Schlecht Johannes, Jooß Kevin, Moritz Bernd, Kiessig Steffen, Neusüß Christian
Department of Chemistry, Aalen University, Beethovenstrasse 1, 73430 Aalen, Germany.
Department of Pharmaceutical and Medicinal Chemistry, Friedrich Schiller University Jena, 07743 Jena, Germany.
Anal Chem. 2023 Feb 28;95(8):4059-4066. doi: 10.1021/acs.analchem.2c04578. Epub 2023 Feb 17.
Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.
电荷异质性的表征是治疗性单克隆抗体（mAb）药物开发和质量控制的重要支柱。含有大量ε-氨基己酸（EACA）的高度选择性且常用的毛细管区带电泳（CZE）方法可提供完整mAb变体详细且可靠的电荷异质性图谱。然而，这些电荷图谱中蛋白质修饰的确切位置仍不明确。电喷雾电离质谱（ESI-MS）是用于此目的的一种有前景的工具；然而，EACA与电喷雾不兼容。在此背景下，我们提出了一种二维CZE-CZE-MS系统，该系统将完整mAb的高效电荷变体分离与选定电荷变体在毛细管内消化后的后续肽段分析相结合。第一维基于熔融石英毛细管中的通用CZE（EACA）方法。在第二维中，使用中性涂层毛细管进行毛细管内还原和用三（2-羧乙基）膦（TCEP）和胃蛋白酶消化，随后对所得肽段进行CZE分离和MS/MS表征。使用应激和非应激mAb对该装置进行了演示，其中碱性、主要和酸性变体的峰以中心切割方式转移、消化并在肽段水平上进行表征。对于四种不同的mAb，重链（HC）和轻链（LC）的序列覆盖率均超过90%，包括低丰度变体（<主峰的2%）。可以检测到并定位常见的修饰（脱酰胺、氧化等）。本研究证明了从CZE电荷异质性图谱中鉴定和定位蛋白质修饰的概念验证，有望以此支持蛋白质药物的开发和质量控制测试。
