In this work, we employ a recently developed biophysical technique that uses diethylpyrocarbonate (DEPC) covalent labeling and mass spectrometry for the identification of mAb binding patches to two multimodal cation exchange resins at different pH. This approach compares the labeling results obtained in the bound and unbound states to identify residues that are sterically shielded and thus located in the mAb binding domains. The results at pH 6 for one mAb (mAb B) indicated that while the complementarity determining region (CDR) had minimal interactions with both resins, the F_C domain was actively involved in binding. In contrast, DEPC/MS data with another mAb (mAb C) indicated that both the CDR and F_C domains were actively involved in binding. These results corroborated chromatographic retention data with these two mAbs and their fragments and helped to explain the significantly stronger retention of both the intact mAb C and its Fab fragment. In contrast, labeling results with mAb C at pH 7, indicated that only the CDR played a significant role in resin binding, again corroborating chromatographic data. The binding domains identified from the DEPC/MS experiments were also examined using protein surface hydrophobicity maps obtained using a recently developed sparse sampling molecular dynamics (MD) approach in concert with electrostatic potential maps. These results demonstrate that the DEPC covalent labeling/mass spectrometry technique can provide important information about the domain contributions of multidomain proteins such as monoclonal antibodies when interacting with multimodal resins over a range of pH conditions.

中文翻译：

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使用生物物理学和模拟相结合的方法探索 IgG1 mAb 与多模式吸附剂的首选结合域

在这项工作中，我们采用了最近开发的生物物理技术，该技术使用焦碳酸二乙酯 (DEPC) 共价标记和质谱法来鉴定不同 pH 下两种多模式阳离子交换树脂的 mAb 结合斑块。该方法比较在结合和未结合状态下获得的标记结果，以识别空间屏蔽并因此位于 mAb 结合域中的残基。一种 mAb (mAb B) 在 pH 6 下的结果表明，虽然互补决定区 (CDR) 与两种树脂的相互作用极小，但 F _C结构域积极参与结合。相比之下，另一种 mAb (mAb C) 的 DEPC/MS 数据表明 CDR 和 F _C结构域均积极参与结合。这些结果证实了这两种 mAb 及其片段的色谱保留数据，并有助于解释完整 mAb C 及其 Fab 片段的保留明显更强。相比之下，mAb C 在 pH 7 下的标记结果表明，只有 CDR 在树脂结合中发挥着重要作用，再次证实了色谱数据。还使用最近开发的稀疏采样分子动力学 (MD) 方法与静电势图相结合获得的蛋白质表面疏水性图来检查从 DEPC/MS 实验中鉴定出的结合域。这些结果表明，DEPC 共价标记/质谱技术可以提供有关多域蛋白（例如单克隆抗体）在一定 pH 条件下与多峰树脂相互作用时的域贡献的重要信息。