Imaging surface plasmon resonance system for screening affinity ligands.
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 (10(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 approximately 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.
Morrill
Paul R
PR
Institute of Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge, UK.
Millington
R B
RB
Lowe
Christopher R
CR
D000345:Affinity Labels / Q000737:chemistry*; D002846:Chromatography, Affinity; D015942:Factor VIIa / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008024:Ligands; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
D000345:Affinity Labels; D007328:Insulin; D008024:Ligands; D015942:Factor VIIa