Recently our lab demonstrated that mAbs have unique domain contributions to binding in multimodal systems, leading to different selectivities. The present work extends this study by investigating how pH impacts mAb retention in multimodal systems. Linear salt gradient experiments were carried out between pH 5-7 for several mAbs in multimodal cation exchange (MM CEX) resins. Avidity was considered by evaluating resins at two different ligand densities. Antibody domain contributions were also evaluated at several pH. A qualitative and quantitative analysis of important surface property patches informed the interpretation of the experimental results. mAb retention was shown to have an inverse, non-linear correlation with pH. Importantly, pH also changed the elution order in these multimodal systems, indicating that it can be used as a lever to create new windows of selectivity. While several of the MM CEX ligands showed similar behavior, a prototype ligand with increased aliphatic hydrophobicity demonstrated distinct selectivity trends, which were connected to unique domain contributions. Interestingly, one mAb showed a pH-dependent spectrum of domain contributions, indicating that pH could be used to tune the relative importance of the (Fab)<sub>2 </sub>and the Fc domains. The difference in retention for mAbs on high and low ligand density resins also showed a pH dependence. These results can facilitate the strategic design of multimodal steps for flow through, bind and elute, or weak partitioning modes and have important implications for creating integrated and/or continuous downstream processes where intermediate buffer exchange steps are minimized or eliminated. Furthermore, this work also has important implications for the purification of powerful mAb-related modalities such as bispecific antibodies and for the design of antibody-based therapeutics for increased biomanufacturability.

Investigating the effect of pH on mAb retention in multimodal chromatography

2019 Spring National Meeting

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