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Reason: Under embargo until Sep 2017. After this date a copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library
Studies with terpyridine-based affinity ligands
thesis
posted on 2017-01-30, 22:06 authored by Lin, Chih-PeiPurification of monoclonal antibodies in the biotechnology industry today is typically achieved by performing the primary capture step using Protein A affinity chromatography. However, the high cost of this downstream process is still a major production bottleneck and many research efforts seek to improve its cost efficiency. Affinity synthetic ligands based on small organic molecules are potential Protein A substitutes due to their comparatively higher robustness, versatility and cheaper preparation. Numerous synthetic ligands have been developed by prominent research teams around the world and have shown significant commercial successes.
This thesis is a study on synthetic ligands based on several terpyridine analogues. The focus is divided between the synthesis, chromatographic purification and molecular mechanistic studies of adsorbents immobilised with the terpyridine ligands (TIGs).
The ligands were prepared using both traditional and green synthetic methodologies. Immobilisation of ligands on epoxide-activated Sepharose 6FF gave eight TIGs. Key binding parameters and antibody loading conditions were established by static binding experiments which utilised an automated robotic workstation extensively. The dynamic binding capabilities of TIG-packed 1 mL columns were characterised on a fast protein liquid chromatography (FPLC) system and adsorbent selectivity for IgG2 was assessed by host cell protein (HCP) clearance levels in eluted fractions. Adsorbents immobilised with chloro-substituted terpyridines showed enhanced IgG2 selectivity where the adsorbent ES-(Cl.S.Cl)tpy in particular demonstrated a HCP clearance comparable to that of other Protein A adsorbents. Mechanistic studies using thermodynamic and molecular modelling techniques revealed, with strong experimental and theoretical evidence, that the terpyridine ligands exert selectivity by targeting a “two proline loops” Fab structure in close proximity to the highly conserved NBS.
The results shown in this thesis encourage further process development and optimisation using chloro-substituted TIGs as they have demonstrated the potential to be viable Protein A substitutes.
History
Campus location
AustraliaPrincipal supervisor
Milton T. W. HearnYear of Award
2014Department, School or Centre
ChemistryCourse
Doctor of PhilosophyDegree Type
DOCTORATEFaculty
Faculty of ScienceUsage metrics
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No categories selectedKeywords
Mixed-mode chromatography1959.1/980475Restricted access and full embargoAffinity chromatographymonash:130690SepharoseAdsorption isothermMolecular dockingthesis(doctorate)Tecan Freedom EvoIsothermal titration calorimetryethesis-20140828-181054Protein purificationMonoclonal antibodyFast protein liquid chromatographyAffinity ligands2014Terpyridine
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