Bietlot H P, Girard M
Bureau of Drug Research, Health Canada, Sir. F.G. Banting Research Centre, Ottawa, Ont., Canada.
J Chromatogr A. 1997 Jan 24;759(1-2):177-84. doi: 10.1016/s0021-9673(96)00767-4.
A high-performance capillary electrophoresis (HPCE) method was developed for the analysis of recombinant human erythropoietin (rhEPO) in final drug preparations. All products examined were formulated with large amounts of human serum albumin (HSA) which is used as a protein excipient. Due to their similar physical characteristics in solution, rhEPO and HSA could not be resolved under HPCE conditions previously developed for the separation of bulk rhEPO. Addition of 1 mM nickel chloride to the electrophoretic buffer allowed complete separation of the two proteins as well as separation of rhEPO into several glycoform populations. The method was linear over the concentration range of 0.03-1.92 mg/ml, with limits of detection and of quantitation of 0.01 and 0.03 mg/ml, respectively. The precision of the method was evaluated from intra- and inter-day replicate injections of both rhEPO standard solution and formulation. Components of within- and between-batch variances were consistently below 5%, which constituted an acceptable level of variation. Products from two manufacturers were analyzed and showed little qualitative but appreciable quantitative lot-to-lot variations for rhEPO content when expressed in terms of units of biological activity. The method also revealed qualitative differences between the two products.
建立了一种高效毛细管电泳（HPCE）方法，用于分析最终药物制剂中的重组人促红细胞生成素（rhEPO）。所有检测的产品都含有大量用作蛋白质辅料的人血清白蛋白（HSA）。由于rhEPO和HSA在溶液中的物理特性相似，在先前开发的用于分离大量rhEPO的HPCE条件下无法分离。在电泳缓冲液中加入1 mM氯化镍可使两种蛋白质完全分离，同时也能将rhEPO分离成几个糖型群体。该方法在0.03 - 1.92 mg/ml的浓度范围内呈线性，检测限和定量限分别为0.01和0.03 mg/ml。通过对rhEPO标准溶液和制剂进行日内和日间重复进样来评估该方法的精密度。批内和批间方差的组成部分始终低于5%，这构成了可接受的变异水平。对来自两个制造商的产品进行分析，结果表明，以生物活性单位表示时，rhEPO含量在质量上差异不大，但在数量上存在明显的批次间差异。该方法还揭示了两种产品之间的质量差异。
