Pharmaceutical Candidate Optimization, Nonclinical Research and Development, Bristol Myers Squibb Company, Princeton, New Jersey 08540, United States.
Protein Engineering, Discovery Biotherapeutics, Bristol Myers Squibb, Redwood City, California 94063, United States.
Anal Chem. 2020 Aug 4;92(15):10709-10716. doi: 10.1021/acs.analchem.0c01876. Epub 2020 Jul 22.
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.
双特异性抗体（BsAbs）具有识别两个不同表位或抗原的独特机制，在各个治疗领域显示出了潜力。BsAbs 表位的分子特征不仅可以深入了解其作用机制，还可以指导候选药物分子的设计和选择。在这项研究中，我们展示了一种综合方法的实际应用，该方法包括尺寸排阻色谱法与多角度光散射和天然质谱（MS）联用，用于双特异性抗体与两个靶抗原（CD3 和 B 细胞成熟抗原）复合物形成的生物物理特性分析。基于 MS 的蛋白质足迹分析策略，包括氢/氘交换 MS、蛋白质快速光氧化和甘氨酸乙酯羧基足迹分析，进一步用于确定 BsAb 的结合表位。这种组合方法提供了 BsAb 与两个不同抗原结合的分子细节，具有快速输出和高分辨率的特点。
