(595d) The Impact of Free Fatty Acids on Interface-Induced Aggregation of a Mab-Surfactant Solution

Protein-based therapeutics such as monoclonal antibodies (mAbs) are inherently prone to aggregation during manufacturing steps or storage periods. One of the reasons for particle formation is that once in contact with hydrophobic interfaces, protein molecules change their higher-order structure, which leads to intermolecular interactions and aggregation. Since the presence of subvisible particles may cause undesirable reactions upon drug administration, surfactants such as polysorbate 20 (PS20) are added to formulations aiming to reduce protein interactions with interfaces. However, PS20 is prone to degradation via hydrolysis, which is usually caused by the presence of copurified enzymes capable of cleaving lateral ester bonds, resulting in free fatty acids (FFAs). Besides the reduced surfactant concentration, another concern is that FFAs present low solubility in aqueous solutions, and FFA particles may serve as nucleation sites for protein aggregation. Further, FFAs are themselves surface active and may compete with the surfactants for the interface. How the presence of FFAs at the interface alters the ability of PS to prevent interface-induced protein aggregation is currently not well understood. In this work, a Langmuir trough is used to record the surface activity of a mAb formulation in the presence of PS20, in the absence and presence of interfacial dilatational stresses. Oleic and lauric acids were added to these formulations to mimic typical FFAs found in degraded formulations. Changes in the surface activity in the presence of these FFA were used to evaluate how degradation byproducts impact interface-induced aggregations in mAb/surfactant formulations. Specifically, measurements of surface tension changes are correlated with the characterization of visible and subvisible particles, both at the interface and in bulk solution. Initially, surface pressures were measured to evaluate the surface activity of protein, surfactants, and FFA molecules at the unstressed interface (when no dilatational stresses were applied). Next, compression/expansion cycles were applied to the interface using Delrin barriers, and surface pressure vs. area isotherms were recorded and analyzed. Lastly, samples from the bulk and the interface were collected and analyzed via microfluid imaging (MFI), to evaluate particle morphology and concentration. Based on the surface pressure vs. time measurements, we concluded that the unstressed interface was dominated by surfactant or FFA molecules, even when surfactant concentration was below CMC. However, after dilatational stresses were applied, we noticed the coadsorption of protein, surfactant, and FFA molecules at the interface, depending on the FFA used. Our dilatational stress studies showed that the addition of lauric acid to protein samples in the presence of surfactant below CMC resulted in higher hysteresis, which may be associated with a higher propensity to protein particle formation. MFI analysis confirmed the observation and showed higher particle counts, especially the formation of larger and elongated aggregates. By contrast, adding oleic acid, which is more soluble in water, resulted in less hysteresis and no significant changes in particle count or morphology. These results suggest that FFA particles may act as nucleation sites for further protein interaction, leading to the formation of proteinaceous aggregates.