Conformational changes of biomolecules in ion-exchange chromatography

Ion-exchange chromatography (IEX) is commonly used as a purification step during biomanufacturing of therapeutic biomolecules. Recent studies have shown evidence of protein conformational changes in IEX, although the charge-based interactions involved are generally considered mild and have relatively little impact on protein stability (e.g., compared to hydrophobic interactions). These conformational changes often lead to complex protein elution behaviors and bring substantial challenges to the manufacturing process and analytical method development for biologics. This chapter first describes two major mechanisms of protein conformational changes in IEX, including (1) interconversion between different conformational states and (2) unfolding and aggregation. Second, the chapter introduces a variety of biophysical methods, which can directly measure and characterize the conformational changes of bound protein. Finally, the chapter discusses the implications of protein conformational changes and the associated key contributing factors, including protein and stationary phase properties, and practical operating conditions. This chapter aims to provide insights into the mechanistic understanding of surface-induced protein structural changes and shed lights on the future directions of protein engineering approach and novel stationary phase design to mitigate undesirable chromatographic performance, which enables robust biomanufacturing and quality control.