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目的建立测定融合蛋白制剂中聚山梨醇酯20(Tween 20)含量的荧光偏振检测方法,并进行验证。方法利用疏水性荧光染料DAF(5-dodecanoylaminofluorescein)与Tween 20胶束内的非极性核心结合,在485 nm激发波长下产生的荧光偏振强度与溶液中胶束浓度呈正相关,来确定样品中的Tween 20浓度。对建立的方法进行专属性、准确性、精密性、拟合度验证。结果该方法的专属性较好,可排除检测体系中蛋白的干扰;该方法重复检测3次高(250μg/ml)、中(150μg/ml)、低(50μg/ml)、定量下限(lower limit of quantitation,LLOQ,25μg/ml)浓度样品的平均回收率均在(100±20)%的可接受范围内,批内和批间变异系数均小于15%;标准曲线拟合度良好。结论立的荧光偏振法专属性、准确性、精密性良好,可用于蛋白制剂中Tween 20含量的测定,并适用于稳定性考察过程中Tween 20有效浓度的监测。
Objective To establish a fluorescence polarization assay for the determination of Tween 20 in a fusion protein preparation. Methods The hydrophobic fluorescence dye DAF (5-dodecanoylaminofluorescein) combined with the non-polar core in Tween 20 micelles was used to determine the intensity of fluorescence polarization at 485 nm excitation wavelength and the concentration of micelles in solution. Tween 20 concentration. The established method of specificity, accuracy, precision, fitness verification. Results The specificity of the method was good and the protein interference in the detection system could be eliminated. The method was repeated for detection of high limit (250μg / ml), medium (150μg / ml), low (50μg / ml) of quantitation, LLOQ, 25μg / ml) were all within the acceptable range of (100 ± 20)%, the intra-assay and inter-assay coefficients of variation were all less than 15%, and the standard curve fitted well. Conclusion The established fluorescence polarization method has the advantages of specificity, accuracy and precision, and can be used for the determination of Tween 20 content in protein preparations and is suitable for the monitoring of the effective concentration of Tween 20 during the stability study.