Analysis of Macromolecular Size Distributions in Concentrated Solutions

Abstract The solution state of macromolecules in concentrated solutions impacts fields ranging from cell biology, to colloid chemistry and engineering of protein pharmaceuticals. Dependent on the interplay between repulsive and weakly attractive forces, proteins may exhibit oligomerization, aggregation, crystallization, liquid‐liquid phase separation, or the formation of multiprotein complexes. The particle size‐distribution is a key characteristic, but difficult to determine when interparticle distances are on the order of their size and macromolecular motion is coupled through hydrodynamic interactions. Here we extend sedimentation velocity analytical ultracentrifugation to measure macromolecular size distributions under these conditions: We apply results from statistical fluid mechanics for the concentration‐dependence of hindered settling and diffusion, embedded in a mean‐field approximation that can resolve coupled sedimentation and diffusion processes of different sized species given experimental sedimentation data. This is combined with a description of transient optical aberrations from lensing in the refractive index gradients associated with sedimentation boundaries (Wiener skewing). We demonstrate this approach in the application to protein solutions with macromolecular volume fractions up to ≈10 %, for example, resolving monomers and dimers of albumin at 140 mg/ml. This enables size‐distribution analysis of proteins at concentrations of therapeutic antibody formations and close to physiological concentration in serum and cells.