title: Dual salt mixtures in mixed mode chromatography with an immobilized tryptophan ligand influence the removal of aggregated monoclonal antibodies.
abstract: In downstream processing of monoclonal antibodies, proper aggregate removal is crucial. Mixed mode ligands such as immobilized tryptophan have been developed to satisfy the need for efficient removal of antibody aggregates. However, method development for mixed mode applications is complicated, since protein binding and elution can be modulated by an increased set of parameters. In the current study, we investigate the effect of different dual salt mixtures on mixed mode chromatography using TOYOPEARL MX-Trp-650M resin, with respect to the dynamic binding capacity, resolution and monomer purity of two different humanized immunoglobulins. Binding capacities varying by more than 50% were observed for different salt mixtures. Furthermore, antibody monomer and aggregate resolution deviated by 30% for different salt mixtures and linear gradient elution. Similar trends were obtained using an immobilized carboxymethyl ligand for the same set of experiments, but the overall resolution was lower. Less kosmotropic salt systems emphasize the electrostatic binding of the relatively hydrophobic mAbs and reduce hydrophobic attraction to a selectivity-determining constraint. Kosmotropic salts such as citrate appear to cause dominating hydrophobic interactions in protein adsorption that hinder electrostatic protein-ligand interactions. This effect may depend on the ionic and hydrophobic site distribution of a protein. The data presented here are important for the further improvement of downstream processing of therapeutic monoclonal antibodies.
authors.affiliation: Tosoh Bioscience GmbH, Stuttgart, Germany. judith.vajda@tosoh.com.
authors.forename: Judith
authors.initials: J
authors.lastname: Vajda
authors.forename: Egbert
authors.initials: E
authors.lastname: Mueller
authors.forename: Eva
authors.initials: E
authors.lastname: Bahret
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002951:Citrates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D019346:Sodium Acetate / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D000077559:Sodium Citrate; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D002951:Citrates; D055767:Immobilized Proteins; D008024:Ligands; D000077559:Sodium Citrate; D018698:Glutamic Acid; D012965:Sodium Chloride; D019346:Sodium Acetate; D014364:Tryptophan
keywords: Dual salts; Dynamic binding capacity; Mixed mode chromatography; Tryptophan; mAb aggregate removal