Effect of storage temperature on the stability of spray dried bacteriophage powders

This study aimed to assess the robustness of using a spray drying approach and formulation design in producing inhalable phage powders. Two types of Pseudomonas phages, PEV2 (Podovirus) and PEV40 (Myovirus) in two formulations containing different amounts of trehalose (70% and 60%) and leucine (30% and 40%) were studied. Most of the surface of the produced powders was found to be covered in crystalline leucine. The powders were stored at 4 °C and 20 °C under vacuum. The phage stability and in vitro aerosol performance of the phage powders were examined on the day of production and after 1, 3 and 12 months of storage. A minor titer loss during production was observed for both phages (0.2-0.8 log₁₀ pfu/ml). The storage stability of the produced phage powders was found to be phage and formulation dependent. No further reduction in titer occurred for PEV2 powders stored at 4 °C across the study. The formulation containing 30% leucine maintained the viability of PEV2 at 20 °C, while the formulation containing 40% leucine gradually lost titer over time with a storage reduction of ∼0.9 log₁₀ pfu/ml measured after 12 months. In comparison, the PEV40 phage powders generally had a ∼ 0.5 log₁₀ pfu/ml loss upon storage regardless of temperature. When aerosolized, the total in vitro lung doses of PEV2 were of the order of 10⁷ pfu, except the formulation containing 40% leucine stored at 20 °C which had a lower lung dose. The PEV40 powders also had lung doses of 10⁶-10⁷ pfu. The results demonstrate that spray dried Myoviridae and Podoviridae phage in a simple formulation of leucine and trehalose can be successfully stored for one year at 4 °C and 20 °C with vacuum packaging.