The effect of geometrical presentation of multimodal cation-exchange ligands on selective recognition of hydrophobic regions on protein surfaces.

The effects of spatial organization of hydrophobic and charged moieties on multimodal (MM) cation-exchange ligands were examined by studying protein retention behavior on two commercial chromatographic media, Capto™ MMC and Nuvia™ cPrime™. Proteins with extended regions of surface-exposed aliphatic residues were found to have enhanced retention on the Capto MMC system as compared to the Nuvia cPrime resin. The results further indicated that while the Nuvia cPrime ligand had a strong preference for interactions with aromatic groups, the Capto MMC ligand appeared to interact with both aliphatic and aromatic clusters on the protein surfaces. These observations were formalized into a new set of protein surface property descriptors, which quantified the local distribution of electrostatic and hydrophobic potentials as well as distinguishing between aromatic and aliphatic properties. Using these descriptors, high-performing quantitative structure-activity relationship (QSAR) models (R(2)>0.88) were generated for both the Capto MMC and Nuvia cPrime datasets at pH 5 and pH 6. Descriptors of electrostatic properties were generally common across the four models; however both Capto MMC models included descriptors that quantified regions of aliphatic-based hydrophobicity in addition to aromatic descriptors. Retention was generally reduced by lowering the ligand densities on both MM resins. Notably, elution order was largely unaffected by the change in surface density, but smaller and more aliphatic proteins tended to be more affected by this drop in ligand density. This suggests that modulating the exposure, shape and density of the hydrophobic moieties in multimodal chromatographic systems can alter the preference for surface exposed aliphatic or aromatic residues, thus providing an additional dimension for modulating the selectivity of MM protein separation systems.