重组人干扰素α2a成品中蛋白含量体积排阻高效液相色谱法的建立

Development of size exclusion-high performance liquid chromatography method for determination of protein content in final product of recombinant human interferon α2a

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 develop and verify a size exclusion-high performance liquid chromatography（SE-HPLC） method for determination of protein content in final product of recombinant human interferon（rhIFN） α2a.Methods The protein contents in reference and test samples of rhIFNα2a were determined by SE-HPLC,based on which the linearity,precision,stability,reproducibility and accuracy of the method were verified.The protein contents in 24 batches of final products of rhIFNα2a from 5 manufacturers were determined by the developed SE-HPLC method.Meanwhile,each batch of samples were determined for biological activity by the method described in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）,and calculated for specific activity（IU / mg）.Results The protein content in rhIFNα2a showed good linear relationship to peak area within a concentration range of 0.364 8 ～ 11.67 μg（r2 = 0.999 9）.The RSD of results of protein content in rhIFN α2a reference tested for 6 times was 2.4%,while that of samples of the same batch tested for 12 times was 2.8%,and that of test results of six samples of the same batch was 2.1%.The average recovery and average specific activity of 24 batches of products from 5 manufacturers were 103.5% and 1.65 × 108 IU / mg respectively.Conclusion The SE-HPLC method for determination of protein content in rhIFNα2a was developed,which was simple,precise,stable,reproducible and accurate,and might be used for determination of protein content and evaluation of specific activity of final product of rhIFN α2a.