Mitigation Strategies against Antibody Aggregation Induced by Oleic Acid in Liquid Formulations.

Abstract

Polysorbates 20 and 80 (PS20 and PS80) are commonly used in the formulations of biologics to protect against interfacial stresses. However, these surfactants can degrade over time, releasing free fatty acids, which assemble into solid particles or liquid droplets. Here, we apply a droplet microfluidic platform to analyze the interactions between antibodies and oleic acid, the primary free fatty acid resulting from the hydrolysis of PS80. We show that antibodies adsorb within seconds to the polar oleic acid-water interface, forming a viscoelastic protein layer that leads to particle formation upon mechanical rupture. By testing two different monoclonal antibodies of pharmaceutical origin, we show that the propensity to form a rigid viscoelastic layer is protein-specific. We further demonstrate that intact PS80 is effective in preventing antibody adsorption at the oleic acid-water interface only at low antibody concentrations and low pH, where oleic acid is fully protonated. Importantly, introduction of the amino acid l-arginine prevents the formation of the interfacial layer and protein particles even at high antibody concentrations (180 mg mL-1). Overall, our findings indicate that oleic acid droplets in antibody formulations can lead to the formation of protein particles via an interface-mediated mechanism. Depending on the conditions, intact PS80 alone might not be sufficient to protect against antibody aggregation. Additional mitigation strategies include the optimization of protein physicochemical properties, pH, and the addition of arginine.