Characterizing Interactions Between Thrombin, Antithrombin III, and An Anti-Thrombin Antibody by Composition-Gradient Multi-Angle Light Scattering

Macromolecular interactions influence all phases of biopharmaceutical development from drug discovery and target validation to the characterization of stable drug formulations at therapeutic doses.  Composition-gradient multi-angle static light scattering (CG-MALS) is a powerful, label-free technique for quantifying reversible interactions and can be applied to many stages of the drug development process.  Because assays are performed in solution, complex interactions can be observed without the influence of tags or surface immobilization.  This not only provides for absolute characterization of affinity and stoichiometry but also enables study of the effects of solvent composition, pH, and molecular conformation on a particular interaction.  Of particular interest are interactions between therapeutic targets, such as enzymes and growth factors, their natural ligands, and therapeutic antibodies.  Using CG-MALS, the interaction between thrombin-α (Thr) and an anti-thrombin antibody (Ab) was measured, yielding the expected 1:2 Ab:Thr stoichiometry as well as a binding affinity of 15 nM at each binding site.  In addition, the time-dependent, irreversible association of thrombin and antithrombin III (AT) was observed and a second-order rate constant calculated, k_on = 6x10^-3 M^-1s^-1.  Finally, CG-MALS was able to identify the recognition of the Ab for the Thr-AT complex to determine whether the antibody bound the thrombin active site.