title: A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.
abstract: Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.
authors.affiliation: Univ. Bordeaux, EA 4135, F-33000 Bordeaux, France; IPB, EA 4135, F-33000 Bordeaux, France; Pall Life Sciences, 48, avenue des Genottes, 95800 Cergy Saint-Christophe, France; Pall Life Sciences, 50 Bearfoot Road, Northborough, MA 01532, USA.
authors.forename: J
authors.initials: J
authors.lastname: Pezzini
authors.affiliation: Univ. Bordeaux, EA 4135, F-33000 Bordeaux, France; IPB, EA 4135, F-33000 Bordeaux, France; Pall Life Sciences, 50 Bearfoot Road, Northborough, MA 01532, USA.
authors.forename: C
authors.initials: C
authors.lastname: Cabanne
authors.affiliation: Pall Life Sciences, 48, avenue des Genottes, 95800 Cergy Saint-Christophe, France; Pall Life Sciences, 50 Bearfoot Road, Northborough, MA 01532, USA.
authors.forename: J-W
authors.initials: JW
authors.lastname: Dupuy
authors.affiliation: Université de Bordeaux, Centre Génomique Fonctionnelle, Plateforme Protéome, Bordeaux, France; Pall Life Sciences, 50 Bearfoot Road, Northborough, MA 01532, USA.
authors.forename: R
authors.initials: R
authors.lastname: Gantier
authors.affiliation: Univ. Bordeaux, EA 4135, F-33000 Bordeaux, France; IPB, EA 4135, F-33000 Bordeaux, France; Pall Life Sciences, 50 Bearfoot Road, Northborough, MA 01532, USA. Electronic address: xavier.santarelli@ipb.fr.
authors.forename: X
authors.initials: X
authors.lastname: Santarelli
mesh_terms: D000327:Adsorption; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D019995:Laboratory Chemicals; D009113:Muramidase / Q000737:chemistry*; D010658:Phenylglyoxal / Q000737:chemistry*
chemical_list: D019995:Laboratory Chemicals; D009113:Muramidase; D010658:Phenylglyoxal
keywords: HEA HyperCel; Lysozyme; Mixed mode chromatography; PPA HyperCel; Phenylglyoxal; Purification