A temperature-triggered phase separation system for stabilizing bioactive protein structure and function

Protein-based pharmaceuticals such as vaccines are susceptible to temperature fluctuation-induced molecular aggregation and denaturation during transport or storage. This loss of structural integrity directly compromises their biological function. This study developed a reversible water-in-water (W/W) protein shielding system triggered by heat shock and featuring a tunable activation temperature to protect against cold chain dysfunction. The aqueous system remains homogeneous until the preset cloud point of 10 °C is surpassed, disrupting the hydrogen bond network and dehydrating polypropylene glycol (PPG) to trigger rapid phase separation. The inner ionic liquids (ILs) phase both enriches and stabilizes the protein hydration layer while preventing diffusion-driven collisions. Meanwhile, the outer PPG phase physically isolates the inner phase from air exposure and prevents protein aggregation at the air-liquid interface. Two protective systems (PPG₃₀/ChCl₁₆ and PPG₃₀/TCC₁₂) were designed and demonstrated the ability to protect a broad range of proteins. Negligible enzymatic activity loss was observed for both lipase and β-galactosidase after five cycles of 1 h of heating at 50 °C. Prior to use, the protein-containing bottom phase can be collected after standing and subsequently retrieved by centrifugation. This facile strategy senses thermal and mechanical stresses to provide act on need protection for ambient storage and transport of proteins.