Mapping a Ternary Carbohydrate Design Space for Stable and Dispersible Protein Dry Powders

Optimizing inhalable protein powders requires simultaneously preserving protein structure and minimizing interparticle cohesion, yet the mechanistic links between excipient composition, drying physics, surface energetics, and aerosol performance remain poorly defined. Here, a constrained ternary mixture design of inulin, trehalose, and mannitol was used to systematically interrogate composition–structure–function relationships in spray-dried lysozyme formulations. Protein secondary structure, glass transition temperature, humidity-dependent surface basicity (via inverse gas chromatography), and aerosol dispersibility (quantified by Wasserstein distance under controlled dispersion conditions) were measured and analyzed using linear and quadratic Scheffé mixture models. Carbohydrate composition strongly governed intermolecular β-sheet content and surface basicity, with trehalose positively associated with β-sheet formation and inulin promoting retention of surface basicity under humidity exposure. Most notably, the relative humidity at which surface basicity reached its minimum exhibited a strong inverse monotonic relationship with dispersibility (Spearman ρ = −0.86, p = 0.013), indicating that early moisture-induced surface reorganization corresponds to greater deviation from complete dispersion under inhalation conditions. These findings support a two-stage mechanistic framework in which drying-stage viscosity evolution defines a finite mobility window for compositional redistribution prior to vitrification, while postdrying surface relaxation dynamics govern susceptibility to moisture-mediated cohesion. Carbohydrate composition thus encodes both protein structural outcomes and aerosol performance through coupled drying- and relaxation-stage processes, providing a mechanistic foundation for rational design of inhalable biologic formulations.