Transforming Waste into Valuables: Preparation and Evaluation of Dual-Ligand Hydrophobic Charge-Induction Chromatography Using Two Poor Performing Ligands

Abstract

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and BET techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against different proteins. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

Keywords: suboptimal ligands, dual-ligand mode, hydrophobic charge-induction chromatography, selectivity factor, adsorption performance