(9d) A Potential Platform Technology for Antibody Purification: Mixed-Mode Chromatography

Mixed-mode chromatography (MMC) is a novel technology for bioproduct separation, especially antibodies. Mixed-mode ligands typically contain a combination of multiple binding modes like hydrophobic interaction, electrostatic forces, hydrogen bonding, etc.. High capacity, salt-tolerance, good stability and relatively low cost are the major advantages of MMC for direct capture processes. However, compared with Protein A capture, the separation selectivity of MMC for antibodies is still limited.

Based on series of tests with MMC using IgG, IgY and IgY(ΔFc), it was found that the control of loading pH or salt addition in the buffer was critical to improve antibody purification from albumin containing feedstocks. The control of caprylate addition might be another effective way for improving antibody purification. High purity and recovery can be obtained after the optimization of operation conditions. To better understand the ligand-protein interactions, a molecular simulation approach was established with combinative methods of molecular docking and dynamic simulation. The results indicated that at neutral conditions the ligand can bind onto some special sites of IgG surface which showed strong hydrophobicity. When the pH changed, the ligand departed quickly from the protein surface due to electrostatic repulsion. In addition, 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 IgG on the ligand-net. The effects of ligand structure, ligand cluster, gel matrix and liquid conditions were also studied. The simulation results could provide useful information on the process development and the design of novel ligands for MMC.

For biopharmaceuticals such as monoclonal antibodies (mAbs), downstream processes require highly selective and robust technologies to achieve extremely high purity. The complexity of the interactions between the mixed-mode ligand and the desired protein might be considered as a challenge to improve the adsorption selectivity. Due to the flexibility of operation development and the innovative design of novel ligands, MMC is likely to become a potential platform for antibody purification with high efficiency and low cost.

* This work was supported by National Natural Science Foundation of China and the Zhejiang Provincial Natural Science Foundation of China.