Separation of monoclonal antibody charge variants using cation exchange chromatography: Resins and separation conditions optimization

摘要

The separation of charge variants is a challenge for the production of monoclonal antibodies (mAbs). Cation exchange chromatography (CEX) is normally used to accomplish this task. In this study, with IgG1-type mAb as the model protein, charge variants separation was investigated under various conditions including resins, elution mode, gradient slope and flow rate. The trade-off between main peak purity and recovery was focused to evaluate the separation performance. The results indicated that the diameter of resin particles was the critical factor. For SP Sepharose Fast Flow (90 mu m diameter resin), the overlap between main peak and charge variants was too serious to meet the separation requirements. For Monomix MC30 SP (30 mu m diameter resin), the purity of main peak was improved to 93.4% under 0.05 pH/CV and 100 cm/h. For Nano SP-15L (15 mu m diameter resin), near 100% purity of main peak could be obtained with a recovery of 56.5% under 0.05 pH/CV and 100 cm/h. Two types of elution gradient, salt gradient and pH gradient, were compared and it was found that there was no obvious difference after the optimization and both showed some improvements with a shallower gradient. In addition, slower flow rate could improve the separation of main peak. In general, smaller resin particle size, shallower gradient slope and slower flow rate could improve the separation performance of main peak and charge variants under both salt gradient and pH gradient elution, which should be selected according to the actual requirements.