Impact of Buffers on Colloidal Property and Aggregation Propensities of a Bispecific Antibody

Abstract

Bispecific antibodies represent a promising avenue whereby 2 different binding specificities of a single-chain antibody can be grafted into a common Fc fragment to generate one antibody-like molecule. Despite the promising efficacy of such modalities, they may lack manufacturability because of stability and aggregation issues. Herein, we performed a systematic buffer screening for an aggregation-prone therapeutic bispecific antibody (BsAb) during early stage development. To this end, various buffers (histidine, glutamate, acetate, and arginine) and buffer combinations, including arginine and glutamate (Arg + Glu), were evaluated for their stabilizing effects on BsAb. Specifically, we identified an equimolar combination of histidine and glutamate (His + Glu at pH 5.0) buffer that showed enhanced colloidal stability as measured by dynamic light scattering interaction parameter (kD). This implies a role of net protein-protein interaction in mediating aggregation propensity of the protein. Two-dimensional nuclear magnetic resonance and multiangle light scattering experiments suggest the formation of a reversible dimer as a potential precursor to overall aggregation process. Furthermore, 1D nuclear magnetic resonance studies suggest a unique mode of interaction of histidine with BsAb that can be modulated by other buffer components or ionic strength.