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 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.

Keywords: Antibiotic resistance; PEV2; PEV40; Phage; Phage dry powder; Pulmonary infections.

Figure 1

SEM images of F1 – F4.

Figure 2

(a) XRD profiles of the spray dried pure leucine and formulations containing 30% (F1, F3) and 40% (F2 and F4) leucine. (b) Raman spectra of reference materials, crystalline trehalose dihydrate (c-Tre), spray dried amorphous trehalose (a-Tre), crystalline l-leucine (c-leu) and amorphous leucine (a-leu), and spray dried phage powders.

Figure 3

(a) DSC and (b) TGA curves for formulations containing 30% (solid lines) and 40% (broken lines) leucine.

Figure 4

(a) Vapor sorption profiles and (b) kinetic of moisture uptake at 60% RH for formulations containing 30% (solid lines) and 40% (broken lines) leucine.

Figure 5

Phage stability in F1 – F4 formulations of phages PEV2 (A) and PEV40 (B) after storage under vacuum at 4 and 20 °C. Titer reductions at 0 (Fresh), 1, 3 6 and 12 months are relative to the titer measured in the spray dryer liquid feedstock. Asterisk (*) denotes a statistically significantly higher (p < 0.05) titer reduction compared with the freshly prepared powders.

Figure 6

The distribution profiles, total mass recovery and FPF of viable PEV2 phage. Data presented as mean ± one standard deviation (n = 3). All formulations were dispersed at 100 L/min for 2.4 s using the Osmohaler™. The aerodynamic cutoff diameter of each stage is quoted in parentheses.

Figure 7

The distribution profiles, total mass recovery and FPF of viable PEV40 phage. Data presented as mean ± one standard deviation (n = 3). All formulations were dispersed at 100 L/min for 2.4 s using the Osmohaler^TM. The aerodynamic cutoff diameter of each stage is quoted in parentheses.

Figure 8

Total fine particle dose of PEV2 (A) and PEV40 (B) phage of powders stored up to 12 months at 4 and 20 °C. Data presented as mean ± one standard deviation (n = 3). Asterisk (*) denotes a statistically significantly higher (p < 0.05) titer reduction compared with the freshly prepared powders.