(580h) Aggregation Propensities of Monoclonal Antibody Formulations Studied through Interfacial Behavior

Monoclonal antibodies (mAbs) are therapeutic proteins that uniquely recognize antigens within the body, making them well-suited for the treatment of various diseases. These amphiphilic molecules readily adsorb to air-solution interfaces to form a viscoelastic network, which can lead to aggregation. One of the major challenges faced in the large-scale manufacture of these antibody molecules is their instability and propensity to form aggregates. This aggregation has an adverse effect on the quality and the immunogenicity of the drug and is triggered primarily due to interfacial stresses. Therefore, understanding the interfacial behavior of these antibody molecules is crucial in determining their aggregation propensity. In the biopharmaceutical industry, surfactants are typically added to mAb formulations as they competitively adsorb to the air-solution interfaces and prevent the antibody from adsorbing and aggregating. We studied mixtures of an antibody prone to aggregation, provided by Genentech, with three pharmaceutically relevant surfactants: polyethylene glycol, poloxamer-188 and polysorbate-20. We performed agitation studies and characterized the aggregation of both smaller (soluble) aggregates (<100 nm) and larger (insoluble) particulate aggregates using size-exclusion chromatography, flow cytometry and light obscuration techniques. The interface was visualized through confocal microscopic imaging of fluorescently-tagged mAbs and co-adsorbed surfactants. Surface tension and mAb relative surface coverage were quantified for these mixtures to get the interfacial characteristics. Moreover, we used a dynamic fluid-film interferometer, built in the lab, to study the thin film drainage as a bubble approached a flat interface in the presence of mAbs and surfactants in the solution. MAbs immobilized the sandwiched thin film, whereas surfactants triggered Marangoni instabilities and dimple washout. The volume of fluid entrapped and the nature of Marangoni surface flows depended on different governing mechanisms - interfacial rheology, surface tension and surface tension gradients for different surfactants. It is shown that the level of aggregation at different length scales correlated with the surface tension, surface relative coverage and interfacial fluid mechanics.