The relevance of protein-based biopharmaceuticals has increased dramatically in the past decades and a variety of products are now available for human therapy. Antibodies in particular are currently the most heavily consumed protein therapeutics, with a current market volume expected to reach 1 trillion US$ in 2015 and a compound annual growth rate (CAGR) of 3-6%. Meeting the increasing demand for these therapeutics at lower prices while complying with increasingly stringent regulatory environments, calls for the development of new technologies and platform approaches for efficient downstream protein purification. Extended use of affinity chromatography holds great promise in meeting the urgent demand for affordable high-quality biological products. This technology, however, is still dependent on the use of biological ligands, such as Protein A, Protein G, and Protein L, that have significant issues associated with their high cost, harsh elution conditions, narrow specificity, low chemical stability, and immunogenicity in patients if they leach into the product stream. Small, robust, synthetic ligands may offer an effective alternative to protein ligands. Peptides in particular combine levels of affinity and specificity similar to those of biological ligands with high chemical and biochemical stability, broader specificity, low immunogenicity and ease of synthesis that can reduce costs.
The work in this thesis aims to discover and characterize novel peptide ligands to produce efficient, robust, and affordable affinity adsorbents for improved downstream purification of biologics. Two main areas have been investigated: (a) the development of linear hexapeptide – based adsorbents for the purification of human antibodies and (b) the design and screening of novel libraries of cyclic peptides for the discovery of novel ligands.
The research conducted on the characterization and development of competitive peptide-based affinity adsorbents comprises: (a.1) testing existing peptide ligands for the purification of antibodies from a variety of sources; (a.2) optimizing the protocol of ligand coupling on chromatographic resins to increase adsorbent binding capacity; (a.3) a method of modification of the resin’s surface chemistry to increase the adsorbent's chemical stability in harsh alkaline conditions; and (a.4.) a combined computational and chemical strategy for the design of protease-stable peptide ligands. The resulting peptide affinity adsorbents compete well with advanced Protein A – based adsorbents in terms of product yield and purity, dynamic binding capacity (~ 50 – 60 g/L), resistance to alkaline cleaning and sanitization, and biochemical stability in the presence of proteolytic enzymes.
In the second part of this work, two methods are presented for the design, synthesis, and screening of libraries of cyclic peptides for the identification of novel affinity ligands. The first method involves the generation and screening of (b.1) a biological mRNA-display library of cyclic peptides, and the second method (b.2) uses a synthetic solid-phase library of “reversible cyclic peptides”. Both libraries have been screened for the identification of ligands for human antibodies. The results of these studies indicate that these libraries are very promising tools for the discovery of robust, selective and affordable peptide ligands.
The methods presented herein offer a new set of tools, not only for affinity ligand discovery, but also for finding new drugs and diagnostic methods. Besides their technological value, these studies also offer insights into the mechanisms of non-covalent interaction that underlie the phenomena of biorecognition and protein activity.
|Advisor:||Carbonell, Ruben G.|
|Commitee:||Flickinger, Michael C., Gurgel, Patrick V., Haugh, Jason M., Khan, Saad A.|
|School:||North Carolina State University|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 75/03(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Organic chemistry, Chemical engineering|
|Keywords:||Bioseparation, Chromatography, Monoclonal antibodies, Peptide ligands, Peptide synthesis, Protein therapeutics|
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