Multiple Heart-Cut Ion-Exchange Chromatography-Reversed-Phase Liquid Chromatography Platform for Online Desalting and Fractionation of Monoclonal Antibody Charge Variants.

Minor deviations in biopharmaceutical manufacturing processes, such as those used for monoclonal antibodies (mAbs), can introduce structural modifications that alter protein efficacy and safety. Monitoring these changes is critical to ensure product consistency and efficacy. Ion-exchange chromatography (IEC) is widely employed to assess product quality by separating charge variants. However, the use of non-volatile salts in IEC makes direct identification and characterization of these variants difficult. In this study, we developed a multi-heart-cut IEC-reversed-phase liquid chromatography (RPLC) platform that enables the separation, online concentration, desalting, and fractionation of mAb charge variants. The system integrates a 10-port valve and a six-column selector valve, allowing automated collection of up to five charge variants within a single analytical workflow. Moreover, the platform can be directly coupled to mass spectrometry for the characterization of charge heterogeneity and glycosylation. Systematic optimization of the capture and buffer-exchange conditions, including evaluation of stationary phase types and mobile phase compositions, was performed to maximize overall recovery. This platform can also be used for other chromatographic modes, such as hydrophobic interaction chromatography or size-exclusion chromatography. This integrated multiple heart-cut IEC-RPLC platform provides a high-resolution and efficient platform for detailed characterization of mAb charge variants, facilitating the characterization of critical product quality attributes.