NovaBioAssays LLC, Woburn, MA, USA.
CMP Scientific Corp, Brooklyn, NY, USA.
Methods Mol Biol. 2022;2500:55-65. doi: 10.1007/978-1-0716-2325-1_6.
Monoclonal antibodies (mAbs) are one of the most widely used types of protein therapeutics. Charge variants are important quality attributes for evaluating developability, activity, and safety for mAb therapeutics. Here, we report a novel online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for mAb charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. Key factors that enable online CIEF-MS include effective capillary electrophoresis-MS (CE-MS) interface with enhanced sensitivity, utilization of MS-friendly electrolytes, beneficial effects of glycerol that reduces non-CIEF electrophoretic mobility and limits band broadening, appropriate ampholyte type and concentration selection for balanced separation resolution and MS detection sensitivity, optimized sheath liquid composition to realize high-resolution CIEF separation and effective MS electrospray ionization, as well as judiciously selected CIEF running parameters. The fundamental premise of CIEF has been verified by the linear correlation between isoelectric point (pI) values and migration time using a mixture of pI markers. By achieving high separation resolutions that are similar as those obtained from imaged CIEF (iCIEF), this method successfully provides highly sensitive MS identification for intact mAb charge variants. Furthermore, a middle-up sample treatment workflow can be adopted to provide in-depth charge variant analysis at subunit level for mAbs with complex charge heterogeneity. The mAb subunit CIEF-MS reveals the source of charge variant with enhanced resolution on both CIEF separation and MS spectra. This novel CIEF-MS method is a valuable tool with distinct advantage for objective and accurate assessment of charge heterogeneity of protein therapeutics.
单克隆抗体（mAbs）是应用最广泛的蛋白治疗药物之一。电荷变异体是评估 mAb 治疗药物开发性、活性和安全性的重要质量属性。在此，我们报道了一种新型的在线毛细管等电聚焦-质谱（CIEF-MS）方法，用于 mAb 电荷变异体分析，该方法在飞行时间（TOF）质谱上采用电动泵送鞘流纳米喷雾离子源，通过压力辅助化学迁移化。实现在线 CIEF-MS 的关键因素包括：具有增强灵敏度的有效毛细管电泳-MS（CE-MS）接口、使用对 MS 友好的电解质、甘油的有益作用，可降低非 CIEF 电泳迁移率并限制谱带展宽、选择合适的两性电解质类型和浓度以实现平衡分离分辨率和 MS 检测灵敏度、优化鞘液组成以实现高分辨率 CIEF 分离和有效的 MS 电喷雾电离，以及明智地选择 CIEF 运行参数。通过使用等电点（pI）标记物混合物，在等电点（pI）值和迁移时间之间建立线性相关性，验证了 CIEF 的基本前提。通过实现与成像 CIEF（iCIEF）获得的相似的高分离分辨率，该方法成功地为完整的 mAb 电荷变异体提供了高灵敏度的 MS 鉴定。此外，可以采用中向上的样品处理工作流程，为具有复杂电荷异质性的 mAb 提供亚基水平的深入电荷变异体分析。mAb 亚基 CIEF-MS 揭示了电荷变异体的来源，在 CIEF 分离和 MS 谱图上都具有增强的分辨率。这种新型的 CIEF-MS 方法是一种具有明显优势的有价值工具，可用于客观、准确地评估蛋白治疗药物的电荷异质性。
