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Published on: March 30, 2017
Development of an inhalable dry powder of mycobacteriophage D29 using thin-film freeze-drying
Benjamin Southard1, Kyoka Melton1, Michael A Sandoval1
1Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA.
Abstract:
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 108 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 °C. The lead powder formulation remained stable in low-humidity storage at 4 °C and 22 °C in a nine-month stability study, with less than 0.5 log reduction in titer over this storage period. However, storage at 40 °C 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.
Insights
This study developed a stable dry powder formulation of anti-tuberculosis mycobacteriophage D29 for pulmonary delivery using thin-film freeze-drying (TFFD). The TFFD method successfully produced inhalable phage powders with high viability and stability for potential tuberculosis treatment.
Area of Science:
- Pharmaceutical Sciences
- Biotechnology
- Microbiology
Background:
- Mycobacteriophage D29, a bacteriophage targeting tuberculosis-causing bacteria, is shear-sensitive and requires specific formulation for pulmonary delivery.
- Pulmonary delivery offers a direct route for phage therapy against lung infections, but requires stable, aerosolizable formulations.
Purpose of the Study:
- To develop a stable dry powder formulation of mycobacteriophage D29 for pulmonary delivery using thin-film freeze-drying (TFFD).
- To optimize formulation and process parameters for phage viability, aerosol performance, and long-term stability.
Main Methods:
- Design-of-experiments (DOE) approach, including screening and Box-Behnken design, to identify optimal formulation excipients (trehalose, leucine, polyvinylpyrrolidone-K25) and process parameters (drum temperature).
- Thin-film freeze-drying (TFFD) technique for generating aerosolizable phage powders.
- Characterization of powder properties using X-ray diffraction (XRD) and differential scanning calorimetry (DSC).
- Stability studies under various temperature and humidity conditions.
- Assessment of phage viability and aerosol performance using a dry powder inhaler (DPI) and nebulization.
Main Results:
- Higher concentrations of trehalose and leucine, along with increased drum temperature, protected phage viability during TFFD.
- Optimized formulations achieved high phage titer recovery (>10^8 PFU/dose) and fine particle fractions up to 70% for aerosol delivery.
- Phage powders exhibited good stability in amorphous form (trehalose, PVP-K25) for 6 months at 4°C, with the lead formulation showing <0.5 log reduction in titer over 9 months at 4°C and 22°C.
- TFFD-delivered phage powder showed significantly better viability (0.33 log loss) compared to nebulized liquid formulation (>2 log loss).
Conclusions:
- Thin-film freeze-drying (TFFD) is a promising method for producing stable, inhalable bacteriophage powders.
- The developed formulation and TFFD process provide a framework for creating shelf-stable phage powders for pulmonary delivery.
- This approach holds potential for advancing phage therapy for respiratory infections like tuberculosis.
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