Developments in Ion Exchange Chromatography–Mass Spectrometry for the Characterization of Intact Proteins and Proteoforms

Ion exchange chromatography (IEC) hyphenated to mass spectrometry (MS) is a powerful non-denaturing technique used for analyzing protein charge heterogeneity (including posttranslational modifications such as deamidation, phosphorylation, and sialylation), as well as for characterizing complex protein mixtures. This review provides an overview of current strategies for implementing IEC–MS, focusing on pH gradient-based methods: chromatofocusing, linear pH gradient elution, and salt-mediated pH gradient elution. First, the fundamental principles, elution modes, and separation mechanisms of IEC are introduced. The review then discusses the limitations of traditional salt gradient elution in IEC, which often relies on nonvolatile additives, and highlights the critical role of pH gradient-based IEC in enabling direct and efficient hyphenation with MS. Key factors influencing protein elution in IEC are summarized to aid method optimization and enhance the understanding of the separation process. Subsequently, recent advancements in IEC–MS are described, covering three key aspects: (i) the selection of appropriate volatile buffer systems and strategies for achieving controlled linear pH gradients, (ii) developments and selection criteria for IEC columns, and (iii) approaches to improve sensitivity and ionization efficiency in IEC–MS. Finally, the review compiles details of recently developed (2010–2025) pH gradient-based IEC–MS methods and summarizes their applications in identifying diverse therapeutic protein variants arising from PTMs, antibody-drug conjugates (ADCs), and other complex biological samples.