HARNESSING THE HIGH-AFFINITY INTERACTIONS BETWEEN SUPRAMOLECULAR POLYMERS AND PROTEINS FOR ANTIBODY SEPARATION AND LONG-ACTING RELEASE
Embargo until
2023-08-01
Date
2022-05-17
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Johns Hopkins University
Abstract
Supramolecular polymers (SPs) are one-dimensional assemblies with a high degree of internal order, representing an emerging class of functional materials for diverse biomedical applications. From a fundamental molecular design perspective, bioactive epitopes, or affinity ligands, can be facilely incorporated into functional SPs to promote highly specific interactions with proteins of interest. One such epitope, Z33, is a two α-helical stranded peptide originating from a subdomain of Protein A, a bacteria-derived protein commonly used in affinity chromatography for industrial monoclonal antibody (mAb) purification. Conjugation of a dodecyl alkyl chain and some additional charged amino acids to the Z33 peptide promotes the self-assembly of the resulting amphiphilic molecules into high-affinity SPs in aqueous environments.
In this dissertation, I first aim to develop a better understanding of mAb capture and phase separation behavior by controlling Z33 presentation on the SP surface through incorporation of either a double glycine or oligoethylene glycol (OEG) linker in the ligand design, separating the epitope from the remaining peptide segment. I use confocal microscopy and turbidity measurements to evaluate mAb capture in the resulting SP networks that form due to mAb’s ability to bind with two ligands on its Fc region, which facilitates SP-mAb crosslinking. My results indicate that SPs containing OEG linkers promote mAb capture further from the nanostructure surface, which also increases SP-mAb crosslinking, enhancing phase separation. Next, I optimize mAb precipitation and recovery from a protein purification standpoint while demonstrating selective antibody capture from clarified cell culture harvest. Furthermore, by modifying SP assemblies, I highlight this system’s adaptability in precipitating mAbs at over 30 g/L titers, surpassing current industrial titer limits. Finally, I tailor the application of designed high-affinity SPs to develop a hydrogel capable of releasing mAbs in a controllable and extended manner. The antibody-loaded hydrogels display tunable mAb release profiles resembling zero-order kinetics in vitro which are strongly influenced by the gel’s initial ligand content and the ligand:protein ratio, while exhibiting excellent biocompatibility and biodegradability in vivo. Overall, this dissertation has laid the framework for engineering supramolecular constructs with an emphasis on mAb capture to generate affinity systems for a multitude of applications.
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Keywords
Supramolecular polymer, self-assembly, monoclonal antibody, coacervation, protein purification, hydrogel