(393j) Molecular Insight Into the Adsorption of IgG in Hydrophobic Charge-Induction Chromatography

The increasing demand of antibodies as therapeutics has led to the development of more efficient, robust and economical processes to purify antibodies from serum, ascitic fluid and cell culture broth. Protein A affinity chromatography is a typical platform technology for antibody purification for its high selectivity. However, it has some disadvantages, such as ligand leakage and CIP difficulty as well as high cost. Currently, hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the functional ligand has been developed as a novel technology for antibody purification. However, compared with protein A capture, the separation efficiency of HCIC still needs to be improved. Better understanding on the separation mechanism of HCIC in molecular scale is necessary for the process development and ligand design. 

In the present work, molecular simulation methods were developed to investigate the interactions between MEP ligand and the Fc fragment of IgG. The forcefield parameters of MEP were first optimized via quantum chemistry, and then the hot sites on the surface of the Fc fragment for MEP binding were explored by molecular docking. The consensus binding site (CBS) was focused and some potential ligand-protein complexes were obtained. These complexes were adopted as the starting structure for the molecular dynamics (MD) simulation. The free binding energies were calculated using the WHAM-umbrella sampling as the reference interaction free energy to refit the linear interaction energy (LIE) parameters. Based on the MD results, four possible binding sites on a single chain and three binding sites on the crossover area of double chains were identified. Furthermore, the ligand-net was constructed to simulate the protein adsorption on the porous surface of chromatographic resins. The MM/PBSA method was used to acquire the binding energy map which was used to scan the most suitable binding orientations of Fc on the ligand-net. It was found that ligand density had significant influence on the binding of Fc, which is in good agreement with experimental results. Several potential binding conformations were taken and evaluated with the MD simulation. The multiple-point binding between Fc and MEP ligand-net was found to have an important role during the capture of Fc. The results indicated that hydrophobic forces dominate the binding of Fc on the ligand-net, and the synergistic actions of consensus binding sites (CBS) with other sites were observed. The simulations provided useful information about the molecular mechanism of IgG binding to the HCIC ligand.

With the computer-aided molecular simulation approaches, it is possible to evaluate the multiple interactions between target proteins and functional ligands at molecular level, which certainly helps to explore the molecular mechanism of bioseparation, and it can be used to optimize the separation conditions and develop new ligands for bio-product recovery and purification.

* This work was supported by National Natural Science Foundation of China, the Zhejiang Provincial Natural Science Foundation of China and special foundation for innovative talents training project of Zhejiang University “985 programs”.