Freeze Induced Protein Instability Driven by Air Water Interfacial Stress

Most ice appears cloudy because tiny air bubbles form as water freezes. The bubbles that form during freezing can damage substances dissolved in the water. This project will focus on damage to substances such as proteins, pharmaceuticals, or vaccines. The project will experimentally control and quantify air bubble formation during freezing and study the impact of reducing air bubble formation on the stability of proteins. The results of this project will improve the shelf life of temperature-sensitive substances. For example, it will allow medicines that are thawed during shipping to be re-frozen without damage. Results of the project will benefit the biotechnology industry by improving resilience of the medical supply chain. Further benefits will accrue from training graduate and undergraduate students in research. The investigators will also conduct outreach at high schools on the interfacial science and engineering of familiar materials such as food, personal care products, or pharmaceuticals. This project will investigate the fundamental mechanisms of cryopreservation-induced damage in biologics. Protein denaturation is often believed to be attributable to denaturation at the ice-water interface. This project will instead focus on the role of gas-liquid interfaces formed in situ during the freezing process. The project will quantify the total air bubble-related stress across a variety of freezing conditions. A library of proteins with diverse surface activities and denaturation tendencies will be studied. Experiments will test the hypothesis that the benefit of deaeration is directly related to a protein's susceptibility to damage due to adsorption at the air-water interface. A key innovation will be the management of a strategic trade-off: introducing mild, controlled stress via deaeration to prevent significantly more destructive interfacial stress during subsequent ice formation. The overall aim is to lower the total burden of interfacial stress across the entire freeze-thaw cycle. If successful, this work will introduce deaeration as a novel third pillar of cryopreservation strategy, alongside the two well-known pillars of formulation optimization and freezing-rate optimization. The results will provide an alternative strategy for stabilizing biologics that are otherwise resistant to current protective methods. The project has potential benefit to biomanufacturing, biotechnology, and to facilitate storage and transport of heat-sensitive biomaterials. Additional benefits derive from training students at college and pre-college levels. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.