The Removal of Endo- and Enterotoxins From Bacteriophage Preparations

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dc.contributor.author Hietala, Ville
dc.contributor.author Horsma-Heikkinen, Jenni
dc.contributor.author Carron, Annelie
dc.contributor.author Skurnik, Mikael
dc.contributor.author Kiljunen, Saija
dc.date.accessioned 2019-10-16T09:16:01Z
dc.date.available 2019-10-16T09:16:01Z
dc.date.issued 2019-07-23
dc.identifier.citation Hietala , V , Horsma-Heikkinen , J , Carron , A , Skurnik , M & Kiljunen , S 2019 , ' The Removal of Endo- and Enterotoxins From Bacteriophage Preparations ' , Frontiers in Microbiology , vol. 10 , 1674 . https://doi.org/10.3389/fmicb.2019.01674
dc.identifier.other PURE: 127458648
dc.identifier.other PURE UUID: 5648cb54-ce39-4e05-978e-78689b91fc7c
dc.identifier.other WOS: 000476737800002
dc.identifier.other ORCID: /0000-0001-8791-9260/work/63349454
dc.identifier.other ORCID: /0000-0003-0461-7270/work/63350062
dc.identifier.uri http://hdl.handle.net/10138/306055
dc.description.abstract The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiMiHyAci03, and Staphylococcus phage vB_SauMiRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_HoEco02 lysate from 3.5 x 10(4) Endotoxin Units (EU)/10(9) plaque forming units (PFU) to 0.09 EU/10 9 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/10(9) PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/10(9) PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay. en
dc.format.extent 9
dc.language.iso eng
dc.relation.ispartof Frontiers in Microbiology
dc.rights cc_by
dc.rights.uri info:eu-repo/semantics/openAccess
dc.subject antibiotic resistance
dc.subject bacteriophage
dc.subject phage therapy
dc.subject endotoxin
dc.subject enterotoxin
dc.subject PHAGE THERAPY
dc.subject PURIFICATION
dc.subject TOXIN
dc.subject 3111 Biomedicine
dc.subject 1183 Plant biology, microbiology, virology
dc.title The Removal of Endo- and Enterotoxins From Bacteriophage Preparations en
dc.type Article
dc.contributor.organization Research Programs Unit
dc.contributor.organization Department of Bacteriology and Immunology
dc.contributor.organization HUMI - Human Microbiome Research
dc.contributor.organization Faculty of Medicine
dc.contributor.organization University of Helsinki
dc.contributor.organization HUSLAB
dc.contributor.organization Helsinki One Health (HOH)
dc.contributor.organization Mikael Skurnik / Principal Investigator
dc.description.reviewstatus Peer reviewed
dc.relation.doi https://doi.org/10.3389/fmicb.2019.01674
dc.relation.issn 1664-302X
dc.rights.accesslevel openAccess
dc.type.version publishedVersion

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