Abstract
The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward—by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 1011 plaque-forming units (PFUs) per milliliter for Pseudomonas, Klebsiella, and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 109 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 106-fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank J. Grose at Brigham Young University for gifting us phages JG265, JG266 and SM219 and the Serratia and Klebsiella strains. We thank R. Schooley at the University of California, San Diego for providing feedback. This research was supported in part by National Institutes of Health grant RC2DK116713 to R.A.E. and San Diego State University startup funds to D.R.R.
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D.R.R. and R.A.E. conceived the concepts and supervised the research and development. T.L. and A.C.S. conducted the in vitro experiments. R.A.E. performed the genomic analyses. All authors wrote and commented on the manuscript.
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Forti, F. et al. Antimicrob. Agents Chemother. 62, e02573-17 (2018): https://aac.asm.org/content/62/6/e02573-17
Roach, D. R. et al. Cell Host Microbe 22, 38–47 (2017): https://doi.org/10.1016/j.chom.2017.06.018
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Luong, T., Salabarria, AC., Edwards, R.A. et al. Standardized bacteriophage purification for personalized phage therapy. Nat Protoc 15, 2867–2890 (2020). https://doi.org/10.1038/s41596-020-0346-0
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DOI: https://doi.org/10.1038/s41596-020-0346-0
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