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.

Influence of Salts on the Adsorption of Lysozyme on a Mixed-Mode Resin

“…Mixed-mode resins can offer high binding capacities for proteins [9] as well as improved selectivities compared to single-mode resins [10,11]. There are several experimental studies on the application of mixed-mode resins in the literature, e.g., [12][13][14][15]. Typically, MMC is thereby applied for challenging separation processes, such as the purification of recombinant proteins, including monoclonal antibodies [15].…”

“…There are several experimental studies on the application of mixed-mode resins in the literature, e.g., [12][13][14][15]. Typically, MMC is thereby applied for challenging separation processes, such as the purification of recombinant proteins, including monoclonal antibodies [15].…”

Separation of recombinant antibodies from DNA using divalent cations

“…Factorial experiment design guided us to the most important factors in our investigation: the use of CaCl 2 and its concentration. Most studies concern the removal of aggregates [29], but DNA and/or other high molecular mass impurities are neglected. 1) motivated a closer investigation of the mechanism of DNA removal.…”

“…Also on-line detection methods are available to check the quality of starting solutions and antibodies [27,28]. Most studies concern the removal of aggregates [29], but DNA and/or other high molecular mass impurities are neglected. We realized that the phosphate content in the harvested supernatants varied quite a bit due to fermentation conditions, cell lines, or media composition.…”