毛细管区带电泳分离重组人红细胞生成素方法的改进

Improvement of the separating method for rhEPO by capillary zone electrophoresis

目的:针对欧洲药典收载的rhEPO毛细管电泳检测方法中pH限制严格,可操作性差的缺点,进行缓冲液组成的优化。方法:实验将响应面分析法(RSM)用于毛细管区带电泳,用最少的实验次数确定了对分离影响最大的因素,并由此确定了最佳缓冲液组成及pH范围。结果:在此优化条件下rhEPO的8个不同糖基化形式在40min内得到了基线分离,各相邻峰间的分离度达1.63～3.29,柱效达1.63×10~5～3.23×10~5理论塔板数·m^(-1),峰形良好。结论:方法具有较宽泛的pH范围,比欧洲药典更为实用。

Objective：The separating method of rhEPO by capillary electrophoresis stipulated in the EP is thought to be of faultiness for its strictly limited buffer pH, which directly leads to be weak in maneuverability. The paper, focusing on mending the shortcoming, carried on the buffer composition optimazation. Method： The optimization process was based on least experiments by employing a central composite design. A pareto chart of standardized effects was performed for estimating effect of each variable. Then the single factor optimization was carried out for the determinant, and finally the optimized buffer composition was obtained. Result ：Putrescine concentration was found to be affecting the biggest and the most efficient separation was achieved with a mixed buffer with 3.0 mmol · L^-1 putrescine at pH value 5. 20 -5. 80. Conclusion：The method with broad pH scope reported in this paper is applicable to sepa- rate the glycoforms of rhEPO and can be used to control the quality of rhEPO,instead that of the EP.