Recent work in our lab has shown the importance of certain domains of a monoclonal antibody (mAb) to be important for binding to different Multimodal (MM) chromatographic systems. Identifying the binding regions on the protein surface is of very high importance for designing efficient bioseparation processes for biologics products. Interaction of certain mAbs was governed by the (Fab)2 domain whereas other mAbs interacted via the FC region of the mAb. In this work, we extend this investigation to study the effect of pH on the binding of mAb to these MM chromatographic systems and evaluate the selectivity trends in these systems. For this study, we used NIST mAb that has been extensively studied by many research labs across the country. First, linear salt gradient chromatography experiments were performed with NIST mAb on various MM cation exchange chromatographic systems. To further investigate the regions on the protein surface that were involved in binding, the NIST mAb was enzymatically digested to get the Fab, (Fab)2 and FC domains. The retention of these domains was evaluated on MM chromatographic resins as a function of pH. There was a shift in the important domain of interaction from pH 5 to 7. While the FC domain was important for binding at pH, the Fab domain appeared to be important for binding at pH 7. By comparing these results with Electrostatic Potential (EP) and Surface Aggregation Propensity (SAP) maps under different conditions, we developed a heuristic understanding of how these shifts were a function of protein surface properties. This work has enabled us to develop a deeper understanding of how different regions on a protein surface interact with the MM chromatographic resins under various conditions and how the pH can be used to modulate the selectivity in these MM systems.

Investigation into the pH effects in multimodal chromatography using NIST mAb and its domains

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.