Shelf-life prediction and storage stability of Aeromonas bacteriophage vB_AsM_ZHF

Phage therapy is a potential alternative to antibiotics for the treatment of bacterial infections. Due to the good antibacterial and therapeutic effects of phages, phage therapy has received attention and the demand for clinical applications has gradually increased. Phage storage stability and shelf life are key aspects of biopharmaceutical development and registration. In this study, Aeromonas salmonicida phage was stored at different temperatures for 12 months. We found that 4 °C was the optimal storage temperature. In the case of cryopreservation, 10% dimethyl sulfoxide (DMSO) was more effective at protecting the phage at -20 °C and -80 °C than 30% glycerin. Indeed, the phage titer decreased by only one order of magnitude within one year when DMSO was added. Hydroxyapatite (HAP) reduced the inactivation of phages by six orders of magnitude during storage at 28ºC for 1 year, significantly lower than that of in SM buffer. In addition, for excipients in lyophilization, tryptic soy broth (TSB) and tryptone or skim milk powder (SMP) in combination with trehalose alleviated phage inactivation during lyophilization and subsequent storage at 28 °C. Furthermore, a model for predicting the phage shelf-life was established with the Accelerated Stability Assessment Program (ASAP) based on the Arrhenius equation. The error of the model was less than 15% by comparing the predicted value with the actual value at 28 °C, indicating high accuracy. The study demonstrated the storage stability and shelf-life model of phage for the first time, which provided a theoretical basis for the development and application of phage products.