Use of Stealth Technology to Enhance Selectivity in the Capture Step for Monoclonal Antibody Production

Todd Przybycien, Chemical Engineering and Biomedical Engineering, Carnegie Mellon University, Pittsburgh, US
Justin Weinberg, Chemical Engineeirng, Carnegie Mellon University, Pittsburgh, Us

The covalent attachment of poly(ethylene glycol) chains to recombinant protein drugs, or “PEGylation,” increases the circulating half-lives of protein drugs by stealth. We have applied this stealth technology to the production of monoclonal antibodies. We have PEGylated protein A chromatography media, used in platform production processes to capture antibodies from clarified cell culture fluids, to enhance its selectivity and to improve its robustness by discouraging the non-specific binding of host cell proteins. We have PEGylated Repligen CaptivA PriMAB media with 5 and 20 kDa, N-terminus selective, aldehyde–activated mono–methoxy PEGs. We used low stoichiometric-ratio modifications to limit reductions in the static and dynamic antibody binding capacities. We found that transport and kinetic resistances were elevated in the modified media relative to the unmodified media, using inverse size exclusion chromatography and van Deemter analyses; the magnitude of the changes in resistance scaled with the volume of PEG immobilized. Confocal laser scanning microscopy was used to visualize the transport of antibodies and contaminant proteins within individual chromatography media particles. Selectivity studies were performed by capturing a monoclonal antibody from industrial cell culture fluids with modified and unmodified protein A columns; a standard bind-wash-elute-regenerate protocol was used throughout. The amount of antibody bound and eluted and the amount of contaminating host cell proteins bound and eluted were assessed in each case and a significant enhancement in selectivity was realized with the PEGylated media. PEGylation also conferred increased protection from proteolytic attack. Ligand PEGylation is a new and generalizable approach to improving the selectivity and longevity of macromolecular affinity chromatography media via stealth.