Zhang Liangyi, Robinson Thomas J, Schmidt Brian D
Analytical Development, Process Sciences, AbbVie Biotherapeutics Inc., 1500 Seaport Boulevard, Redwood City, CA 94063 United States.
Analytical Development, Process Sciences, AbbVie Biotherapeutics Inc., 1500 Seaport Boulevard, Redwood City, CA 94063 United States.
J Chromatogr A. 2014 Nov 7;1367:109-17. doi: 10.1016/j.chroma.2014.09.051. Epub 2014 Sep 30.
Analytical cation-exchange chromatography (CEX) is widely used to profile the charge heterogeneity of therapeutic monoclonal antibodies (mAbs). However, the consistency of CEX profiles of a mAb can be significantly reduced by metal ion impurities from sample, mobile phase or leachates from the stainless steel components of the pumping system. In this work, we have developed a new CEX method that dynamically removes metal ions during sample analysis by incorporating the use of chelating agents (1-5mM) in HPLC mobile phases. Among four different chelating agents that were evaluated, EDTA and oxalic acid showed excellent capability of removing metal ions and provided consistent CEX chromatograms for mAb1. Furthermore, the use of oxalic acid in mobile phases not only improved the reproducibility of CEX chromatograms, but also increased the resolution of charge isoforms. Oxalic acid appears capable of binding to mAbs and reducing the positive surface charge density, resulting in a modulation of chromatographic separation. Due to this modulation effect, the CEX resolution was dependent on the concentration of the chelating agent. Optimal resolution for mAb1 was obtained with 2mM of oxalic acid. The oxalic acid modulated CEX method was shown to be capable of monitoring the degradation of mAb1. We further qualified this method according to International Committee on Harmonization (ICH) guidelines and demonstrated that the oxalic acid modulated CEX method is precise and robust at different chromatographic conditions and is suitable for use in a development and/or GMP setting.
分析型阳离子交换色谱法（CEX）被广泛用于分析治疗性单克隆抗体（mAb）的电荷异质性。然而，样品、流动相中的金属离子杂质或泵系统不锈钢部件的沥出物会显著降低mAb的CEX图谱的一致性。在本研究中，我们开发了一种新的CEX方法，通过在高效液相色谱流动相中加入螯合剂（1-5mM），在样品分析过程中动态去除金属离子。在评估的四种不同螯合剂中，乙二胺四乙酸（EDTA）和草酸表现出优异的去除金属离子的能力，并为mAb1提供了一致的CEX色谱图。此外，在流动相中使用草酸不仅提高了CEX色谱图的重现性，还提高了电荷异构体的分辨率。草酸似乎能够与mAb结合并降低正表面电荷密度，从而调节色谱分离。由于这种调节作用，CEX分辨率取决于螯合剂的浓度。使用2mM草酸时，mAb1获得了最佳分辨率。结果表明，草酸调节的CEX方法能够监测mAb1的降解情况。我们根据国际协调会议（ICH）指南对该方法进行了进一步验证，结果表明，草酸调节的CEX方法在不同色谱条件下精确且稳健，适用于研发和/或GMP环境。
