Investigating the Role of Ligand Hydrophobicity and Ionization on Protein Retention in Mixed-Mode Chromatography

Mixed-mode chromatography can offer superior selectivity and separation performance over its single-mode counterparts; however, many aspects of mixed-mode ligand design remain poorly understood. Here, a series of increasingly hydrophobic amino acid analogs of the mixed-mode cation exchanger Nuvia cPrime are investigated using a library of proteins spanning a range of isoelectric points, hydrophobicities, and molecular sizes. Although methyl and dimethyl substitutions produce no appreciable increase in protein retention, replacing the dimethyl substituent with an isobutyl group increases retention by approximately 50%, revealing a step change upon reaching a threshold ligand hydrophobicity. Notably, selectivity remains largely similar across the entire ligand series despite substantial differences in ligand structure. Because the hydrophobic substituents are also found to substantially alter ligand pKa, experiments across multiple pH conditions are used to distinguish direct hydrophobic effects from changes in effective ligand charge. Protein retention is found to be much more strongly correlated with ligand hydrophobicity than with ionic capacity, indicating that hydrophobic interactions dominate over electrostatic contributions across the ligands. The effects of protein isoelectric point and hydrophobicity are subsequently examined to further understand how protein properties influence the relative contributions of electrostatic and hydrophobic interactions on this ligand series. Highly hydrophobic proteins overcome electrostatic repulsion once a critical hydrophobic threshold is reached, with this threshold governed by both ligand properties and the intrinsic hydrophobicity of the protein. Further, a selectivity crossover is observed between proteins with markedly different electrostatic and hydrophobic characteristics, highlighting the importance of matching ligand chemistry to the properties of the target protein. Collectively, these findings inform the design of new mixed-mode ligands by elucidating how ligand and protein hydrophobicity and electrostatic interactions govern protein retention.