Molecular Simulation Of Antibody Separation With Hydrophobic Charge-Induction Chromatography

Hydrophobic charge-induction chromatography (HCIC) is a new technology for bioseparation which has been applied for antibody purification. However, it is still quite limited to understand the molecular interactions between HCIC ligands and antibody. The mechanism of hydrophobic binding and electrostatic-repulsion elution are used conceptually to interpret the experimental observations, lacking of the molecular binding information, such as binding sites on the protein, binding modes, etc. Recently the developments of molecular simulation methods offer the possibility to investigate the nature of the interaction between proteins and ligands. Therefore, in the present work the molecular interaction mechanism of antibody separation with HCIC is studied with some molecular simulation tools. Main results were listed as follows.Firstly, combining the methods of molecular docking and molecular dynamic (MD) simulations, the interaction between a typical HCIC ligand—4-mercaptoethyl-pyridine (MEP) and IgG was investigated. Molecular docking was first used to identify the potential binding sites around the protein surface of Fc Chain A of IgG (Fc-A), and 12 potential binding sites are found. Then 6 sites are further studied using the molecular dynamics simulations. The results revealed that at neutral conditions MEP ligand can bind stably on the site around TYR319 and LEU309 of Fc-A, which shows obviously a pocket structure with strong hydrophobicity, and MEP ligand form hydrogen bonds with the former two amino acid residues. When the pH lowers to 4.0, it can be found that MEP bound formerly on the Fc-A departs quickly due to the electrostatic repulsion between the positively charged ligand and protein. With the aids of molecular simulations, the separation mechanism of HCIC is verified from the view of molecular interactions:the binding with hydrophobic interactions at neutral condition and the desorption with electrostatic repulsion at acid condition.Secondly, the influences of spacer, thioether sulfur atom and heterocycles of the HCIC ligand for IgG binding were explored by the molecular simulations. It was found that the spacer containing sulfone group could form the additional hydrogen bonds with amino acid residues on the surface of Fc-A. Those hydrogen bonds enhanced the binding at neutral conditions, but became unfavorable for the desorption of protein at acid conditions. No special contributions of the thioether sulfur atom in the binding were observed in the simulations, and it could be replaced by either nitrogen or oxygen atom. Different heterocycles showed diverse binding behaviors. The van der Waals interaction energies between the heterocycles and Fc-A were in the order of 2-mercapto-benzimidazole (MBI)>MEP>2-mercapto-l-methyl-imidazole (MMI)> 3-Mercapto-1H-1,2,4-triazole (MTR), corresponding to the same order of the hydrophobicity of heterocycles. At pH 4.0, the positively-charged MMI and MBI ligands departed from the surface of Fc-A, while the non-charged ligands still bind stably on the hydrophobic pocket of Fc-A. To explore the molecular models more close to the real chromatographic situations, a cellulose panel was introduced as the matrix model. The model system of "cellulose panel—immobilized ligand" were established and the binding of Fc-A on the immobilized MEP ligand was studied by MD simulations. During 4 ns simulated time span, Fc-A was stably captured on the immobilized ligand through the hydrophobic pocket, either in the case of single MEP ligand or in the case of ligands net. The simulation results could be helpful to elucidate some experimental observations.Finally, the adsorption ofγ-globulin of human serum, which was mostly composed of IgG, on the commercial HCIC adsorbent MEP HyperCel was studied with the adsorption isotherm and thermodynamics method of isothermal titration calorimetry. The adsorption enthalpy (-1.68 kcal/mol), entropy (7.56 cal/(mol·K)) and Gibbs free energy (-3.93 kcal/mol) were determined. The results indicated that the main force driving the adsorption was the hydrophobic interaction. The van der Waals interaction and hydrogen bonds also contributed in the adsorption. These experimental results confirmed some conclusions of molecular simulation as mentioned above.Generally, in the present work, the molecular simulation methods were introduced to investigate the interactions between ligand for HCIC and IgG, as well as the influences of pH and ligand structure on the binding. The mechanism of antibody separation with HCIC was verified from an atomistic point of view. Furthermore, the simulation results were confirmed with the thermodynamic analysis. The results revealed that the molecular simulation methods could be effective tools in exploring the protein-ligand interaction, which would facilitate the design of novel ligand and improve the application of new chromatographic technique.