Evalyne M. Jansen, Henderik W. Frijlink and Wouter L. J. Hinrichs
Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute of Pharmacy, Faculty of Science and Engineering, University of Groningen, Groningen, The Netherlands.
Summary
Pulmonary delivery is a promising route for biologics, especially for respiratory diseases. Spray drying is commonly used to produce inhalable dry powder formulations. Inulin, a high glass transition temperature (Tg) polysaccharide, can be used to stabilize biologics during spray drying. However, at elevated relative humidity (RH), moisture uptake lowers the Tg below ambient temperature. This leads to an increased molecular mobility, causing aggregation, plasticization and protein instability. Leucine, a hydrophobic amino acid, is frequently added to improve powder dispersibility, but its role in mitigating moisture-induced destabilization during storage is less well understood. In this study, inulin-based powders with and without 4 wt-% leucine were spray dried and stored at 43%, 58%, 69%, and 75% RH at ambient temperature. Both formulations displayed similar physicochemical properties after spray drying. However, during storage, inulin-only powders exhibited pronounced aggregation from 58% RH onwards, whereas leucine-containing powders did not show particle aggregation. Protein stability was assessed using β-galactosidase (β-gal) and lactate dehydrogenase (LDH). β-gal remained enzymatically active for 20 days at 75% RH regardless of leucine, consistent with stabilization via water replacement. By contrast, LDH showed ~50% loss of activity within 5 days at 75% RH, indicating reliance on vitrification, which is compromised under humid conditions. These findings demonstrate that leucine enhances the physical stability of spray-dried powders by reducing moisture-induced aggregation, though it does not directly improve protein stability under humid conditions. This insight supports the rational design of more robust inhalable biologic formulations for use in humid climates.