Ion exchange chromatography of biotherapeutics: Fundamental principles and advanced approaches.

Abstract

Ion exchange chromatography (IEX) is an important analytical technique for the characterization of biotechnology-derived products, such as monoclonal antibodies (mAbs) and more recently, cell and gene therapy products such as messenger ribonucleic acid (mRNA) and adeno-associated viruses (AAVs). This review paper first outlines the basic principles and separation mechanisms of IEX for charge variant separation of biotherapeutics, and examines the different elution modes based on salt or pH gradients. It then highlights several recent trends when applying IEX for the characterization of biotechnology-derived products, including: i) the effective use of pH gradients, ii) the improvement of selectivity by using organic solvents in the mobile phase, multi-step gradients, or by combining ion pairing and ion exchange, and iii) the increase in analytical throughput using ultra-short columns or automated screening of conditions. The review also discusses the incorporation of IEX into multidimensional liquid chromatography setups, integrating it with other chromatographic dimensions for the analysis of complex biotherapeutic products. It also covers the coupling of IEX with mass spectrometry (MS), ion mobility spectrometry (IMS), and multi-angle light scattering (MALS) to identify the various species contained in complex biotherapeutic samples. In conclusion, IEX is considered today as an essential technique in the analytical toolbox for the characterization and quality control of biotechnology-derived products. It offers a unique separation mechanism and can be coupled with highly informative detectors, such as MS and MALS.