Cyclodextrin Displacement Chromatography And Its Application In Antibody Purification

Monoclonal antibody drugs have become the most industrially valuable biological molecules, and the proportion of antibody drugs'sales in biopharmaceutical has been increasing year by year. With the continuous expansion of the market and the productivity of the antibody, how to improve the current chromatography techniques to achieve better efficiency and capacity for protein separation is in urgent need. To solve the problems of hydrophobic charge induction chromatography and conventional hydrophobic interaction chromatography, which are associated with acid elution and high concentration of salt for protein adsorption, this paper puts forward a new strategy for elution. This method allows the recovery of bound proteins through cyclodextrin-based displacement elution. The research contents of this paper include the following aspects:1. Design of the MEP-HCIC supramolecular displacement chromatography:first of all, combining the technology of molecular docking with the actual chromatographic process, it shows that ?-cyclodextrin is the best displacer. In the process of optimum conditions, we found that cyclodextrin elution has a high salt and pH tolerance. Under physiological conditions, the 15 mM (3-cyclodextrin can get effective elution of the hIgG from the MEP-HCIC, and the recovery of hIgG is 87%. When this elution strategy was used to separate antibody directly from human serum, substantial elution of bound IgG could be obtained at pH 7.4, with product purity comparable to traditional method with an acidic buffer.2. Design of salt-independent hydrophobic displacement chromatography using cyclodextrin as supermolecular displacer:the host-guest interaction between ?-cyclodextrin and the benzoic acid revealed a modest binding strength (Ka= 4.1 × 103 M-1) by using isothermal titration calorimetry, which proved that the feasible in benzoic acid-HIC media replacement elution of cyclodextrins. Preparing with different density of benzoic acid-HIC and testing for the dynamic adsorption capacity of hIgG, we found that IgG dynamic binding capacity and ligand density was a positive linear correlation. However,15 mM ?-cyclodextrin replacement elution efficiency and ligand density was a negative linear correlation. Taking these two factors, the optimal ligand density is 62 ?mol/mL. Eventually, we used the serum as separation taget, and the operation of loading and elution are under physiological conditions, which also can separate IgG effectively from the serum.3. The development of the new ligand of cyclodextrin displacement chromatography: With the technology of isothermal titration calorimetry, we analysed the binding force of ?-cyclodextrin and candidate ligands, then we choose the ibuprofen as the ligand and synthesis medium, IgG as a model protein, we evaluate its dynamics and thermodynamic properties. Under physiological conditions, ibuprofen-HIC is of a high hIgG binding capacity, which achieved 90% protein recovery by cyclodextrin elution.The results of the study are beneficial to expand the application of hydrophobic charge induced chromatography and hydrophobic interaction chromatography as economical chromatography in the field of antibody separation. In addition, a new concept of supermolecular displacement chromatography was proposed, and a new screening strategy for the hydrophobic ligands were established.