Highly concentrated therapeutic monoclonal antibody (mAb) solutions are desired for subcutaneous delivery in patients. However, at these high concentrations, protein-protein interactions (PPI) including short-range anisotropic attractions may cause prohibitively high viscosities and poor storage stability. The understanding of this behavior has been limited by the lack of characterization techniques that are applicable at these extreme concentrations. Here, we determine the effects of pH and co-solute on the strength and geometry of short-range anisotropic attractions for a set of mAbs using structure factors (S(q)) from small angle x-ray scattering (SAXS) and coarse-grained molecular dynamics (MD) simulations. We show that that the S(q) at intermediate q measures the extent of clustering and is a better metric for high concentration viscosity than measures of net interaction strength at low or high concentration (k<sub>D</sub> or S(0) respectively). By fitting S(q) to MD simulations, we quantify the strength of short-range, anisotropic attraction (K) and cluster size distribution directly at high concentration. At low pH and ionic strength, weak K, no clustering, and low viscosity suggest that the propensity of positive charges reduces the likelihood of anisotropic attractions. By raising pH and/or ionic strength, the effect of these positive charges is reduced, and the greater attractions are captured by a larger K and increased clustering and viscosity. Adding the binding cosolute arginine adds positive charges on the mAb surface and disrupts anisotropic attractions, weakening clusters and reducing viscosity. The importance of cluster size and morphology, which are obtained from SAXS/simulation, on viscosity is further highlighted in an improved viscosity model. The ability to characterize short-range, anisotropic attractions and identify the presence of oligomers has great utility in designing and formulating low viscosity, stable mAb solutions.

Protein-protein interactions, self-association, and viscosity in antibody solutions via small angle x-ray scattering (SAXS) and coarse-grained molecular dynamics simulations

ACS Spring 2022

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