Introduction: During their production, processing, storage and delivery to patients, therapeutic proteins are exposed to various interfaces, such as the interface between the silicone oils that are used to lubricate glass syringes and aqueous solutions in which the proteins are formulated. Proteins may adsorb to these interfaces, which in turn can result in aggregation of the protein. Protein aggregation in pharmaceutical formulations is associated with changes in potency, risks of increased immunogenicity, and shortened shelf life, and hence is a major contributor to the estimated $1.2 billion required for development of a new protein-based therapeutic. Interfacial damage of proteins is a particular problem at fluid-fluid interfaces, where the dynamic nature of the interface (e.g., in response to shear forces experienced during shipping and handling of a protein formulation) may offer increased exposure of interfaces to proteins. This project examines the behavior of therapeutic proteins as they interact with silicone oil/water interfaces. The mechanisms of such interactions are probed with advanced spectroscopic and physical techniques, with a goal of developing rational design strategies to prevent interfacial protein damage and reduce associated costs and health risks.
Intellectual Merit: Protein adsorption and aggregation at interfaces is ubiquitous, but the fundamental mechanisms leading to protein adsorption at interfaces and consequent generation of aggregates remain poorly understood. This project, a collaboration between two research groups with expertise in protein interfacial science, protein conformational thermodynamics and aggregation kinetics will address both the microscale mechanisms that lead to protein adsorption and unfolding at interfaces, and the kinetic processes that result in macroscopically observable protein aggregation.
To characterize the kinetics of adsorption and interfacial aggregation at oil-water interfaces, a combination of several state-of-the-art experimental techniques will be developed and applied. These include single-molecule tracking micro-rheology (using fluorescence microscopy), emulsion adsorption, fluorescence-activated cell sorting (FACS) and front-face fluorescence quenching of adsorbed protein. Molecular conformation of proteins at silicone oil-water interfaces will be measured using Forster resonant energy transfer (FRET) in order such as to establish direct connections between protein conformation and dynamic processes such adsorption, desorption, interfacial mobility, aggregation. Likewise, protein adsorption at the air-water interface will be measured using dynamic pendant bubble tensiometry, emulsion depletion experiments and FACS, and the resulting protein aggregation monitored by flow microscopy, chromatography and FACS. The links between interfacially-induced protein damage and formulation conditions will be explored by determining the effects of interfacial area change, protein concentration, thermodynamic conditions, and excipients. By manipulating the thermodynamic stability of the protein's native state structure (e.g., with stabilizing excipients), microscopic protein unfolding processes will be linked to interfacial phenomena and macroscopic measurements of agitation-induced-aggregation kinetics.
Broader Impacts: The project will have several broad impacts. First, a detailed understanding of protein adsorption at oil-water interfaces will aid the design of formulations that provide protection against interfacially-induced protein aggregation, reduce development costs and offer increased patient safety. Second, the many new techniques that will be tested and developed will be of use to a wide variety of academic and industrial scientists. Furthermore, protein adsorption and aggregation at interfaces is of critical importance to several other scientific endeavors, such as vaccinology, applications of microfluidics and nanotechnology in the diagnostics arena and the development of implantable medical devices. By linking two research groups with diverse expertise in interfacial science and protein formulation, the graduate and undergraduate students who participate in this research will receive a broad, cross-disciplinary training. In addition, because of the groups close ties with the biopharmaceutical industry, the results of this research will be rapidly disseminated so as to afford maximum impact in practical applications of the proposed new, fundamental scientific studies.