Improving Selectivity in the Chromatographic Analysis of Monoclonal Antibodies (mAbs) Through the Use of Multi-Isocratic Elution Mode

Abstract

When characterizing monoclonal antibodies (mAbs) with reversed-phase liquid chromatography (RPLC), it is often challenging to attain sufficient selectivity between mAbs and their related variants. A new strategy, referred to as multi-isocratic elution mode, has recently been developed. It is based on setting a series of consecutive isocratic steps and very short steep gradient segments at solute elution. This elution mode offers several advantages compared to the usually applied linear gradient mode. Large biomolecules can benefit the most because of their “on/off” elution behavior. Arbitrary selectivity can be set between closely related protein variants while maintaining sharp peaks because of the strong band compression effects occurring at elution within the steep gradient segments.