Stability of therapeutic bacteriophages in clinically approved crystalloid infusion solutions

ABSTRACT The growing burden of antimicrobial resistance underscores the need for effective alternatives to conventional antibiotics, with bacteriophage therapy representing a promising approach. However, insufficient product stability during storage and administration may reduce the delivered dose and confound assessments of clinical efficacy. Clinically approved crystalloid infusion solutions could facilitate phage preparation and administration, but their suitability for phage storage remains poorly characterized. The stability of four virulent bacteriophages targeting Pseudomonas aeruginosa or Staphylococcus aureus was evaluated in sodium chloride 0.45%, sodium chloride 0.9%, Ringer’s lactate, Ringer’s acetate, and saline magnesium (SM) buffer. Phage preparations containing 10⁹ plaque-forming units per mL were stored at 4°C, −20°C, or −80°C, with or without 10% glycerol, for up to 6 months. Phage infectivity was highly dependent on the phage, solution, and storage temperature. Buffered solutions generally preserved infectivity better than saline, particularly under frozen conditions. Glycerol improved stability during freezing, although the magnitude of this effect varied among phages and was most apparent in saline solutions. Storage at −80°C with glycerol provided the most consistent preservation across all four phages, with titers remaining close to baseline. Ringer’s solutions supported high stability at −80°C also without glycerol. By contrast, storage at 4°C resulted in greater variability and reduced stability for several phages. These findings demonstrate that clinically approved crystalloid solutions can serve as vehicles for both phage storage and administration and suggest a need for phage-specific stability testing. IMPORTANCE Reliable bacteriophage therapy requires formulations that preserve phage activity during storage and clinical administration. Yet, standardized approaches to phage formulation and storage remain lacking, and many clinical applications still rely on empirical practices rather than evidence-based pharmaceutical principles. This study addresses an important translational barrier by examining the stability of therapeutically relevant phages targeting Staphylococcus aureus and Pseudomonas aeruginosa in crystalloid solutions routinely used for intravenous administration. Comparison of distinct phages across different formulations, temperatures, and storage durations demonstrates that stability depends strongly on the individual phage and storage conditions. These findings expand the limited evidence on phage stability in clinically applicable formulations and reinforce the rationale for individual phage stability testing during product development to ensure the delivery of an active and consistent therapeutic dose.