Fab–Fc and Fab–Fab interactions of variable strength and valency contribute to the high concentration viscosity of IgG ₁ antibodies

The variable domains in Fab regions are well-established contributors to high concentration viscosity of IgG, primarily through charge and hydrophobic interactions. In contrast, the roles of the Fc and the number of self-interacting sites (valency) are less well understood. Here, we investigate the relative contributions of Fab-Fab and Fab-Fc interactions to high concentration viscosity for a diverse panel of 20 IgG₁ antibodies, by rheometry, coarse-grained simulations, and molecular surface property analysis. Strikingly, fragmentation of IgG₁ into F(ab')₂ plus Fc reduced viscosity (-11 to -93%) for all antibodies tested, demonstrating prevalent contributions of Fc to viscosity. Coarse-grained simulations with one site per Fab and two sites per Fc qualitatively tracked trends in experimental rheometry data for 20 parental antibodies and their fragments. In these simulations Fab-Fab and Fab-Fc interaction strengths were independently varied to capture possible interaction differences arising from parental sequences or any mutations. These coarse-grained simulations suggest that Fab-Fc attractions generate branched IgG₁ networks and disproportionately larger clusters relative to Fab-Fab interactions of comparable strength. This study suggests that a four-site self-interaction model, previously proposed for a single antibody (omalizumab), is broadly applicable to diverse IgG₁. Beyond well-established variable domain engineering, this self-interaction model predicts that Fc engineering may reduce IgG₁ viscosity, a much sought after goal to enable subcutaneous delivery. Clinically validated Fc mutations are demonstrated here to substantially reduce the viscosity for multiple IgG₁ (-33 to -91% reduction, n = 6), supporting this emerging antibody design concept.