Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696, United States.
J Chromatogr A. 2021 Oct 11;1655:462499. doi: 10.1016/j.chroma.2021.462499. Epub 2021 Aug 26.
This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.
这项工作描述了阳离子交换色谱（CEX）与紧凑、易用的台式飞行时间质谱仪（ToF/MS）的直接联用，用于单克隆抗体（mAb）的分析特性描述。为此，选择了广泛的商业 mAb 产品（包括过期样品和 mAb 生物类似物），以得出可靠的结论。从色谱角度来看，测试了各种缓冲液和柱尺寸。考虑到 pH 响应、缓冲液随时间的稳定性和 MS 兼容性，最佳折衷方案由以下配方代表：50 mM 乙酸铵，调至 pH 5.0（流动相 A）和 160 mM 乙酸铵，调至 pH 8.5（流动相 B）。尽管 2.1 mm id CEX 柱观察到的峰较宽，但仍优先选择用于 CEX-MS 操作，因为柱外分散引起的效率损失仍然可以接受，同时 MS 兼容性得到了很大增强（由于低流速）。就 MS 而言，避免使用玻璃瓶装的流动相、实验室玻璃器皿和玻璃小瓶很重要，以尽量减少由于金属污染物导致的 MS 分辨率、灵敏度和质量精度的损失。使用这种新的 CEX-MS 装置，可以进行简单快速的电荷变体分析（不到 10 分钟），允许分离和鉴定几种电荷变体。
