We explored the hypothesis that covalently attaching poly(ethylene glycol), or PEGylating, protein A chromatography media could increase its selectivity and its robustness by reducing non-specific protein-protein interactions via an excluded volume effect. We modified a commercial protein A (ProA) chromatography resin with 5 and 20 kDa aldehyde-activated PEG and characterized antibody static and binding capacity, mass transport behavior, selectivity and resistance to proteolytic attack as a function of the volume of immobilized PEG. Antibody binding capacity decreased roughly linearly with the volume of immobilized PEG; decreases in static capacity were attributed to binding site blockage while decreases in dynamic binding capacity reflected decreased pore diffusivity and slower binding kinetics for antibodies in addition to the loss in binding sites. Digestion of the PEGylated media with chymotrypsin showed biphasic binding capacity loss kinetics and that the PEGylation chemistry used provides a significant resistance to proteolytic attack, thereby increasing robustness relative to unmodified media. PEGylated media had increased resistance to non-specific protein binding relative to unmodified media, including resistance to “hitchhiker” proteins with a tendency to bind already bound antibody. Challenge of our most heavily modified protein A media with Chinese hamster ovary harvested cell culture fluids gave a 37% increase in antibody selectivity with respect to host cell proteins relative to unmodified media when using the same, standard load, wash, elute, regenerate protocol. This study suggests that the excluded volume effects can be exploited to improve the performance of affinity chromatography media based on immobilized macromolecular ligands.

PEGylating protein A chromatography media increases selectivity and robustness

255th National Meeting (2018)

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