Solution-Phase Kinetics of Ranibizumab Binding VEGF Using Solid-Phase Surface Plasmon Resonance.

Achieving sufficient pathway suppression for low-abundance cytokines like VEGF may require depletion to femtomolar concentrations using high-affinity antagonists. Understanding the kinetics of this pharmacodynamically complex process is important for the development of anti-VEGF therapeutic molecules like Ranibizumab and requires experimental determination of the kinetic binding constants of the VEGF-antagonist complex. These measurements are challenging due to extremely high binding affinities, long residence times, and a need to mimic the in vivo environment. We introduce a mixed-phase surface plasmon resonance assay that reports the solution-phase association rate constant, combined with an optimized "chaser" assay that reports the dissociation rate constant. Running both assays at 37 °C under physiologically relevant buffer conditions allows solution-phase affinity measurements under near in vivo conditions. Our analysis of Ranibizumab binding VEGF provided precise measurements of residence time (19 days) and affinity constant (1.9 pM). These extreme interaction constants are not measurable by solution-phase equilibrium-based methods since (a) an equilibrium time of 26 days would be required, and (b) a reasonable signal-to-noise ratio is impractical since the concentration of detected species must remain less than the affinity constant. We explore the limits of solution-phase assays using simulations and demonstrate that without strict adherence to the basic assumptions of the bimolecular solution-phase model then recorded dose-response curves are prone to ambiguous model fits that are easily misinterpreted. In contrast, we show that kinetic methods can be applied more broadly for mechanistic characterization of interactions exhibiting extremely high affinity and stability.