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While there is need for clear regulatory pathways for bacteriophage approval for mainstream use in clinical practice, practitioners and patients have been able to access bacteriophage therapy under compassionate grounds and through magistral preparations. However, there is currently no standard for purifying these bacteriophages to ensure safety, and good manufacturing practice certification may not be achieved in these emergency uses. In this study, we employed an Interleukin Receptor Associated Kinase (IRAK) 3 knockout monocyte-based assay to evaluate the endotoxin removal efficacy of three common bacteriophage purification methods: Triton X-100 exposure, CsCl density gradient ultracentrifugation, and Pierce\u2122 High-Capacity Endotoxin Removal Resin spin columns. In our experiments we tested these purification methods on three different bacteriophage morphotypes: siphovirus, podovirus and myovirus. We showed that the lowest endotoxin levels and immune responses were achieved when purifying bacteriophages with Triton-X treatment. The results from purifying with CsCl density gradient ultracentrifugation were comparable, and these were both significantly better than purification with Pierce\u2122 High-Capacity Endotoxin Removal Resin spin columns. We also showed that Triton X-100 purification resulted in the lowest loss of bacteriophage titres. Finally, of the bacteriophages tested here, it did not appear that virus morphology affected efficacy of endotoxin removal.<\/jats:p>","DOI":"10.1186\/s12985-024-02580-y","type":"journal-article","created":{"date-parts":[[2024,12,18]],"date-time":"2024-12-18T23:30:21Z","timestamp":1734564621000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Evaluation of effectiveness of bacteriophage purification methods"],"prefix":"10.1186","volume":"21","author":[{"given":"Siti Saleha","family":"Binte Mohamed Yakob Adil","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph","family":"Tucci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helen","family":"Irving","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cassandra","family":"Cianciarulo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mwila","family":"Kabwe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,12,19]]},"reference":[{"issue":"1","key":"2580_CR1","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1002\/bies.201400152","volume":"37","author":"EC Keen","year":"2015","unstructured":"Keen EC. 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All protocols were approved by the La Trobe University Biosafety committee, Approval No. IBC22009.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"318"}}