(660i) Supramolecular Engineering of Self-Assembling High Affinity Polymers for Binding-Triggered Phase Separation and Antibody Purification.

The current industry standard for monoclonal antibody (mAb) purification, Protein A chromatography, has become a major downstream bottleneck of the purification process due to its limited resin capacity and production constraints. Affinity precipitation is a promising alternative that combines the high selectivity of a Protein A resin with an operationally feasible precipitation technique. We report here a cost-effective and efficient purification alternative using a supramolecular polymeric immunofiber (IF) system for high affinity mAb capture and separation. These IFs consist of co-assembled filler and ligand peptide amphiphiles with rationally chosen stoichiometric ratios for optimal antibody precipitation and recovery. By using an improved linker design on a high affinity Z33 peptide ligand, we can capture and precipitate mAbs with yields of over 97%, leading to an overall mAb recovery yield of 88%. Additionally, we have demonstrated the feasibility of this IF system when transitioning to mAb purification from clarified cell culture harvest, with a mAb precipitation yield of over 90% and recovery of 63% without the aid from high ionic strength solutions.

Our efforts to better understand the correlation between ligand design optimization and antibody precipitation yield improvements have led us to examine how polymer-protein coacervation is facilitated by multivalent mAb binding with a rationally chosen ligand possessing a flexible linker. We found that the ability of each protein to bind to two different ligands is essential for crosslinking different supramolecular polymers and forming a network of polymer-protein complexes. More importantly, improvements in ligand design through the addition of a flexible linker promotes mAb capture by supramolecular polymers and induces phase separation of densely packed polymer-protein coacervates, leaving the mAb-deficient polymers in the supernatant. Overall, these results enhance our understanding of supramolecular polymer-protein interactions and highlight the promising potential of peptide-based affinity systems for mAb purification in a salt-free environment.