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Dual salt mixtures in mixed mode chromatography with an immobilized tryptophan ligand influence the removal of aggregated monoclonal antibodies
Author(s) -
Vajda Judith,
Mueller Egbert,
Bahret Eva
Publication year - 2014
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201300230
Subject(s) - chemistry , elution , salt (chemistry) , ligand (biochemistry) , monomer , monoclonal antibody , chromatography , hydrophobic effect , downstream processing , resolution (logic) , tryptophan , hydrophilic interaction chromatography , adsorption , amino acid , organic chemistry , antibody , polymer , high performance liquid chromatography , biochemistry , receptor , artificial intelligence , computer science , immunology , biology
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.