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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 establish a HPLC method for the determination of recombinant human interferon α1b (rhIFNα1b) protein content. Methods The content of IFNα1b protein was determined by HPLC external standard method. The linearity and precision of the method were also verified. The established HPLC method was used to determine the protein content of three batches of rhIFNα1b samples and compared with the results of Lowry method. Results The protein showed a good linear relationship with the peak area in the range of 5 ~ 50μg (r = 0.998 1). The protein content of the rhIFNα1b reference solution was detected by repeated injections 6 times. The relative standard deviations RSD) was 0.92%, <2%. The protein contents of 0812001, 0812003 and 0812006 batch rhIFNα1b samples were 0.465,0.503 and 0.496 mg / ml, respectively. The protein concentration determined by HPLC external standard method and Lowry method was The difference between the two methods is that RSD of HPLC method is less than 2%, RSD of Lowry method is more than 4%. The precision of HPLC method is better than that of Lowry method. Conclusion A HPLC external standard method was established for the determination of the protein content of rhIFNα1b. The method is simple, accurate and can be used to detect the protein content of IFNα1b.