The present paper builds upon previous work on mAb domain contributions to multimodal (MM) chromatography by examining how pH can impact mAb surface properties and retention in these systems. Linear salt gradient experiments were carried out between pH 5-7 for several mAbs with different pI and surface hydrophobicities in four different MM CEX resins at two ligand densities. mAb retention showed an inverse, non-linear correlation with pH. Changing pH affected the elution order, creating unique windows of selectivity in each of the MM CEX resins. One mAb showed a pH-dependent spectrum of domain contributions, demonstrating that pH can be used to tune the relative importance of the (Fab)2 and Fc domains for some mAbs in MM systems. Positive, negative, and hydrophobic patches were calculated between pH 5-7 for the mAbs. Visualizing these patches on the protein surface demonstrated that each mAb showed a unique distribution of surface charge and hydrophobicity that changed with pH. The sum of patch areas was tracked across this pH range to quantitatively understand how pH impacted these important surface properties. The quantitative analysis then was narrowed to consider only patches in the CDR loops, which were hypothesized to be an important interaction site for some mAbs in these systems. Interestingly, differences in the titration of CDR loop patches for each mAb were shown to be a result of Histidine titrations and patches in this region were qualitatively correlated with experimental trends including the observed elution order reversals. These results indicate that pH potentially can be employed as a lever for the strategic design of multimodal steps to create flow through, bind and elute, or weak partitioning operations with important implications for the design of integrated and/or continuous downstream purification processes. Furthermore, the ability to tune domain contributions in MM separations using pH creates intriguing possibilities for current downstream challenges such as the removal of product-related impurities, as well as the purification of bispecific mAbs.

中文翻译：

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pH对多峰阳离子交换色谱系统中抗体保留的影响。

本文通过检查pH值如何影响mAb表面特性和在这些系统中的保留度，以先前关于mAb域对多峰（MM）色谱分析的贡献为基础。在四种配体密度不同的四种MM CEX树脂中，对几种具有不同pI和表面疏水性的mAb在pH 5-7之间进行了线性盐梯度实验。mAb保留率与pH呈反比，非线性关系。改变pH值会影响洗脱顺序，从而在每种MM CEX树脂中产生独特的选择性窗口。一个mAb显示了pH依赖的域贡献谱，表明pH可以用于调整MM系统中某些mAb的（Fab）2和Fc域的相对重要性。计算mAb的pH 5-7之间的阳性，阴性和疏水性斑点。可视化蛋白质表面上的这些斑块表明，每个mAb均具有随pH改变的独特的表面电荷和疏水性分布。在此pH范围内跟踪贴片面积的总和，以定量了解pH如何影响这些重要的表面性能。然后缩小了定量分析的范围，只考虑了CDR环中的补丁，这些补丁被认为是这些系统中某些mAb的重要相互作用位点。有趣的是，每个mAb的CDR环补片滴定度的差异显示为组氨酸滴定的结果，该区域中的补片与实验趋势（包括观察到的洗脱顺序颠倒）定性相关。这些结果表明，pH可以用作多峰步骤策略设计的杠杆，以创建流过，结合和洗脱或弱分配操作，这对集成和/或连续下游纯化工艺的设计具有重要意义。此外，使用pH调节MM分离中的结构域贡献的能力为当前的下游挑战（例如去除产物相关杂质以及纯化双特异性mAb）创造了诱人的可能性。