重组蛋白中吐温80含量硫氰酸铵钴显色检测法的改进及验证

Improvement and verification of cobaltthiocyanate ammonium color-developing method for determination of Tween 80 content in recombinant protein

目的改进重组蛋白中吐温80含量的硫氰酸铵钴显色检测方法,并进行验证。方法将《中国药典》四部(2020版)中收录的吐温80含量检测方法,即硫氰酸钴铵比色法中的二氯甲烷调整为4.00 g,硫氰钴铵溶液浓度提高3倍,并对改进后的方法进行专属性、线性范围、定量限、重复性、日间精密性、准确性验证。采用该方法检测3批重组人源抗hTNF-α单克隆抗体注射液中吐温80的含量。结果改进的方法具有良好的专属性;吐温80标准品浓度在0~400μg/mL范围内与A620呈良好的线性关系,回归方程为y=0.000 6 x-0.003 4,R2=0.999;重复性及日间精密性RSD均<5%;定量限为22.69μg/mL;加入80、180、320μg吐温80标准品的加样回收率分别为100.00%、98.77%、99.31%。3批重组人源抗hTNF-α单克隆抗体注射液中吐温80含量分别为0.95、1.00、1.02 mg/mL。结论改进后的硫氰酸铵钴显色法具有良好的专属性、精密性及准确性,可用于重组人源抗hTNF-α单克隆抗体注射液中吐温80含量的检测。本研究为其他重组蛋白中吐温80含量的检测提供了实验依据。

Objective To improve and verify a cobaltthiocyanate ammonium color-developing method for determination of Tween 80 content in recombinant protein.Methods The cobaltthiocyanate ammonium color-developing method for determination of Tween 80 content in Chinese Pharmacopoeia(Vol Ⅳ,2020 edition) was improved by adjusting the dichloromethane content to 4.00 g and increasing the concentration of ammonium cobalt thiocyanate solution by 3 times,and verified for specificity,linear range,limit of qunatitation(LOQ),reproducibility,inter-precision and accuracy,by which the Tween 80 contents in three batches of recombinant human monoclonal antibody(McAb) against hTNF-αfor injection were determined.Results The improved method showed good specificity.The standard Tween 80 at concentrations of 0~400 μg/mL showed good linear relationship to the A620 value,with a regression equation of y = 0.000 6 x-0.003 4(R2= 0.999).The RSDs in tests for reproducibility and inter-precision were less than 5%.The LOQ was22.69 μg/mL.The recovery rates of standard Tween 80 solution at concentrations of 80,180 and 320 μg/mL were 100.00%,98.77% and 99.31% respectively.The Tween 80 contents in three batches of recombinant human McAb against TNF-α for injection were 0.95,1.00 and 1.02 mg/mL respectively.Conclusion The improved cobaltthiocyanate ammonium color-developing method showed good specificity,precision and accuracy, and might be used for determination of Tween 80 content in recombinant human McAb against TNF-α for injection, which provided an experimental basis for determination of Tween 80 content in other recombinant proteins.