Protein Stability At the Silicone Oil-Water Interface Using Single Molecule TIRF and Interfacial Tensiometry

Therapeutic protein solutions have become a popular strategy in disease treatment.  However, protein aggregation is a significant problem that must be understood and minimized in order to develop safer and more effective treatments. Aggregation at the silicone oil-water interface is of particular interest due to increased aggregation seen within prefilled glass syringes coated with silicone oil. In this presentation, the authors investigate the aggregation behavior of three model proteins (lysozyme, insulin, and IgG) at the silicone oil-water interface. Single molecule total internal reflection fluorescence microscopy (SMTIRFM) and interfacial tensiometry were used to identify the aggregation mechanics of the three proteins on the molecular level as well as demonstrate a connection between the microscopic data and bulk solutions. SMTIRFM captures molecular interactions by directly observing fluorescently-labeled proteins within a developing non-fluorescent protein layer allowing the collection of molecular interfacial diffusion rates as well as absolute adsorption kinetics. By directly capturing molecular interactions, SMTIRFM delves into within the regime where interfacial tensiometry does not measure any change in interfacial tension, known as the lag phase of protein adsorption.