ActRⅡB-Fc融合蛋白生物学活性测定方法的建立

Method for Determination of the Biological Activity of ActRⅡB-Fc Fusion Protein

目的:建立ActRⅡB-Fc融合蛋白的生物学活性检测方法。方法:利用A204-CAGA12-LUC细胞系,通过荧光素酶检测系统进行ActRⅡB-Fc融合蛋白的生物学活性检测,根据四参数拟合分析计算样品相对效价,并对该方法的专属性、精密性和准确性进行验证。结果:ActRⅡB-Fc融合蛋白在该方法中存在量效关系,且符合4-参数方程:y=(A-D)/[1+(x/C)^(B)]+D。该方法具有良好的专属性;6批ActRⅡB-Fc融合蛋白样品经3次测定,相对效价平均值在(86.74±6.44)%~(108.81±15.07)%,RSD均小于15%;2批回收率样品经3次测定,回收率分别为(114.99±12.42)%和(81.19±7.35)%;8次独立测定重复性较好。结论:研究建立的ActRⅡB-Fc融合蛋白生物学活性检测方法专属性强,准确性高,精密度好,可作为ActRⅡB-Fc融合蛋白生物学活性的常规检测方法。

Objective:To establish a method to detect the biological activity of ActRⅡB-Fc fusion protein.Methods:The biological activity of ActRⅡB-Fc fusion protein was determined by ONE-Glo;Luciferase Assay System with the use of A204-CAGA12-LUC cell line,and the relative potency of the samples was calculated by four-parameter fitting analysis.The developed method was validated in specification,precision and accuracy.Results:ActRⅡB-Fc fusion protein shows a dose-effect ralationship in this method and it was in accordance with the four-parameter equation:y=(A-D)/[1+(x/C)^(B)]+D.The method has good specificity.Six batches of samples of the ActRⅡB-Fc fusion protein were tested for 3 times,and the average relative potencies ranged from(86.74±6.44)%to(108.81±15.07)%with relative standard deviation less than 15%.Two batches of recovery samples were detected for 3 times,the recoveries of which were(114.99±12.42)%and(81.19±7.35)%respectively.The results of 8 independent determinations showed good reproducibility.Conclusion:A method for detecting biological activity of ActRⅡB-Fc fusion protein has been successfully developed,which shows good specification,precision and accuracy and can be used as a routine method for biological activity detection of ActRⅡB-Fc fusion protein.