Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting.,Analytical Chemistry

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Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-07-08 , DOI: 10.1021/acs.analchem.0c01876
Richard Y-C Huang ₁ , Feng Wang ₂ , Matthew Wheeler ₃ , Yun Wang ₁ , Robert Langish ₁ , Bryant Chau ₂ , Jia Dong ₂ , Winse Morishige ₂ , Natalie Bezman ₃ , Pavel Strop ₂ , Arvind Rajpal ₂ , Olafur Gudmundsson ₁ , Guodong Chen ₁

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Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs’ epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA). MS-based protein footprinting strategies, including hydrogen/deuterium exchange MS, fast photochemical oxidation of proteins, and carboxyl group footprinting with glycine ethyl ester, were further applied to determine BsAb’s binding epitopes. This combination approach provides molecular details on the binding mechanisms of BsAb to the two distinct antigens with rapid output and high resolution.

中文翻译：

表征双特异性抗体/抗原复合物和通过SEC / MALS，天然质谱和蛋白质印迹法绘制结合表位的综合方法。

具有识别两种不同表位或抗原的独特机制的双特异性抗体（BsAbs）在各种治疗领域均显示出潜力。BsAbs表位的分子表征不仅可以详细了解其作用机理，还可以指导候选药物分子的设计和选择。在这项研究中，我们说明了一种集成方法的实际实用性，包括采用多角度光散射的尺寸排阻色谱法和天然质谱（MS）来对具有两种靶抗原，分化簇3（CD3）的BsAb的复合物形成进行生物物理表征。）和B细胞成熟抗原（BCMA）。基于质谱的蛋白质足迹策略，包括氢/氘交换质谱，蛋白质的快速光化学氧化，进一步使用甘氨酸乙酯的羧基和羧基足迹，来确定BsAb的结合表位。这种组合方法提供了有关BsAb与两种不同抗原的结合机制的分子细节，且输出迅速且具有高分辨率。

更新日期：2020-08-04

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