Mixed-mode ligands can have process advantages over traditional chromatographic media based on the combination of orthogonal electrostatic and hydrophobic interactions that simultaneously act to absorb and resolve proteins. However, the theoretical chemical diversity of a mixed-mode ligand library can be vast, resulting in a prohibitively large design space for each target product.<br/>A homologous ligand library based on the commercially-available Nuvia cPrime ligand was generated to systematically explore the various features of a mixed-mode cation-exchange ligand. By measuring the retention of a library of proteins in a salt-based linear gradient elution step, structural variants were identified that had significantly altered the chromatographic selectivity of the original system. While the substitution of the polar amide bond with more hydrophobic chemistries was found to selectively enhance affinity for hydrophobic regions with aromaticity, increasing the solvent exposure of the aromatic ring enhanced the ligand affinity for both types of hydrophobic residues. Certain structural changes raised the pKa of the ligand, which consequently lowered the charge density of the resin surface at low pH and increased the importance of hydrophobic association. Finally, a Quantitative Structure-Activity Relationship (QSAR) model was trained on this experimental data, which identified numerical descriptors that quantified critical ligand properties for mixed-mode resins and could correctly predict the retention of proteins on mixed-mode and ion-exchange resins that were not included in the original model. This model could be used further as an <i>in silico </i>approach to screen novel mixed-mode ligands and select the optimal media for particularly separating separation challenges.

Systematic exploration of homologous ligand library leads to improved design principles and tools for the prediction of protein selectivity in mixed-mode chromatographic systems

249th National Meeting (2015)

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