Enhancing the performance limits of hydrophobic charge-induction chromatography with the introduction of a second ligand

Abstract

Traditional chromatography often faces issues such as high cost and insufficient selectivity, driving a continuous demand for improved technologies. Current chromatography primarily relies on single-ligand modes, and the methods for its performance optimization are gradually encountering limitations. This study validated the efficacy of integrating a second ligand into the system to boost chromatographic performance, focusing on hydrophobic charge-induction chromatography. Initially, optimal resin (4FF-MMI, 109 µmol/g resin) was targeted, with various densities of the second ligand (4FF-MMI⁺) assessed for performance enhancement, achieving optimal values at (110+11) µmol/g resin. Characterization, including Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy and Brunauer-Emmett-Teller, was conducted. Subsequently, Optimization effects under different pH, salt, and flow rate conditions were studied. Results showcase significant chromatographic enhancement with the second ligand. In the final phase, both 4FF-MMI and 4FF-MMI⁺ were utilized for human IgG separation from human serum, employing pH 8.0 and pH 7.0 as the loading pH, pH 5.0 as the elution pH, and a flow rate of 0.75 mL/min. Compared to 4FF-MMI, 4FF-MMI⁺ achieved 96.6 % purity and 91.6 % recovery, representing increases of 10.6 % and 20.4 %, respectively. Additionally, the resin with the second ligand exhibits commendable repeatability and stability. In conclusion, this study highlights the potential of adding a second ligand to improve chromatographic capabilities, offering a novel strategy to augment existing ligand libraries.