Binding and Elution Properties of Mixed-Mode Chromatography and Its Applications for Purification

Mixed-mode (multimodal) chromatography now occupies an important place in biopharmaceutical purification. It adds a new dimension to such conventional chromatographies as ion-exchange, hydrophobic interaction, reverse-phase and size-exclusion. Mixed-mode chromatography resins are composed of multiple functional groups that help protein binding and elution. These groups on resins confer hydrophobic, aromatic, electrostatic and hydrogen-bonding interactions. Earlier version of mixed-mode resins was composed of aliphatic hydrophobic groups and used extensively for extraction and purification of small organic compounds, but is not useful for proteins, as the protein binding is too strong to elute without application of organic solvents. Namely, such aliphatic mixed-mode resins are essentially identical to the operational procedure of reverse-phase chromatography. Later version of mixed-mode resin is composed of aromatic and charged groups, which make protein binding salt-tolerant: namely, protein binding occurs in the presence of salt. Such multiple binding mechanisms of mixed-mode chromatography offer various advantages, which are a topic of this special issue. Halan et al. provide an overview of various mixed-mode ligands and their application for separation of peptides, proteins, nucleic acids and small molecules [1]. Santarelli and Cabanne describe application of mixed-mode chromatography for antibody purification and its selectivity [2], followed by Cabanne and Santarelli who describe the development of a high-throughput screening to fully utilize mixed-mode chromatography technique [3]. Arakawa et al. [4] describe solvents to selectively elute the bound proteins during MEP chromatography, as an alternative to the conventional low pH elution. Solvents, or elution modifiers, play a critical role in separation and recovery during mixed-mode chromatography. Arakawa and Kita describe fundamental mechanism of the effects of solvents on macromolecular interactions based on protein-solvent interaction analysis. Among the elution modifiers used, arginine has been extensively used for washing and elution in mixed-mode chromatography [5]. Hirano et al. describe the mechanism by which arginine exerts its effects on disrupting interactions between proteins and mixed-mode ligands [6]. Nucleic acids also have both electrostatic and hydrophobic properties and hence are potential candidates for purification by mixed-mode chromatography, in particular due to their different conformational states. Matos and Bülow compare mixed-mode chromatography with ion-exchange or hydrophobic interaction chromatography for nucleic acid purification [7]. In two papers Arakawa et al. and Arakawa demonstrate the usefulness of mixed-mode chromatography in separation of protein isoforms [8, 9]. He et al. report use of mixed-mode chromatography in removal of contaminants, which have similar properties to the target antibody, IgM [10]. Yoshimoto et al. report the selectivity of hydroxyapatite chromatography utilizing its electrostatic and metal affinity properties. I believe that these papers provide readers comprehensive views of mixed-mode chromatography [11]. I wish to thank the staff of Current Peptide and Protein Science for their assistance in developing this Hot Topics issue and Professor Ben Dunn, Editor-in-Chief of CPPS, for his encouragement