Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein-protein interactions and self-association

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient D c , as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D 0 / D s (equal to the microscopic relative viscosity η eff / η 0 ) was found to be smaller than η / η 0 . Smaller ratios of the microscopic to macroscopic viscosity ( η eff / η ) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D 0 / D s with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from η eff / η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics. Measurement and interpretation of self-diffusion of a highly concentrated mAb with different formulations in context of viscosity and protein self-interactions.