Biologics and Genetic Therapies Directorate, Health Canada, 251-Sir Frederick Banting Driveway, Ottawa, K1A 0K9, Canada.
Anal Chim Acta. 2012 Feb 3;713:7-22. doi: 10.1016/j.aca.2011.11.041. Epub 2011 Nov 25.
Human erythropoietin (hEPO), a hormone involved in the formation of red blood cells, is a 30 kDa glycoprotein with a high carbohydrate content. The production of recombinant hEPO has made possible its widespread therapeutic use and its banned use in competition sports. Methods to analyze EPO and other erythropoiesis stimulating agents (ESAs) are necessary for the characterization and quality control of these biopharmaceuticals and also for doping control. In this paper, high resolution separation methods, namely high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with special attention to CE-coupled mass spectrometry, are reviewed. The usefulness of these techniques when applied in different modes to separate the glycoprotein isoforms, aggregates or excipients are detailed. In addition, sample preparation methods that have been applied to ESA samples for subsequent determination by HPLC or CE, as well as the potential compatibility of other preparation methods, are discussed. Applications of the HPLC and CE methods regarding regulatory considerations for biopharmaceuticals analysis, with emphasis on biosimilars, and doping control are also included. Finally, limitations of the present methods and their possible solutions are considered.
人红细胞生成素（hEPO）是一种参与红细胞生成的激素，是一种 30 kDa 的糖蛋白，碳水化合物含量很高。重组 hEPO 的生产使其在治疗上得到广泛应用，并禁止在竞技体育中使用。分析 EPO 和其他促红细胞生成素刺激剂（ESAs）的方法对于这些生物制药的表征和质量控制以及兴奋剂控制都是必要的。本文综述了高分辨率分离方法，即高效液相色谱（HPLC）和毛细管电泳（CE），特别关注 CE 与质谱的联用。详细说明了这些技术在不同模式下用于分离糖蛋白异构体、聚集体或赋形剂的有用性。此外，还讨论了用于随后通过 HPLC 或 CE 测定的 ESA 样品的样品制备方法，以及其他制备方法的潜在兼容性。还包括了 HPLC 和 CE 方法在生物制药分析（重点是生物类似药）和兴奋剂控制方面的监管考虑因素的应用。最后，考虑了当前方法的局限性及其可能的解决方案。
