Engineering High-Affinity Supramolecular Polymers for Antibody Capture and Purification
Embargo until
2021-08-01
Date
2020-07-16
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Publisher
Johns Hopkins University
Abstract
Monoclonal antibodies (mAbs) have received considerable attention over the past three decades for the treatment of many diseases. With the significant titer improvement in cell culture processes, the mAb capture step using protein A chromatography has become one of the major downstream bottlenecks due to limited resin capacity and high production cost. As such, affinity precipitation has been increasingly explored as a promising alternative to purify mAbs and other therapeutic proteins. Despite recent advances in new affinity precipitants, challenges still remain in achieving high capture efficiency and complete removal of the precipitants. Supramolecular polymers formed by self-assembly of peptides and peptide derivatives are attractive biomaterials due to their inherent biodegradability and biocompatibility and have been widely explored for use in regenerative medicine, drug delivery, and disease diagnostics. Importantly, the selective presentation of various bioactive epitopes on a supramolecular substrate enables specific biology interfacing and molecular recognition. Furthermore, the multilevel reversible transitions within a supramolecular system make it uniquely suited for use as effective affinity agents for mAb precipitation and purification.
The aim of this dissertation is to develop peptide-based supramolecular polymers as affinity agents for efficient capture and purification of mAbs. First of all, Z33, a protein A-derived peptide with two α-helical strands, was selected from literature as the mAb binding ligand due to its high binding affinity and short sequence. I discovered that the alkylation strategy plays an important role in preserving the α-helical conformation of the peptides within its supramolecular assemblies. Second, I designed and constructed self-assembling immuno-amphiphiles (IAs) via the direct conjugation of the Z33 peptide to linear hydrocarbons. The resulting IAs can effectively associate under physiological conditions into immunofibers (IFs) while preserving their native α-helical conformation and mAb binding affinity. However, the mAb precipitation efficiency was found to be very modest, which was attributed to the steric hindrance among tightly packed ligands that prevents their efficient interactions with mAbs.
Third, to reduce the steric hindrance among Z33 ligands, I developed co-assembled IFs formed by a Z33-containing amphiphile with a rationally designed filler molecule to modulate the distribution of Z33 on IF surfaces. Under optimized conditions, IFs can specifically precipitate mAbs with a yield greater than 99%. I also demonstrated the feasibility of capturing and recovering mAbs from clarified cell culture harvest. Importantly, the added IFs can be easily removed via membrane separation, without introducing new contaminants. However, this system was limited by the high ammonium sulfate concentration necessary to trigger mAb precipitation. Lastly, to minimize the usage of salt, a series of IF building blocks with OEG (or PEG) linkers was designed to optimize the presentation of Z33 on IF surfaces. Results reveal that the mAb-IF interactions could be significantly improved as the linker length increases; however, too long a linker has an adverse impact on the function of the resultant IFs. I demonstrated that the desired IF system was able to precipitate mAbs without the help of additional salt and promising yields were obtained, especially when using a sequential precipitation strategy.
These findings shed important light on the engineering of supramolecular polymers for specific molecular recognition and capture. I envision that the peptide-based supramolecular IF system can be potentially scaled up, serving as an efficient alternative for the purification of mAbs and other proteins of interest.
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Keywords
Supramolecular polymers, Self-assembly, Peptide immunofiber, Affinity binding, Precipitation, Antibody purification