Room-Temperature Aerosol Dehydration of Green Fluorescent Protein

Rapid Room-Temperature Aerosol Dehydration (RTAD) is a novel, scalable drying technology for powderization and thermal stabilization of pharmaceutical drug products. Compared to conventional spray drying processes, typically using droplets of 10–200 μm in diameter generated by high-shear spraying, RTAD uses much smaller droplets with diameter 0.1 to 20 μm produced in modified liquid atomization processes. These fine droplets evaporate rapidly within 10–100 ms under room-temperature conditions, thereby reducing drying-induced stresses for thermally sensitive biologics. In this study, we used Green Fluorescent Protein (GFP) as a model biological molecule to optimize the design of the RTAD system and the process parameters. We experimentally investigated the effects of droplet size, multiphase flow patterns in the drying chamber, and application of polysorbate 20 as a model surfactant on GFP fluorescence after drying and powder reconstitution. The experiments demonstrated that the presence of surfactant in the formulation significantly influenced the GFP fluorescence intensity, especially for smaller droplets. The numerical studies using Computational Fluid Dynamics simulations revealed that the drying of droplets was dependent on the patterns of multiphase flow in the drying chamber, which can impact the intensity of GFP fluorescence in the produced dry powders. Non-axisymmetric flows and closed circulating streamlines near the drying gas inlet resulted in considerably longer particle residence times, which we infer means that GFP molecules were subjected to excess stress that negatively impacted the GFP fluorescence intensity. Through iterative optimization of the chamber design, process parameters and feedstock formulation, we achieved recovery of the GFP fluorescence intensity that exceeded 96% in the obtained dry powders. This work establishes GFP as a sensitive model biologic and its fluorescence intensity as a powerful tool to rapidly assess process efficiency and the ability to preserve bioactivity after dehydration. The study has broad implications for the design and scale-up of drying technologies, which can potentially transform the production of dry powder biopharmaceuticals.