Rule-based mitigation of charge asymmetry-triggered monoclonal antibody self-assembly

When evaluating the proclivity of a monoclonal antibody (mAb) for non-specific self-assembly, it is common to restrict charge distribution analyses to the variable region of mAbs, leaving out possible contributions from the constant region to the observed sticky behavior. Here, the aim was to study the relationship between charge asymmetry over the entire mAb surface and self-assembly propensity. To do so, we selected three mAbs with decreasing levels of charge asymmetry and evaluated their ability to engage in attractive self-interaction as a function of mAb concentration and ionic strength, using small-angle X-ray scattering, dynamic light scattering and micro-flow imaging. We show that the mAbs with oppositely charged Fab and Fc domains are characterized by overall attractive protein-protein interactions in solution amounting to diverse sub-visible morphologies, which vary non-linearly with mAb concentration and ionic strength. As a proof of concept, we also report the absence of any of such assemblies for the mAb with like-charged Fab and Fc domains, resulting in an overall repulsive behavior in solution. Altogether, we show how to utilize charge distribution analyses of full-length mAbs to rationally develop formulations that prevent unwanted self-assembly.