重组人干扰素α1b蛋白质含量HPLC检测方法的建立

Development of high performance liquid chromatography for determination of recombinant human interferon α1b content

目的建立测定重组人干扰素α1b(Recombinant human interferonα1b,rhIFNα1b)蛋白质含量的HPLC检测方法,并进行验证。方法应用HPLC外标法测定IFNα1b对照品的蛋白质含量,并对方法的线性及精密度进行验证。采用建立的HPLC外标法测定3批rhIFNα1b样品的蛋白质含量,并与Lowry法的检测结果进行比较。结果蛋白质在进样量5～50μg范围内,与峰面积呈良好的线性关系(r=0.998 1);用建立的方法重复进样6次,检测rhIFNα1b对照品溶液的蛋白质含量,相对标准偏差(RSD)为0.92%,<2%;用建立的方法检测0812001、0812003、0812006批rhIFNα1b样品溶液的蛋白质含量分别为0.465、0.503和0.496 mg/ml;HPLC外标法与Lowry法测定的蛋白质浓度有一定的差异,HPLC法的RSD<2%,Lowry法的RSD>4%,HPLC法的精密度较Lowry法好。结论建立了测定rhIFNα1b蛋白质含量的HPLC外标法,该方法操作简便,精密度较好,可用于IFNα1b原液蛋白质含量的检测。

Objective To develop and verify the high performance liquid chromatography （HPLC） for determination of recombinant human interferon cob （rhlFNotlb） content. Methods HPLC external standard method was used for determination of protein content in rhlFNαlb reference, and verified for linearity and precision. Three batches of rhlFNαlb samples were determined for protein content by the developed HPLC method, of which the result was compared with that by Lowry method. Results The determination result showed good linearity to peak area within a sample load range of 5 50μg （r = 0. 998 1 ）. The relative standard deviation （RSD） of protein contents in rhlFNctlb reference determined by the developed method for 6 times was 0. 92% （〈 2%）. The protein contents of three batches of rhlFNαlb samples, with Lot No. of 0812001, 0812003 and 0812006, were 0. 465, 0. 503 and 0. 496 mg/ml, respectively. The protein contents determined by the developed HPLC method and Lowry method showed a certain difference, of which the RSDs were less than 2% and more than 4% respectively. However, the precision of HPLC method was higher than that of Lowry method. Conclusion The HPLC external standard method for determination of rhlFNαlb content was developed, which was simple, precise, and suitable for determination of rhlFNαlb content in bulk.