Bispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and their involvement in IgG4 mediated autoimmune diseases. While the production of BsAbs is getting more accessible, their analytical characterization remains challenging. We explored the potential of sheathless CE‐MS for monitoring exchange efficiency and stability of in‐house produced bispecific antibodies. Two IgG4 bispecific antibodies with different molecular characteristics were prepared using controlled Fragment antigen binding (Fab)‐arm exchange. Separation of BsAbs from their parent monospecific antibodies was achieved using a polyethyleniimine (PEI)‐coated capillary and acidic background electrolytes permitting reliable assessment of the exchange efficiency. This was especially valuable for a Fab‐glycosylated BsAb where the high glycan heterogeneity resulted in an overlap of masses with the monospecific parent antibody, hindering their discrimination by MS only. The method showed also good capabilities to monitor the stability of the generated BsAbs under different storage conditions. The levels of degradation products were different for the studied antibodies indicating pronounced differences in stability. Overall, the proposed method represents a useful analytical tool for exchange efficiency and stability studies of bispecific antibodies.

中文翻译：

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无鞘 CE-MS 作为监测双特异性抗体交换效率和稳定性的工具。

双特异性单克隆抗体 (BsAbs) 因其作为生物药物的广泛益处以及参与 IgG4 介导的自身免疫性疾病而受到极大关注。虽然 BsAb 的生产越来越容易获得，但它们的分析表征仍然具有挑战性。我们探索了无鞘 CE-MS 在监测内部生产的双特异性抗体的交换效率和稳定性方面的潜力。使用受控片段抗原结合 (Fab) 臂交换制备了两种具有不同分子特征的 IgG4 双特异性抗体。使用聚乙烯亚胺 (PEI) 涂层的毛细管和酸性背景电解质实现了 BsAb 与其亲本单特异性抗体的分离，从而可以可靠地评估交换效率。这对于 Fab 糖基化 BsAb 尤其有价值，因为高聚糖异质性导致与单特异性亲本抗体的质量重叠，阻碍了它们仅通过 MS 进行区分。该方法还显示出在不同储存条件下监测生成的 BsAb 稳定性的良好能力。所研究抗体的降解产物水平不同，表明稳定性存在显着差异。总体而言，所提出的方法代表了用于双特异性抗体的交换效率和稳定性研究的有用分析工具。该方法还显示出在不同储存条件下监测生成的 BsAb 稳定性的良好能力。所研究抗体的降解产物水平不同，表明稳定性存在显着差异。总体而言，所提出的方法代表了用于双特异性抗体的交换效率和稳定性研究的有用分析工具。该方法还显示出在不同储存条件下监测生成的 BsAb 稳定性的良好能力。所研究抗体的降解产物水平不同，表明稳定性存在显着差异。总体而言，所提出的方法代表了用于双特异性抗体的交换效率和稳定性研究的有用分析工具。