荧光偏振法测定融合蛋白制剂中聚山梨醇酯20的含量

Determination of polysorbate 20 content in fusion protein injection by fluorescence polarization

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 develop a fluorescence polarization（FP）assay for polysorbate 20（Tween 20）content in fusion protein injection. Methods The Tween 20 content in samples was determined based on the principle that the intensity of fluorescence polarization at an excitation wavelength of 485 nm,produced by binding of hydrophobic fluorescent dye 5-dodecanoylaminofluorescein（DAF） to the nonpolar core in Tween 20 micelle,was positively related to the micelle concentration in solution. The developed method was verified for specificity,accuracy,precision and goodness-of-fit. Results The developed method showed high specificity,by which the interference of protein in detection system was eliminated. The samples at high（250 μg / ml）,moderate（150 μg / ml）,low（50 μg / ml）and LLQQ（lower limit of quantitation, 25 μg / ml）concentrations were determined by the developed method for 3 times,of which the mean recovery rates were within the acceptable range of（100 ± 20）%,both the coefficients of variation in intra- and inter-assays were less than 15%. The standard curve showed a high goodness-of-fit. Conclusion The developed FP method showed high specificity, accuracy and precision,which might be used for determination of Tween 20 content in protein products and was suitable for monitoring the effective concentration of Tween 20 during investigation on stability.