Leucine Protects Dry Powders for Inhalation Against Irreversible Moisture-Induced Aggregation

Abstract

Background: Pulmonary administration offers a promising route for certain biologics, particularly when treating respiratory diseases. Spray drying is widely employed to produce inhalable powders with the biologics incorporated in a stabilizing amorphous sugar. Hydrophobic amino acids such as leucine are frequently added to improve dispersibility. Objectives: While the aerodynamic benefits of leucine are well established, its influence on irreversible moisture-induced dry powder particle aggregation and protein stability during storage remains less evaluated. Methods: In this work, inulin-based powders with and without 4 wt-% leucine were spray dried and stored at 43%, 58%, 69%, and 75% relative humidity (RH) at 20 ± 2 °C. Results: Immediately after drying, both formulations displayed comparable physicochemical characteristics. However, during storage of inulin-only formulations, dry powder particles showed viscous flow and formed big irreversible aggregates after storage at an RH of 58% and above, whereas leucine-containing powders remained intact across all tested conditions up to 20 days. Protein stability was assessed using lactate dehydrogenase (LDH) and β-galactosidase (β-gal) as model proteins. At 43% RH, the Tg remained above the storage temperature, and both LDH and β-gal retained their enzymatic activity for up to 20 days. At 75% RH, however, the Tg dropped to below storage temperature, resulting in a loss of stability for LDH, consistent with its reliance on vitrification. In contrast, β-gal maintained its activity at 75% RH, indicative of stabilization through water replacement. Conclusions: Overall, these results demonstrate that leucine enhances the physical stability of inulin powders by preventing irreversible aggregation under humid conditions. However, this effect does not extend to protecting proteins reliant on vitrification. These findings highlight the potential of inulin and leucine to reduce the need for stringent storage conditions of biologics.