Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography

•Spacer technologies in biochromatography are described.•Comparison of two different Protein A resins.•Immobilized recombinant Protein A oligomers increases the dynamic binding capacity of IgG.•BET shaped isotherms for IgG equilibrium adsorption on Protein A resin.•Multilayer IgG binding has no influence on protein aggregation, purity and recovery.

Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130 mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100 mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9 mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.