title: A comparative study of different strategies for removal of endotoxins from bacteriophage preparations.
abstract: Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 1012-1013pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer.
authors.affiliation: Laboratory Bacteriology Research, Department of Clinical Chemistry, Microbiology and Immunology, University Ghent, Medical Research Building II, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: van.belleghem.jonas@gmail.com.
authors.forename: Jonas D
authors.initials: JD
authors.lastname: Van Belleghem
authors.affiliation: Laboratory for Molecular and Cellular Technology (LabMCT), Queen Astrid Military Hospital, Nederoverheembeek, Belgium; Laboratory Bacteriology Research, Department of Clinical Chemistry, Microbiology and Immunology, University Ghent, Medical Research Building II, De Pintelaan 185, 9000 Ghent, Belgium.
authors.forename: Maya
authors.initials: M
authors.lastname: Merabishvili
authors.affiliation: Rousselot (Northern, Central and Eastern Europe, UK and Ireland), Meulestedekaai 81, 9000 Gent, Belgium.
authors.forename: Bjorn
authors.initials: B
authors.lastname: Vergauwen
authors.affiliation: Laboratory of Gene Technology, KULeuven, Kasteelpark Arenberg 21, Box 2462, 3001 Leuven, Belgium.
authors.forename: Rob
authors.initials: R
authors.lastname: Lavigne
authors.affiliation: Laboratory Bacteriology Research, Department of Clinical Chemistry, Microbiology and Immunology, University Ghent, Medical Research Building II, De Pintelaan 185, 9000 Ghent, Belgium.
authors.forename: Mario
authors.initials: M
authors.lastname: Vaneechoutte
mesh_terms: D001435:Bacteriophages / Q000254:growth & development*; D002498:Centrifugation; D002586:Cesium / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D015169:Colony Count, Microbial; D003902:Detergents; D004731:Endotoxins / Q000302:isolation & purification*; D017830:Octoxynol / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000821:virology*; D012997:Solvents; D013211:Staphylococcus aureus / Q000821:virology*; D014776:Virus Cultivation
chemical_list: D002712:Chlorides; D003902:Detergents; D004731:Endotoxins; D012997:Solvents; D002586:Cesium; D017830:Octoxynol; C028019:cesium chloride
keywords: Bacteriophages; Endotoxin quantification; Endotoxin removal; Endotoxins; Lipopolysaccharides (LPS); Pseudomonas aeruginosa; Staphylococcus aureus