and validate the size exclusion-high performance liquid chromatography (SE-HPLC) for the determination of interferon α2a in recombinant human interferon α2a. Methods SE-HPLC method was used to determine the recombinant human interferon α2a and its protein content. The linearity, precision, stability, repeatability and accuracy of the method were verified. The established method of SE-HPLC was used to detect the interferon α2a protein content in 24 batches of samples produced by 5 enterprises. Meanwhile, the biological activity of each batch of samples was determined according to the Chinese Pharmacopoeia (2010 edition), and the specific activity (IU / mg). Results The recombinant human interferon α2a showed a good linear relationship with the peak area (r2 = 0.999 9) in the range of 0.364 8 ~ 11.67 μg. The established method of continuous injection of 6 times, the detection of reference substance solution interferon α2a protein content, RSD was 2.4%; the same batch of sample injection 12 times to detect the test solution interferon α2a protein content, RSD was 2.8% ; The same batch of samples taken 6 injections to detect the test solution interferon α2a protein content, RSD was 2.1%; 24 batches of samples produced by the five companies, the average recovery was 103.5%, the average specific activity of 1.65 × 108IU / mg. Conclusion The method of SE-HPLC for the determination of recombinant human interferon α2a protein content is established. The method is simple, accurate, stable, reproducible and accurate. It can be used to determine the content of interferon α2a in recombinant human interferon α2a And specific activity evaluation.