Improved lyophilization conditions for long-term storage of bacteriophages

Prasanth Manohar,Nachimuthu Ramesh

doi:10.1038/s41598-019-51742-4

Prasanth Manohar, Nachimuthu Ramesh

Open Access

https://doi.org/10.1038/s41598-019-51742-4

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Journal: Scientific Reports	Publication Date: Oct 23, 2019
Citations: 56	License type: open-access

Affiliation: Vellore Institute of Technology University

Abstract

Phage therapy is one of the promising alternatives to combat the increasing problem of antibiotic resistance. Lyophilization is used for the preparation of pharmaceutical products to improve their stability in long-term storage. The aim of this study was to improve the stability of lyophilized bacteriophages using different excipients. Three lytic bacteriophages Escherichia phage ECP311, Klebsiella phage KPP235 and Enterobacter phage ELP140 were subjected to lyophilization using six different excipients: glucose, sucrose, gelatin, mannitol, polyethylene glycol and sorbitol. The lyophilized phages were stored at 4 °C and 37 °C and rehydrated using biological saline to test their viability at 5 months interval up to 20 months. The results showed that the use of sucrose, gelatin and their combination was beneficial in maintaining the viability of phages post-lyophilization. When lyophilized phages were stored at 4 °C, their viability was maintained up to 20 months, but at 37 °C there was a reduction in activity after 10 months. This is one of the few studies to report the lyophilization of phage cocktails to have viability for up to 10 months. Our study identified promising lyophilization excipients to effectively lyophilize bacteriophages for pharmaceutical applications and long-term storage.

Highlights

Phage therapy is one of the promising alternatives to combat the increasing problem of antibiotic resistance
For the three tested phages, ECP311 (MG972768), KPP235 (MG983840) and ELP140 (MG999954), sucrose (0.5 M and 1.0 M), gelatin (1% and 2%) and the combination sucrose plus gelatin (0.5 M and 1%) were found to be beneficial in restoring www.nature.com/scientificreports the phage activity post-lyophilization (
Though earlier studies showed the use of polymer such as Polyethylene glycol (PEG) as a good excipient[2,3], this study showed the use of gelatin to have a more improved activity as an excipient

Summary

Introduction

Phage therapy is one of the promising alternatives to combat the increasing problem of antibiotic resistance. Our study identified promising lyophilization excipients to effectively lyophilize bacteriophages for pharmaceutical applications and long-term storage. To maintain stability during long-term storage of phages, preparation of lyophilization powders is one of the effective mechanisms that need more exploration. Earlier studies showed the use of skimmed milk, gelatin, peptone, sodium glutamate, polyethylene glycol, glycerol and other sugars (mannitol, sucrose, and trehalose) as effective excipients for phage lyophilization[3]. The majority of the studies on phage lyophilization showed less than 2 log reduction in phage titer when stored at 4 °C but it is important to study the viability of phages at ambient temperatures (35–37 °C) for long-term storage. We evaluated the viability of phages for long-term storage up to 20 months at 4 °C and 37 °C, and lytic activity of these phages was determined before and after lyophilization. A phage cocktail was lyophilized and the viability was evaluated up to 20 months at 4 °C and 37 °C

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