A Reappraisal of Sedimentation Nonideality Coefficients for the Analysis of Weak Interactions of Therapeutic Proteins.

The study of weak or colloidal interactions of therapeutic proteins in different formulations allows prediction and optimization of protein stability. Various biophysical techniques have been applied to determine the second osmotic virial coefficient B as it reflects on the macromolecular distance distribution that governs solution behavior at high concentration. In the present work, we exploit a direct link predicted by hydrodynamic theory between B and the nonideality of sedimentation, commonly measured in sedimentation velocity analytical ultracentrifugation through the nonideality coefficient of sedimentation, k. Using sedimentation equilibrium analytical ultracentrifugation for independent measurement of B, we have examined the dependence of k on B for model proteins in different buffers. The data exhibit the expected linear relationship and highlight the impact of protein shape on the magnitude of the nonideality coefficient k. Recently, measurements of k have been considerably simplified allowing higher throughput and simultaneous polydispersity assessment at higher protein concentrations. Thus, sedimentation velocity may offer a useful approach to compare the impact of formulation conditions on weak interactions and simultaneously on higher-order structure of therapeutic proteins.