A certain impurity may bind weaker or tighter to a particular type of chromatography column (e.g., ion exchange, hydrophobic interaction or mixed-mode) than the target protein, and this forms the basis for separation. For impurities that bind weaker, they can be removed by an appropriate pre-elution wash. However, we previously showed that wash-enabled impurity clearance is usually sensitive to loading density, causing poor robustness of the process. In this work, with a bispecific antibody case study, we compared the relative robustness of anion exchange and the corresponding mixed-mode chromatography, which mediates both anion exchange and hydrophobic interactions, on removing a weakly-bound byproduct by wash. It was learned that under a fixed appropriate wash condition, the latter achieves consistent byproduct clearance and good yield over a much wider range of loading density than the former. As wide loading density range is highly desirable for large-scale manufacturing, the above finding suggests that for a chromatography step that employs stepwise elution and relies on pre-elution wash for removing weakly-bound impurities, mixed-mode chromatography could be a better choice than the corresponding monomode ion exchange chromatography.

某种杂质可能比目标蛋白质更弱或更紧密地与特定类型的色谱柱（例如离子交换、疏水相互作用或混合模式）结合，这构成了分离的基础。对于结合较弱的杂质，可以通过适当的预洗脱洗涤将其除去。然而，我们之前表明，启用洗涤的杂质清除通常对负载密度敏感，导致该过程的稳健性差。在这项工作中，通过双特异性抗体案例研究，我们比较了阴离子交换和相应的混合模式色谱法（介导阴离子交换和疏水相互作用）在通过洗涤去除弱结合副产物方面的相对稳健性。据了解，在固定适宜的洗涤条件下，与前者相比，后者在更广泛的装载密度范围内实现了一致的副产品清除率和良好的产量。由于大规模生产非常需要宽负载密度范围，因此上述发现表明，对于采用逐步洗脱并依靠预洗脱洗涤来去除弱结合杂质的色谱步骤，混合模式色谱可能是更好的选择比相应的单模离子交换色谱法。