Imaging surface plasmon resonance system for screening affinity ligands

摘要

A surface plasmon resonance (SPR) system for screening ligands for application in affinity chromatography is described. A combinatorial library of 13 ligands was synthesised, characterised and immobilised to agarose beads and gold SPR devices. Binding and elution behaviour and a range of K_(AX) values (13~3 to 10~5 M~(-1)) were measured against two target proteins, an insulin analogue (MI3) and a recombinant clotting factor (rFVIIa), in order to create a relational database between the traditional chromatographic format and the new SPR screening system. The SPR transducer surface was fabricated with affinity ligands in a two-dimensional, spatially addressable format, which was durable (> 100 cycles) and stable over 6 months. The imaging SPR system comprised a direct optical, CCD-based, instrument capable of imaging the change in refractive index created by biochemical interactions and allowed affinity ligands to be evaluated 15-fold faster with 130-fold less target protein than conventional chromatographic methods. The binding and elution data from both the SPR and chromatographic systems for both target proteins were comparable, with the K_(AX) value generating a nearly linear correlation (R~2 = 0.875) and a slope bias of ～2.5±0.25-fold higher for the SPR system. The imaging SPR system has proven capable of screening and evaluating affinity ligands for potential use in the recovery of biopharmaceutical proteins.