Diffusion and Sedimentation Interaction Parameters for Measuring the Second Virial Coefficient and Their Utility as Predictors of Protein Aggregation

The concentration-dependence of the diffusion and sedimentation coefficients ( k D and k s , respectively) of a protein can be used to determine the second virial coefficient ( B 2), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B 2 under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k D and k s via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B 2 values for HEWL. In contrast to the assumptions necessary for determining k D and k s via SV alone, k D and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k D and k s assumed opposite signs; and 2), k D ≥ k s . Further, we demonstrate the utility of k D and k s as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B 2, k D , and k s , thus establishing the potential for k D to serve as a high-throughput predictor of protein aggregation.