Surface patches induce nonspecific binding and phase separation of antibodies

Nonspecific interactions are a key challenge in the successful development of therapeutic antibodies. The tendency for nonspecific binding of antibodies is often difficult to reduce by rational design, and instead, it is necessary to rely on comprehensive screening campaigns. To address this issue, we performed a systematic analysis of the impact of surface patch properties on antibody nonspecificity using a designer antibody library as a model system and single-stranded DNA as a nonspecificity ligand. Using an in-solution microfluidic approach, we find that the antibodies tested bind to single-stranded DNA with affinities as high as K(D) = 1 µM. We show that DNA binding is driven primarily by a hydrophobic patch in the complementarity-determining regions. By quantifying the surface patches across the library, the nonspecific binding affinity is shown to correlate with a trade-off between the hydrophobic and total charged patch areas. Moreover, we show that a change in formulation conditions at low ionic strengths leads to DNA-induced antibody phase separation as a manifestation of nonspecific binding at low micromolar antibody concentrations. We highlight that phase separation is driven by a cooperative electrostatic network assembly mechanism of antibodies with DNA, which correlates with a balance between positive and negative charged patches. Importantly, our study demonstrates that both nonspecific binding and phase separation are controlled by the size of the surface patches. Taken together, these findings highlight the importance of surface patches and their role in conferring antibody nonspecificity and its macroscopic manifestation in phase separation.