Understanding and enhancing the selectivity of Multimodal protein Chromatography

Prof. Steven Cramer, Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute

14 November 2013 at 10:30

Location: JHE 326H


In this work a detailed investigation into the engineering of multiple weak interactions to create selective multimodal protein separation systems was carried out. This research seeks to determine what conditions are required to achieve selective separations of similar protein variants and to provide fundamental insight into the mechanisms underlying these separations.  The retention of protein libraries on several multimodal cation-exchange systems, including Capto MMC and Nuvia cPrime was first under a range of fluid phase modifier conditions. While these ligands are constructed from similar functional groups (a phenyl ring and carboxylic acid), the retention of many proteins proved to be sensitive to subtle changes in the ligand chemistry and geometrical presentation that affected the exposure of the phenyl ring to the surrounding solvent.  Further, the effects of fluid phase modifiers were found to be quite different for the adsorption of various proteins in the two MM systems. All-atom explicit Molecular Dynamics (MD) simulations were then carried out to shed light on the multiple weak interactions that resulted in the unique selectivities achieved in these multimodal chromatographic systems. The knowledge from these simulations was also used to deconvolute synergistic MM interactions into its key contributors. This was coupled with protein surface characterization techniques to evaluate the strength and importance of electrostatic and hydrophobic interactions in these systems. Simulations were also performed to evaluate the interactions of fluid phase modifiers with proteins and to study how they enhance/reduce protein-ligand binding.  A range of biophysics techniques is also employed to study the energetics, kinetics and thermodynamics of protein binding to self-assembled monolayers (SAMs) of MM ligands. This work provides fundamental understanding of the nature of these interactions at the molecular level and insight into the design of MM ligands, the roles of synergy and the modulation of selectivity using fluid phase modifiers with important implications for addressing challenging problems in downstream bioprocessing.

Return to list of all seminars