Studies with terpyridine-based affinity ligands
2017-01-30T22:06:28Z (GMT) by
Purification 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.