Cotham Victoria C, Liu Anita P, Wang Shunhai, Li Ning
Analytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA.
Analytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA.
J Pharm Biomed Anal. 2023 May 10;228:115337. doi: 10.1016/j.jpba.2023.115337. Epub 2023 Mar 14.
Affinity chromatography coupled with native mass spectrometry has emerged as a powerful tool for the analysis of therapeutic monoclonal antibodies (mAbs). Exploiting the specific interactions between mAbs and their ligands, these methods not only provide orthogonal means to study the highly complex mAb attributes, but also offer insights on their biological relevance. Despite the great promise, application of affinity chromatography - native mass spectrometry in routine mAb characterization has been limited, largely due to the complicated experimental set up. In this study, we introduced a generic platform to facilitate the online coupling of different affinity separation modes with native mass spectrometry. Built upon a recently introduced native LC-MS platform, this new strategy can accommodate a wide range of chromatographic conditions, and therefore, allow greatly simplified experimental set up and facile swapping of affinity separation modes. The utility of this platform was demonstrated by successful online coupling of three affinity chromatography methods (protein A, FcγRIIIa, and FcRn) with native mass spectrometry. The developed protein A-MS method was tested both in a "bind-and-elute" mode for rapid mAb screening and in a high-resolution resolving mode to study mAb species with altered protein A affinity. The FcγRIIIa-MS method was applied to achieve glycoform-resolved analyses of both IgG1 and IgG4 subclass molecules. The FcRn-MS method was demonstrated in two case studies, where specific post-translational modifications and Fc mutations were known to alter FcRn affinities.
亲和色谱与原生质谱联用已成为分析治疗性单克隆抗体（mAb）的强大工具。这些方法利用单克隆抗体与其配体之间的特异性相互作用，不仅提供了研究高度复杂的单克隆抗体特性的正交方法，还能深入了解其生物学相关性。尽管前景广阔，但亲和色谱 - 原生质谱在常规单克隆抗体表征中的应用一直有限，主要原因是实验设置复杂。在本研究中，我们引入了一个通用平台，以促进不同亲和分离模式与原生质谱的在线联用。基于最近推出的原生液相色谱 - 质谱平台，这种新策略可以适应广泛的色谱条件，因此，可大大简化实验设置，并方便地切换亲和分离模式。通过成功地将三种亲和色谱方法（蛋白A、FcγRIIIa和FcRn）与原生质谱在线联用，证明了该平台的实用性。所开发的蛋白A - 质谱方法在“结合 - 洗脱”模式下用于快速单克隆抗体筛选，并在高分辨率解析模式下用于研究具有改变的蛋白A亲和力的单克隆抗体种类。FcγRIIIa - 质谱方法用于实现IgG1和IgG4亚类分子的糖型解析分析。FcRn - 质谱方法在两个案例研究中得到了验证，其中已知特定的翻译后修饰和Fc突变会改变FcRn的亲和力。
