Molecular mechanism of hydrophobic charge-induction chromatography: interactions between the immobilized...

Hydrophobic charge-induction chromatography (HCIC) is a novel bioseparation technology, especially for antibody purification. In order to better understand the molecular mechanism of HCIC, the typical ligand of 4-mercaptoethyl-pyridine (MEP) was coupled onto the cellulose matrix, and the binding and departing of IgG were studied with the molecular dynamics simulation. Based on the previous work with free MEP ligand (J. Phys. Chem. B, 116 (4) (2012) 1393-1400), the pocket around TYR319 and LEU309 on the CH2 domain of IgG was selected as the potential binding site for the Fc fragment of IgG (Fc-A), and the complex of matrix-ligand-Fc-A was formed for the molecular simulation. Both single ligand and ligand net were investigated in the present work. It was found that the MEP ligand immobilized on the cellulose matrix could capture the Fc-A at neutral pH during the simulation, and the Fc-A would depart quickly when pH was changed to 4.0. The hydrophobic interactions and hydrogen bonds controlled the binding of Fc-A on the immobilized ligands at neutral pH and the electrostatic repulsion caused the departing of Fc-A at acid condition. For the ligand net, multipoint binding was found, while one ligand dominated the binding of Fc-A and other ligands might enhance the adsorption of protein. In addition, the adsorption isotherm and the isothermal titration calorimetry (ITC) were used to evaluate the molecular interactions. The experimental results indicated that the hydrophobic interaction is the major driving force for the adsorption of IgG on the MEP resin, which was in good agreement with those findings of molecular simulation. The molecular simulation and thermodynamic results verified strongly the molecular mechanism of HCIC--the hydrophobic interactions for binding and the charge-induction repulsion for elution. Better understanding on the molecular interactions would be beneficial to design new HCIC ligands for improving the efficiency of antibody separation.