(36e) Combination of Mixed-Mode Chromatography and Expanded-Bed Adsorption for Antibody Recovery

Mixed-mode chromatography (MMC) is a novel technology for bioproduct separation, especially antibodies, which combines multiple binding modes like hydrophobic interaction, electrostatic forces, hydrogen bonding, etc.. High capacity, salt-tolerance, good selectivity and relatively low cost are the major advantages of MMC for direct capture process. Expanded-bed adsorption (EBA) was developed in 1990s as an innovative technology that allows capturing target proteins directly from unclarified feedstock, e.g., culture suspensions and cell homogenates. EBA technology combines solid-liquid separation with an adsorption step in a single-unit operation, aiming at increased overall yield, reduced operational time, and less requirements for capital investment and consumables. Two chromatographic techniques, MMC and EBA, would be integrated in the present work to develop new separation technology, mixed-mode EBA, improving the capture efficiency and reducing the pretreatments on the feedstock, such as clarification, dilution and salt-adjustment.

MMC ligands (such as 4-Pyridylethylmercaptan (MEP), 2-mercapto-1-methylimidazole (MMI) and 5-amine-1,3-benzimidazol (ABM)) were coupled onto the matrix for EBA (3% crosslinked agarose containing tungsten carbide). The density of matrix is up to 3.0 mg/ml with a suitable size distribution, which is very beneficial to operate at high velocity in an expanded bed. The matrix activation and the ligand coupling were optimized, and the ligand density could be controlled at the range of 40~100 μmol/ml resin. The saturated adsorption capacities of IgG could reach more than 60 mg IgG/ml resin. The effects of pH and salt addition on the adsorption of IgG were investigated, and the adsorption showed high pH-sensitivity and low salt-dependence. In addition, high dynamic binding capacity was found even at high operation velocity (>400cm/h), and the efficient elution could be achieved at a mild acidic condition. The adsorption in expanded-bed mode could match that in packed bed. Finally, new technology was challenged to capture IgG directly from the crude cell-containing feedstock with relative high conductivity, and good performance and high separation efficiency was obtained.

For biopharmaceuticals such as monoclonal antibodies (mAbs), downstream processes require highly productive and robust technologies to improve the process efficiency. The combination of MMC and EBA has been demonstrated to be a potential new platform for antibody capture with reduced feedstock pretreatments, high efficiency and relative low cost, which can be expanded to other bioproduct separation.

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