Mixed-mode chromatography (MMC) is new technology for bioproduct separation, especially for antibody purification. The mixed-mode ligands typically combine multiple binding modes, including hydrophobic interaction, electrostatic forces and hydrogen bonding, resulting in a combination of protein-ligand interactions. 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 binding selectivity of mixed-mode ligands to antibodies still needed to be improved with a rational design strategy.
Based on the evaluation of the binding modes between some natural Fc-specific ligands and Fc fragment of IgG with molecular simulation, novel MMC ligands was designed for antibody purification, including amino-benzimidazole (ABI), a combining of amino acid and amino-benzimidazole (AA-ABI) and some tetrapeptide ligands. The hydrophobic groups could provide the orientated binding to target protein and the charged groups would induce the electrostatic repulsion for the efficient elution. New MMC ligand was coupled to the agarose gel, and the adsorption behaviors were investigated. High affinity to IgG was found with high static adsorption capacity at the neutral pH, and the flow rate had little impact on the dynamic binding capacity of IgG. In addition, the elution could be achieved at mild acidic pH with high recovery. After the optimization of operation pH and salt addition, IgG could be separated from the albumin containing feedstock and monoclonal antibodies (mAbs) could be purified from CHO cell culture supernatant with high purity and recovery. The results indicated that the resins developed were better than commercial MMC resins and comparable to Protein A resin.
For biopharmaceuticals, 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 can improve the binding selectivity. MMC with the specially-designed ligands would be a potential alternative to Protein A-based capture for the cost-effective purification of antibody.
This work was supported by National Natural Science Foundation of China and the Zhejiang Provincial Natural Science Foundation of China.