Room-Temperature Aerosol Dehydration of Green Fluorescent Protein

Abstract

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, RTAD utilizes sub-20 {micro}m droplets that evaporate rapidly at room temperature, thereby reducing drying-induced stresses for thermally sensitive biologics. In this study, we employed Green Fluorescent Protein (GFP) as a model biological molecule to optimize the RTAD system design and process parameters. We investigated the effects of droplet size, multiphase flow patterns in the drying chamber, and usage of polysorbate 20 as a model surfactant on GFP fluorescence after drying and powder reconstitution. The experiments demonstrated that the presence of polysorbate 20 in the formulation significantly influenced GFP fluorescence intensity, especially for smaller droplets. The numerical studies using Computational Fluid Dynamics simulations revealed that the intensity of GFP fluorescence in the produced dry powders was dependent on the patterns of multiphase flow in the drying chamber. Non-axisymmetric flows and closed circulating streamlines near the drying gas inlet negatively impacted the GFP fluorescence intensity. Through iterative optimization of chamber design, process parameters, and feedstock formulation, we achieved the recovery (compared to the initial samples) of GFP fluorescence intensity exceeding 96% in the obtained dry powders. This work establishes GFP as a sensitive model biologic and its fluorescence intensity as a powerful tool for rapidly assessing bioactivity following dehydration. The insights gained from this study have broad implications for the design and scale-up of room-temperature drying technologies, which can potentially transform the production of dry powder biopharmaceuticals.