Research On Tetrapeptide Biomimetic Chromatography For Antibody Purification

Monoclonal antibodies (mAb), acting as an important role among biopharmaceutical drugs, have a broad application prospect. Nowadays, Protein A based affinity chromatography is the platform in the antibody purification due to its high specificity. However, Protein A chromatography still has some drawbacks, such as high cost, harsh elution condition, protein ligand leakage, etc. Therefore, exploring an economical and efficient purification technique will be of great importance. Currently, mixed mode chromatography and short peptide biomimetic chromatography are two novel techniques for antibody purification, but the understanding of them is not enough. With the aim of efficient purification of antibody, the dissertation started with the antibody purification by mixed mode chromatography and comparatively compared purification mechanisms and results of different mixed mode ligands. Then mixed mode ligands and short peptide ligands were compared in terms of antibody binding by molecular docking, which showed short peptide ligands had better selectivity. The interaction mechanisms between short peptide ligand and antibody were analyzed using molecular simulation, which was the basis of the design of tetrapeptide library. After screening, a novel tetrapeptide ligand was found. One resin with this novel tetrapeptide as ligand was prepared in an attempt to improve the antibody purification performance.First, four commercial mixed mode resin (Bestarose Diamond MMA, Bestarose Diamond MMC, MEP HyperCel and PPA HyperCel) were evaluated and compared in the aspect of antibody separation. The artificial protein mixture of human immunoglobulin (hlgG) and bovine serum albumin (BSA) was used as the feedstock. Anionic mixed mode resin, Bestarose Diamond MMA, was determined as the most appropriate resin by pH gradient elution, and would be further investigated. After optimization of pH of step elution, pH 5.0 was adopted for the first step elution. As a result, the purity and recovery of hIgG could reach 97.9% and 92.8% respectively. Even the loading volume was further increased from 1 mL to 10 mL, satisfying purity and recovery could still be remained. On the other hand, it was also found antibody could be purified efficiently by salt modulation. When adding 0.5 M sodium chloride to equilibrium buffer, a purity of 95.4% and a recovery of 90.1% could be obtained for hIgG. The results demonstrated the feasibility of antbody purification by mixed mode chromatographic resin and laid the foundation for the design of novel small molecule ligand.Second, short peptide biomimetic ligand was shown to be more specific to antibody than mixed mode resin via molecular docking. So a short peptide ligand, DAAG, was investigated, and the interaction mechanisms between DAAG and Fc fragment was discussed by combining molecular docking and molecular dynamics simulation. The results of molecular docking showed that DAAG could bind to the consensus binding site with a favorable orientation, namely geometry-favorable and energy-favorable. The results of molecular dynamics simulation under neutral condition showed that DAAG bound to Fc-A fragment via multiple interactions, like ionic bond, hydrophobic interaction, hydrogen bond, van der Waals interaction, pi-cation interaction and so on, which endowed the ligand with strong affinity and high selectivity. Then the results suggested that attractions would be counterbalanced by repulsions under acidic condition, leading to the departure of ligand from protein surface. At last, the short peptide biomimetic ligand model for antibody purification was put forward based on the analysis of molecular interactions, which could guide the design of novel short peptide biomimetic ligand.Third, according to molecular simulation results and literatures, a tetrapeptide library was designed, which featured the model of Ac-HN-Aro-Aro-R-Aro/Ali-COOH. The optimal ligand was determined as Ac-YFRH by double screening of Flexible Docking and molecular dynamics simulation. With the self-made amine-activated matrix, tetrapeptide ligand could be coupled efficiently by HATU chemistry. The adsorption isotherm results showed that the resin adsorption to IgG decreased with the drop of pH. On the other hand, Ac-YFRH resin was shown to have good salt-dependency. For BSA containing artificial feedstock, when 1 M NaCl was applied under neutral condition for adsorption and pH 5.0 for elution, the purity and recovery could reach 98.4% and 89.4% with the resin, indicating good antibody separation performance for Ac-YFRH resin.Finally, the hIgG adsorption performances and purification performances of Ac-YFRH resin were examined. Resins with varied ligand densities were prepared to investigate the effects of salt concentration on the adsorption performances, including static binding capacity, adsorption dynamics and dynamic binding capacity. The results showed the static binding capacity increased with the increase of ligand density, and the apparent desorption constant changed in the opposite direction. The adsorption dynamics experiments showed the curve could be fitted well with pore diffusion model, but the surface binding resistance should not be neglected. The results also showed that the dynamic binding capacity increased with the increase of ligand density. The dynamic binding capacity for hIgG was relatively low at high flow rate, and varied little at low-middle flow rates. The ratio of dynamic binding capacity to static binding capacity could reach 41.2%. Further, a tetrapeptide resin with ligand density of 101 μmol/g was prepared and evaluted for the purification process for complex feedstock, including bovine whey and CHO cell culture supernatant. Ac-YFRH resin had dynamic binding capacities of 1.6 mg/mL and 12.5 mg/mL for bovine IgG and mAb respectively with the retention time of 3 minutes. With the optimization of loading volume and elution condition, the purified bovine IgG had a purity of 84.3% and a recovery of 86.9%, and the purified mAb (anti-IF-12) had a purity of 95.7% and a recovery of 86.2%, showing high performance of Ac-YFRH resin for antibody purification.In the present work, focusing on efficient purification of antibody, the work began with mixed mode chromatography. Short peptide ligand was introduced after comparing the antibody selectivity with mixed mode ligand. Then ligand-antibody interactions were analyzed in order to design and screen a tetrapeptide ligand with high affinity. At last, the novel ligand would be evaluated for its binding performance to IgG and purification performance of IgG from complex feedstocks. The results would help better understand the separation modes of mixed mode chromatography and short peptide biomimetic chromatography, as well as give appropriate theory guidance for the design and optimization of novel ligand.