Anal Chem. 2020 Jul 7;92(13):9312-9321. doi: 10.1021/acs.analchem.0c01641. Epub 2020 Jun 19.
Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.
双特异性抗体因其高特异性和能够将免疫细胞靶向肿瘤而备受关注，是一种很有前途的免疫治疗药物。然而，由于在细胞培养中生产时可能会产生多种形式的抗体，因此对双特异性抗体的分析具有挑战性。大多数双特异性抗体的分析都依赖于带有质谱（LC-MS）的液相色谱，而如果这些形式没有物理分离，那么这种方法可能会漏检或定量能力降低。在这里，我们报告了一种新的、灵敏的混合模式排阻色谱（MM SEC）与多角度光散射（MALS）相结合的方法，可在天然条件下分析不同形式的双特异性 IgG 分子。该方法具有出色的分离各种抗体形式的能力，峰分辨率超过我们测试过的其他方法，可分离出所需的双特异性分子、亲本同二聚体、半分子以及轻链和重链错配的抗体。通过在线 MALS 对每个峰进行分析，然后通过对分离形式的完整和还原 LC-MS 进行鉴定和确认。本研究中的 MM SEC 通过与分布在轻链 CDR 上的蛋白质表面疏水区簇的相互作用来发挥作用，这是一种新颖的机制。这种新型 MM SEC 允许对低丰度形式进行定量检测，并为筛选细胞培养克隆的表达谱、监测纯化和评估药物物质纯度提供了新工具。
