Development of an inhalable dry powder of mycobacteriophage D29 using thin-film freeze-drying.

Publication date: Jul 05, 2025

This study aimed to develop a stable dry powder formulation of anti-tuberculosis mycobacteriophage D29 for pulmonary delivery using Thin-Film Freeze-Drying (TFFD), which uses rapid freezing and sublimation to generate highly aerosolizable powders. D29 is a shear-sensitive phage with a long, non-contractile tail from the family Siphoviridae, the most common morphotype for mycobacteriophages. Following a design-of-experiments approach, we first screened formulation and process variables for their influence on phage stability. Higher concentrations of trehalose and leucine and a higher drum temperature during freezing were identified as protective factors for the phage. A Box-Behnken design was then used to optimize levels of trehalose, leucine, Polyvinylpyrrolidone (PVP)-K25, and the drum temperature, and a model was constructed to enable prediction of optimal formulations. The predicted optimal formulation for titer recovery did retain high phage viability after drying and could deliver 10 or more plaque-forming units per dose from a dry powder inhaler. Additional formulations optimized for aerosol performance achieved fine particle fractions up to 70 % without further loss of titer. X-ray diffraction and differential scanning calorimetry of the phage powders showed that mannitol and leucine exhibited some degree of crystallinity while trehalose and PVP-K25 remained amorphous throughout 6 months of storage at 4 ^0C. The lead powder formulation remained stable in low-humidity storage at 4 ^0C and 22 ^0C in a nine-month stability study, with less than 0. 5 log reduction in titer over this storage period. However, storage at 40 ^0C led to rapid loss of phage activity. Delivery of the phage powder via a dry powder inhaler resulted in only 0. 33 log reduction in titer compared to delivery of a liquid phage formulation by nebulization, which showed over a 2-log loss. These findings support TFFD as a promising method for producing shelf-stable, inhalable bacteriophage powders and establish a formulation framework for developing similar powders of other therapeutic bacteriophages.

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