A high-throughput method for detection of protein self-association and second virial coefficient using size-exclusion chromatography through simultaneous measurement of concentration and scattered light intensity

To characterize protein self-association along with second virial coefficient (a measure of solution nonideality) using size-exclusion chromatography (SEC) utilizing a novel flow cell that is capable of simultaneously measuring protein concentration and scattered light intensity. beta-lactoglobulin A (beta Lg), known to exhibit NaCl-dependent monomer-dimer equilibrium at pH 3.0, was used as the model protein. A range of concentrations and corresponding scattered light intensities, obtained in the eluting peak from a single protein injection, in different solution conditions, were used to generate the Debye plots (Kc/R theta vs c). The Debye light scattering equation was modified to include the monomer-dimer equilibrium model and the second virial coefficient to analyze the data obtained. Debye plots of beta Lg, while linear at pH 2.3, 0 M NaCl (pure monomer) and at pH 3.0, 1 M NaCl (pure dimer), showed curvature at pH 3.0, for varying NaCl concentrations (0.02-0.5 M). The curvature was indicative of the association behavior of this protein. The modified Debye light scattering equation, when fit onto the nonlinear Debye plots, yielded apparent K-a values ranging from 10(2) to 10(5) M-1 stop under various solution conditions. The apparent K-a values obtained from this method followed similar trend to those reported in literature. SEC combined with simultaneous detection of scattered light intensity and concentration provides a rapid means of detection of protein self-association. The short duration of sample detection and analysis combined with SEC makes this method a useful tool for high-throughput characterization of protein association during early stages of protein formulation.