毛细管区带电泳方法评价曲妥珠单抗类似物的电荷异质性

目的：建立毛细管区带电泳（CZE）方法用于评价曲妥珠单抗类似物的电荷异质性。方法：实验中选用甲基纤维素（MC）和尿素作为缓冲液添加剂以改善曲妥珠单抗类似物电荷变体间的分离；0.1 mol·L^-1盐酸溶液与0.1 mol·L^-1氢氧化钠溶液前处理冲洗以有效减少单抗吸附。本文考察了运行缓冲液pH及浓度、分离电压、温度、添加剂对主峰与酸、碱变体峰分离度的影响。优化电泳条件为：5 kPa进样10 s，分离温度为15 ℃，分离电压为20 kV，运行缓冲液为600 mmol·L^-1 ε-氨基己酸（EACA）pH 5.4，包含0.1% MC和4 mol·L^-1尿素溶液。结果：在优化的实验条件下，曲妥珠单抗类似物与其电荷变体能够得到较好分离，并且主成分的相对百分含量与迁移时间的日内精密度均小于2%。结论：本方法操作简单，分离度良好，成本较低，可以满足曲妥珠单抗类似物电荷异质性的分析要求。

Objective: To develop a method of capillary zone electrophoresis to analyze the charge heterogeneity of trastuzumab biosimilar. Methods: Resolution can be improved among trastuzumab biosimilar charge variants after adding methyl cellulose(MC)and urea.Utilizing 0.1 mol·L^-1 hydrochloric acid and 0.1 mol·L^-1 sodium hydroxide to flush capillary prior to loading each sample can reduce adsorption of antibodies.A number of separation parameters were investigated and optimized,including pH,concentration of the separation buffer,concentration of urea,voltage and temperature.And the optimized conditions were as follows:the sample was injected under pressure of 5 kPa for 10 s;the separation temperature and voltage were 15 ℃ and 20 kV,respectively.The running buffer was 600 mmol·L^-1ε-amino-caproic acid(EACA)containing 0.1% MC,4 mol·L^-1 urea,pH 5.4. Results: Under the optimal conditions, the trastuzumab biosimilar showed good resolution between the main peak and the peaks for the acid and basic variants.Intraday precision studies performed on the CZE method with three charge variants yielded good separation,and the relative percentage contents and migration time for the main component had a RSD of less than 2%. Conclusion: The method has several advantages including simple operation,good separation resolution,and low cost(uncoated capillary),and can meet the requirements of trastuzumab biosimilar charge heterogeneity analysis.