Enhancing Size-Based Selectivity in Hydrophobic Charge-Induction Chromatography: Transitioning from Planar to Three-Dimensional Recognition

Abstract

The separation and purification of protein-based therapeutics from complex feedstocks pose significant challenges, particularly in achieving high selectivity for proteins with varying sizes and structures. Hydrophobic Charge-Induction Chromatography (HCIC) has emerged as a promising platform for protein purification. This study investigates the impact of transitioning from planar to three-dimensional (3D) recognition in HCIC on protein adsorption preferences. By engineering ligands with 3D spatial recognition capabilities, we aimed to modulate protein adsorption behavior and enhance size-based selectivity. Comparative studies were conducted using ungrafted single-ligand resins (4FF-S), grafted single-ligand resins (4FF-G-S), and grafted dual-ligand resins (4FF-G-D), with bovine immunoglobulin G (bIgG, 153.1 kDa) and bovine serum albumin (BSA, 66.4 kDa) serving as model large and small proteins, respectively. Resin characterization, including functional groups, morphology, and pore features, was analyzed using FTIR, SEM, and BET techniques. The results demonstrated that the introduction of grafting and dual-ligand systems significantly altered the pore structure, with average pore sizes of 4FF-S, 4FF-G-S, and 4FF-G-D being 13.1, 20.5, and 19.8 nm, respectively. Static adsorption experiments evaluated the impact of ligand density (80-600 μmol/g resin) and grafting degree (80-130 μmol/g resin), while the effects of pH (4.0-8.0) and salt conditions (0-0.8 M NaCl and (NH4)2SO4) on adsorption performance were systematically investigated. Under optimal conditions (pH 7.0, no salt), selectivity factors (α = QbIgG/QBSA) reached 1.5, 3.7, and 6.8 for 4FF-S, 4FF-G-S, and 4FF-G-D, respectively, indicating 146% and 353% enhancements in bIgG selectivity for 3D-recognition systems. Adsorption kinetics revealed accelerated adsorption rates for bIgG on 3D-recognition resins, while BSA adsorption rates decreased significantly. Breakthrough experiments investigated the effects of flow rate and competitive adsorption on the dynamic adsorption capacities of the three types of resins. The results demonstrated that while 3D recognition enhanced the dynamic adsorption capacity for bIgG, it reduced that for BSA, thereby shifting the resin’s adsorption preference toward larger-sized proteins. Finally, chromatographic separation using bovine serum was carried out to confirm that 3D recognition enhanced adsorption selectivity for large proteins under actual raw material conditions. The recovery of bIgG by 4FF-S increased from 69.5% to 85.5% (for 4FF-G-S) and 92.7% (for 4FF-G-D). These findings highlight the advantages of 3D-recognition resins in improving binding efficiency, achieving size-based selectivity, and maintaining superior performance under dynamic conditions. This work provides a foundation for advancing chromatographic techniques to meet the growing demands for precise and efficient protein purification in biopharmaceutical applications.

Keywords: Size-based selectivity, Three-dimensional recognition, Hydrophobic charge-induction chromatography, Grafted dual-ligand resin, Protein purification