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Title: Novel affinity ligands for immunoglobulins based on the multicomponent Ugi reaction
Author: Haigh, Jonathan Michael
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2008
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A novel use of the four-component Ugi reaction to generate a solid-phase, immunoglobulin-binding library is described herein. An aldehyde-functionalised Sepharose solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate the final tri-substituted scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and fragmented (Fc and Fab) molecules. A number of putative lead candidates emerged for whole IgG, in addition to highly specific Fab and Fc-binding ligands. A Fab-specific ligand (A3C1I1) comprising an Ugi scaffold substituted with 1-amino-2-naphthol (A3), glutaric acid (C1) and isopropyl isocyanide (I1) was selected based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human plasma showed 100% of serum IgG was adsorbed from the 20 mg ml-1 crude stock and subsequently eluted with a purity of 81.0% under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH1-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions. The immobilised ligand was subjected to a series of studies to define an optimised affinity adsorbent which binds 73.06 mg IgG ml-1 moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1 ± 0.25 mg Fab ml-1 moist resin displaying an affinity constant Kd: (2.6 ± 0.3) x 10-6 M.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available