Multimodal (MM) Chromatography offers highly selective protein separation systems by taking advantage of hydrophobic, electrostatic and Van der Waals interactions. Due to the complexity of such protein-ligand interactions, there is a need for the molecular level understanding of the nature of proteins binding to different MM chromatographic systems. In addition, it is important to understand the thermodynamics of these interactions, particularly the relative contributions of various entropic and enthalpic components under different conditions. Chromatographic retention data has demonstrated unique selectivity trends between two monoclonal antibodies (mAbs) on various MM CEX resins. In particular, we observed a selectivity reversal between Capto MMC and Nuvia cPrime, which differ in the presentation of functional groups on the chromatographic surface. The objective of this work is to obtain fundamental insights into thermodynamic driving forces involved in the interactions of these mAbs with MM ligands. Isocratic chromatography experiments were carried out with the two mAbs on Capto MMC and Nuvia cPrime at different temperatures and salt concentrations. Interestingly, mAb retention decreased with an increase in temperature, phenomena generally not observed in hydrophobic interaction chromatography. The chromatographic retention data was then evaluated using non-linear Van’t Hoff model to determine the changes associated with enthalpy, entropy, heat capacity and free energy upon adsorption. The governing thermodynamic driving force was observed to shift from enthalpic to entropic at various salt concentrations and temperatures. The isocratic data was also fit to the Melander model to obtain insights into relative electrostatic and hydrophobic contributions towards binding at different temperatures. Further, coarse-grained simulations were used to provide insights into the molecular nature of these interactions. The results in this presentation demonstrate how thermodynamic analysis of chromatographic data can be used in concert with molecular dynamic simulations to more deeply understand the connection between unique protein surface properties and the interactions of proteins to various multimodal surfaces.

Investigation into the thermodynamics of mAb: Ligand interactions in MM chromatography

255th National Meeting (2018)

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