(320a) Separation of Monoclonal Antibody Charge Variants with Continuous Chromatography and Mixed-Mode Resins

For monoclonal antibodies (mAbs) expressed by mammalian cells, the generation of charge variants is inevitable, which would lead to lower potency and stability. It is necessary to separate charge variants. However, it is quite difficult to obtain target mAb with high purity and high yield due to the similarity of charge variants with mAb.
To improve the resolution and trade-off between purity and recovery, two new bioseparation technologies, mixed-mode chromatography and twin-column continuous chromatography, were combined. The mode of multi-column counter-current solvent gradient purification (MCSGP) was investigated with the recycle of the peak overlap between target mAb and charge variants. With cation exchange resins as the control, various factors including mixed-mode ligands, resin particle sizes, elution modes, gradient slopes and flow rates were tested. The results indicated that the resolution of charge variant with mixed-mode resins was higher than that with normal cation exchange resins due to the combination of different molecular interactions. In addition, MCSGP with mixed-mode chromatography could improve the recovery significantly with high purity. Near 100% purity of target mAb could be obtained after the process optimization. The chromatographic models were used to aid the process development. The results demonstrated that twin-column continuous chromatography with mixed-mode resins is a promising technology for mAbs charge variants separation with high purity and high recovery.

Keywords: Monoclonal antibody, Charge variants, Continuous chromatography, Mixed-mode chromatography, Cation exchange chromatography