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

Abstract

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 log 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 log pfu/ml measured after 12 months. In comparison, the PEV40 phage powders generally had a ∼ 0.5 log pfu/ml loss upon storage regardless of temperature. When aerosolized, the total in vitro lung doses of PEV2 were of the order of 10 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 10-10 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.

Inhalable bacteriophage powders: Glass transition temperature and bioactivity stabilization

“…Delivering them as aerosols can achieve direct targeting in the lungs. The feasibility of bioengineering phages into inhalable powders by spray drying has been demonstrated in vitro 1‐6 along with efficacy studies in vivo 7,8 . In the production process, it is essential to preserve phage bioactivity in the dry state by suitable excipients.…”

“…In the production process, it is essential to preserve phage bioactivity in the dry state by suitable excipients. Disaccharides such as trehalose, sucrose, and lactose are reported to provide protection to phages during drying and subsequent storage 1‐6 . This concept of sugar‐imparted phage protection was originated from protein stabilization mechanisms 9,10 .…”

“…There are two hypotheses on the mechanism of stabilizing proteins by sugars in the solid state, namely, water replacement and vitrification 9,10 . Since proteins are a major constituent of phages, these stabilization hypotheses may also be applicable to phages 3,4,11 …”

“…Many other studies have attempted to understand protein stabilization mechanism in the solid state 10,12,19‐21 . However, only a limited number of studies have explored phage stabilization in spray dried powders 3,4,22 …”

Implications of Bacteriophage- and Bacteriophage Component-Based Therapies for the Clinical Microbiology Laboratory

“…Given the proteinaceous composition of viruses, shear forces or variations in the temperature and pH to which phages are subjected during handling may impact titers as a result of protein misfolding or denaturation. Their shelf life may vary by bacteriophage species, as a result of phage size and morphology (29,(36)(37)(38)(39). The storage methods utilized in the Republic of Georgia, where phage therapy is used clinically, have included freezedrying, freezing, refrigeration, and short-term ambient storage (Marina Tediashvili, personal communication), though some studies suggest that phase changes, including freezing or drying, may result in drops in titer, at least among certain phages (37).…”