Determination of Protein-Protein Interactions at High Co-Solvent Concentrations Using Static and Dynamic Light Scattering

Abstract

Protein-protein interactions are commonly measured in terms of the second osmotic virial coefficient, B₂₂ from static light scattering (SLS) or the interaction parameter, k_D from dynamic light scattering (DLS). Often these measurements are carried out at high co-solvent compositions, where correction factors are required for the light scattering analysis. For lysozyme in aqueous solutions containing the co-solvents NaCl, arginine chloride, urea, sucrose or guanidine chloride, we show that B₂₂ determination requires using in the light scattering equation the refractive index increment of the protein measured at constant solvent chemical potential. Because the increment decreases with increasing co-solvent composition, using a constant value can lead to mis-interpretation of protein-protein interaction trends deduced from the B₂₂ measurements. Furthermore, there is a contribution to the intensity auto-correlation function measured by dynamic light scattering due to co-solvents. This effect is removed by including longer delay times when fitting the cumulant analysis to determine the diffusion coefficients. We show that an experimentally observed correlation between B₂₂ and k_D is recovered once these correction factors have been applied. The findings are particularly relevant to biopharmaceutical industry, where B₂₂ and k_D measurements are used for screening excipient effects in liquid formulations.

Keywords: Arginine; Biopharmaceuticals; Excipients; Guanidine hydrochloride; Lysozyme; Osmotic second virial coefficients; Urea.