Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation : Part II: Concentrated protein solutions.

Abstract

Protein-protein interactions in monoclonal antibody solutions are important for the stability of a therapeutic drug and directly influence viscosity in concentrated protein solutions. This study describes the use of small-angle scattering to estimate protein-protein interactions at high concentrations of the IgG1 NISTmAb reference material and validate colloidal models for interacting molecules. In particular, we studied the colloidal stability of the NISTmAb at high protein concentrations and analyzed protein-protein interactions upon adding sodium chloride and its effect on viscosity. Isotropic colloidal models for interacting molecules were combined with an ensemble of atomistic structures from molecular simulation to account for the flexibility of the NISTmAb in solution. In histidine formulation buffer, net repulsive electrostatic interactions are important for the colloidal stability of the NISTmAb at high concentrations. Addition of sodium chloride increased the viscosity of the NISTmAb and decreased the colloidal stability due to charge screening of the repulsive interactions. The interactions at high concentrations (up to ~ 250mg/mL) were consistent with those from light scattering at low concentrations (below ~ 20mg/mL). However, in the presence of sodium chloride, the screening of charges was less pronounced with increasing protein concentration and the interactions approached those of the repulsive hard-sphere models. Additionally, we studied the NISTmAb under frozen conditions using in situ neutron scattering to analyze the crowded state as proteins are excluded from the water-rich phase. In the frozen samples, where protein concentration can reach hundreds of mg/mL in the protein-rich phase, sodium chloride did not affect the molecular spacing and crowding of the NISTmAb. Graphical Abstract Net repulsive interactions in concentrated NISTmAb solutions assessed by small-angle neutronscattering.