Stabilization of bacteriophage during freeze drying

With preliminary clinical trials completed for the treatment of antibiotic resistant infections using bacteriophages, there is a need to develop pharmaceutically acceptable formulations. Lyophilization is an established technique for the storage of bacteriophage, but there is little consensus regarding drying cycles, additives and moisture content specific to phage. Here, the addition of sucrose or poly(ethylene glycol) 6000 yielded stable freeze-dried cakes only from high concentrations (0.5 M and 5%, respectively), with addition of bacteriophage otherwise causing collapse. Gelatin, which is added to storage media (a solution of salts), played no role in maintaining bacteriophage stability following lyophilization. A secondary drying cycle was most important for maintaining bacteriophage activity. The addition of high concentrations of PEG 6000 or sucrose generally caused a more rapid fall in bacteriophage stability, over the first 7–14 d, but thereafter residual activities for all phage formulations converged. There was no distinct change in the glass transition temperatures (Tg) measured for the formulations containing the same additive. Imaging of cakes containing fluorescently labeled bacteriophage did not show gross aggregation or phase separation of bacteriophage during lyophilization. However, the moisture content of the cake did correlate with lytic activity, irrespective of the formulation, with a 4–6% moisture content proving optimal. We propose that residual moisture is followed during lyophilization of bacteriophage from minimal concentrations of bulking agent.