Separation and Analysis Technology Team , Bristol-Myers Squibb Research and Development , Post Office Box 4000, Princeton , New Jersey 08543 , United States.
Anal Chem. 2018 Dec 18;90(24):14527-14534. doi: 10.1021/acs.analchem.8b04396. Epub 2018 Dec 4.
Previously, we reported a new online capillary isoelectric focusing/mass spectrometric (CIEF/MS) method for intact monoclonal antibody (mAb) charge variant analysis that uses an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. The direct online CIEF/MS method exhibited excellent resolution of charge variants conforming to those of imaged capillary isoelectric focusing with ultraviolet detection (iCIEF/UV). However, for complex mAbs, CIEF/MS spectra of the intact charge variant peaks may be too convoluted to be effectively interpreted. In the present study, we implemented a middle-up approach to enhance the capability of the CIEF/MS method for characterizing complex mAb charge variants by reducing sample complexity. To demonstrate such a strategy, we fragmented cetuximab through IdeS enzymatic cleavage and dithiothreitol (DTT) reduction. For the first time, online CIEF/MS resolved the complex charge variants of cetuximab at subunit level, corroborating the profiles obtained by iCIEF/UV. Furthermore, high-resolution TOF mass spectra with high mass accuracy were obtained for the eight charge variants separated by CIEF/MS after IdeS cleavage and for the 11 charge variants after IdeS digestion with subsequent DTT reduction. In-depth analyses revealed the identities of all charge variants and pinpointed the causes of charge heterogeneity, which are in accord with those reported in the literature. The main sources of charge heterogeneity of cetuximab were identified as terminal lysine on the Fc domain (up to one on each single-chain Fc), glycolylneuraminic acid residues on the Fd' domain (up to two on each Fd'), and likely several deamidation species on the Fd' domain. No charge heterogeneity contribution was found from light chain. The in-depth characterization of complex charge variants for cetuximab demonstrates the remarkable capability of this middle-up CIEF/MS approach. This novel workflow holds great potential for detecting and elucidating charge variants to help understand proteins with complex charge heterogeneity.
此前，我们报道了一种新的在线毛细管等电聚焦/质谱（CIEF/MS）方法，用于完整单克隆抗体（mAb）电荷变异体分析，该方法在带有压力辅助化学迁移的飞行时间（TOF）MS 上使用电动力学泵送鞘流纳米喷雾离子源。直接在线 CIEF/MS 方法对符合成像毛细管等电聚焦与紫外检测（iCIEF/UV）的电荷变异体表现出优异的分辨率。然而，对于复杂的 mAb，完整电荷变异体峰的 CIEF/MS 光谱可能过于复杂，难以有效解释。在本研究中，我们采用中向上方法来增强 CIEF/MS 方法对复杂 mAb 电荷变异体进行表征的能力，从而降低样品的复杂性。为了证明这种策略，我们通过 IdeS 酶切和二硫苏糖醇（DTT）还原使西妥昔单抗发生片段化。首次在线 CIEF/MS 解析了西妥昔单抗的复杂电荷变异体，达到亚基水平，与 iCIEF/UV 获得的图谱一致。此外，在 IdeS 切割后通过 CIEF/MS 分离的 8 种电荷变异体和 IdeS 消化后随后进行 DTT 还原的 11 种电荷变异体均获得了高分辨率的 TOF 质谱，具有高质量精度。深入分析揭示了所有电荷变异体的身份，并确定了电荷异质性的原因，这与文献中报道的一致。西妥昔单抗电荷异质性的主要来源被鉴定为 Fc 结构域末端赖氨酸（每个单链 Fc 上多达一个）、Fd'结构域糖基神经氨酸残基（每个 Fd'上多达两个），以及可能存在多个 Fd'结构域上的脱酰胺化产物。未发现轻链对电荷异质性有贡献。对西妥昔单抗复杂电荷变异体的深入表征证明了这种中向上 CIEF/MS 方法的卓越能力。这种新的工作流程具有很大的潜力，可用于检测和阐明电荷变异体，以帮助理解具有复杂电荷异质性的蛋白质。
