†Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg 67000, France.
‡Centre d'Immunologie Pierre Fabre, Saint-Julien-en-Genevois 74164, France.
Anal Chem. 2015 Jun 16;87(12):6240-50. doi: 10.1021/acs.analchem.5b00928. Epub 2015 May 22.
Monoclonal antibodies (mAbs) are highly complex glycoproteins that present a wide range of microheterogeneities that requires multiple analytical methods for full structure assessment and quality control. Capillary zone electrophoresis-mass spectrometry (CZE-MS) couplings, especially by electrospray ionization (ESI), appear to be really attractive methods for the characterization of biological samples. However, due to the presence of non- or medium volatile salts in the background electrolyte (BGE), online CZE-ESI-MS coupling is difficult to implement for mAbs isoforms separation. Here, we report an original strategy to perform off-line CZE-ESI-MS using CZE-UV/fraction collection technology to perform CZE separation, followed by ESI-MS infusion of the different fractions using the capillary electrophoresis-electrospray ionization (CESI) interface as the nanoESI infusion platform. As the aim is to conserve electrophoretic resolution and complete compatibility with ESI-MS without sample treatment, hydroxypropylcellulose (HPC) coated capillary was used to prevent analyte adsorption and asymmetric CZE conditions involving different BGE at both ends of the capillary have been developed. The efficiency of our strategy was validated with the separation of Cetuximab charge variant by the middle-up approach. Molecular weights were measured for six charge variants detected in the CZE separation of Cetuximab subunits. The first three peaks correspond to Fc/2 variants with electrophoretic resolution up to 2.10, and the last three peaks correspond to F(ab')2 variants with average electrophoretic resolution of 1.05. Two Fc/2 C-terminal lysine variants were identified and separated. Moreover, separation of Fc/2 fragments allowed the glycoprofiling of the variants with the characterization of 7 different glycoforms. Regarding the F(ab')2 domain, 8 glycoforms were detected and separated in three different peaks following the presence of N-glycolyl neuraminic acid residues in some glycan structures. This work highlights the potential of CZE technology to perform separation of mAbs especially when they carry sialic acid carbohydrates.
单克隆抗体（mAbs）是高度复杂的糖蛋白，呈现出广泛的微观不均一性，需要多种分析方法来全面评估结构并进行质量控制。毛细管区带电泳-质谱（CZE-MS）联用技术，特别是通过电喷雾电离（ESI），似乎是用于生物样品特性描述的极具吸引力的方法。然而，由于背景电解质（BGE）中存在非挥发性或中等挥发性盐，在线 CZE-ESI-MS 联用对于 mAbs 异构体分离来说很难实现。在这里，我们报告了一种原始策略，使用 CZE-UV/馏分收集技术进行离线 CZE-ESI-MS，先进行 CZE 分离，然后使用毛细管电泳-电喷雾电离（CESI）接口作为纳喷雾电离（nanoESI）进样平台对不同馏分进行 ESI-MS 注入。由于目的是在不进行样品处理的情况下保留电泳分辨率并与 ESI-MS 完全兼容，因此使用羟丙基纤维素（HPC）涂层毛细管防止分析物吸附，并开发了涉及毛细管两端不同 BGE 的不对称 CZE 条件。通过中间向上方法分离西妥昔单抗的电荷变异体来验证我们策略的效率。在西妥昔单抗亚基的 CZE 分离中检测到六种电荷变异体，测量了它们的分子量。前三个峰对应于 Fc/2 变异体，电泳分辨率高达 2.10，后三个峰对应于 F(ab')2 变异体，平均电泳分辨率为 1.05。鉴定并分离了两个 Fc/2 C 末端赖氨酸变异体。此外，Fc/2 片段的分离允许对变体进行糖基化分析，对 7 种不同的糖型进行了表征。对于 F(ab')2 结构域，在一些糖结构中存在 N-糖基神经氨酸残基的情况下，检测到并分离了 8 种糖型，它们在三个不同的峰中呈现。这项工作突出了 CZE 技术在分离 mAbs 方面的潜力，尤其是当它们携带唾液酸碳水化合物时。
