The effect of kosmotropic salts on aggregate removal from monoclonal antibodies in mixed-mode anion exchange chromatography

Abstract

Efficacious and safe application of recombinant proteins in humans as drugs depends on their high purity accomplished by downstream processing. The present study evaluates the effects of kosmotropic salts used in the flow-through mode of mixed-mode anion exchange (MMAEX) chromatography to remove high molecular weight (HMW) impurities efficiently from monoclonal antibody (mAb) products. For this purpose, the impact of different kosmotropic salts at varying concentrations along with changes of the pH were systematically assessed by high throughput screens on a set of mAbs differing in their relative hydrophobicity. In contrast to the more hydrophobic mAbs, for the more hydrophilic mAbs, a clear and consistent removal of HMW species to values > 80% (achieving up to 2.5-fold improvement) occurred with the addition of increasing amounts of a kosmotropic salt (sodium chloride, sodium sulfate, ammonium sulfate, potassium chloride and potassium sulfate) to the loads applied to MMAEX chromatography. Overall, the herein described effect on the removal of HMW species by MMAEX resins was specific for kosmotropic salts, independent of conductivity, and facilitated by slightly basic pH conditions. These findings were confirmed at lab-scale operation achieving > 99% purity, indicating the potential to implement MMAEX in combination with kosmotropic salts as a polishing step for hydrophilic mAbs.