Mitigation\nStrategies against Antibody Aggregation\nInduced by Oleic Acid in Liquid Formulations

Polysorbates 20 and 80 (PS20 and PS80) are commonly used\nin the\nformulations of biologics to protect against interfacial stresses.\nHowever, these surfactants can degrade over time, releasing free fatty\nacids, which assemble into solid particles or liquid droplets. Here,\nwe apply a droplet microfluidic platform to analyze the interactions\nbetween antibodies and oleic acid, the primary free fatty acid resulting\nfrom the hydrolysis of PS80. We show that antibodies adsorb within\nseconds to the polar oleic acid–water interface, forming a\nviscoelastic protein layer that leads to particle formation upon mechanical\nrupture. By testing two different monoclonal antibodies of pharmaceutical\norigin, we show that the propensity to form a rigid viscoelastic layer\nis protein-specific. We further demonstrate that intact PS80 is effective\nin preventing antibody adsorption at the oleic acid–water interface\nonly at low antibody concentrations and low pH, where oleic acid is\nfully protonated. Importantly, introduction of the amino acid l-arginine prevents the formation of the interfacial layer and\nprotein particles even at high antibody concentrations (180 mg mL<sup>–1</sup>). Overall, our findings indicate that oleic acid\ndroplets in antibody formulations can lead to the formation of protein\nparticles via an interface-mediated mechanism. Depending on the conditions,\nintact PS80 alone might not be sufficient to protect against antibody\naggregation. Additional mitigation strategies include the optimization\nof protein physicochemical properties, pH, and the addition of arginine.