Antibody Separation By Hydrophobic Charge-Induction Chromatography And Novel Ligand Design

Antibody has been widely used as the therapeutic drug with a broad market demand and prospect. The high cost of separation processes based on the Protein A (ProA) affinity chromatography has seriously limited the further development of antibody industry. Exploring economical and efficient techniques for antibody separation has great significance for the antibody industry. As a new bioseparation technology, hydrophobic charge-induction chromatography (HCIC) has the potential for antibody purification. Howerver, the separation processes with HCIC are immature, the higher performance of HCIC ligand would be demanded, and the molecular-level knowledge on the separation mechanism of HCIC still remains unclear. Combining the experimental and molecular simulation methods, this work is to understand separation mechanism of HCIC, design novel HCIC ligand, and then develop new antibody separation processes with low cost and high efficiency,.The adsorption and separation performance of commercial HCIC resin MEP HyperCel and ProA affinity adsorbent were compared. The influence of solution pH on the static adsoption capacity and the effects of flowrate on dynamic adsorption capacity were investigated. On the basis of the purification of monoclonal antibody (mAb) from cell culture supernatant (CCS), it was found that the performace of MEP HyperCel was comparable to ProA affinity adsorbent. HCIC has the promising potential to be an alternative to ProA affinity chromatograpy. With bovine serum albumin (BSA) as the model comtaminant, the influence of pH, salt and caprylate addition on the adsorption and desorption were studied and the protein-ligand interactions were discussed. It was found that modification of pH or ionic strength, or adopting caprylate as the albumin-selective modifier could effectively remove BSA from the MEP resin with little influence on IgG adsorption, and improve the process of antibody purification with the HCIC.The interactions between HCIC ligand and antibody were investigated with molecular simulation to reveal the separation mechnism of HCIC on the molecular scale. There are several possible binding sites on the surface of Fc domain of IgG for MEP ligand binding. Mutiple interactions, including hydrophobic interaction, pi-pi interaction, pi-cation interaction and hydrogen bonds, play an important role in the protein-ligand binding. High ligand density is necessary for HCIC adsorption. During the binding process, the comformation change of protein and the cooperaction between multple binding sites were observed. For the protein elution, the electrostatic repulsion between the basic residues of Fc and the protonated-pyridine of MEP ligand is the main driven force.Molecular simulation methods were used to investigate molecular interactions between the consensus binding site (CBS) of the Fc domain and seven natural Fc-specific ligands. The analysis on the binding energy of Fc-ligand complex indicated that hydrophobic interactions provide the main driving force for the Fc-ligand binding processes. The hot spots on the ligands and Fc were identified with the computational alanine scanning approach. It was found that the residues of tryptophan and tyrosine on the ligands have significant contributions for the Fc-ligand binding, while Met252, Ile253, Asn434, His435, and Tyr436are the key residues of Fc. Moreover, two binding modes based on tryptophan or tyrosine were summarized and constructed according to the pairwise interaction analysis. Some guidelines for the rational design of CBS-specific ligands with high affinity and specificity were proposed.On the basis of the understanding of adsorption behavior and the protein-ligand binding mode, two HCIC ligands5-amino-benzimidazo (ABM) and tryptophan-5-amino-benzimidazo (W-ABM) were designed, and new HCIC resins were prepared successfully. The performance for antibody separation was evaluated. The resins with ABM and W-ABM as the functional ligands had high adsorption capacity for IgG, while the adsorption capacity to BSA was quite low. In addition, two resins had the typical salt-independent property for the adsorption of IgG. The dynamic capacity of new resins was little affected by the flowrate and high dynamic capacity could be maintained even at high flowrate. The IgG could be effectively eluted at weak acidic condition. ABM and W-ABM resins were used to separate IgG from mimetic serum or CCS, and excellent performance was obtained. The results indicated that new HCIC resins developed would be promising for antibody separation and purification.