A comparative study of different strategies for removal of endotoxins from bacteriophage preparations

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...

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Bibliographic Details
Published in:Journal of microbiological methods 2017-01, Vol.132, p.153-159
Main Authors: Van Belleghem, Jonas D, Merabishvili, Maya, Vergauwen, Bjorn, Lavigne, Rob, Vaneechoutte, Mario
Format: Article
Language:English
Subjects:
Bacteriophages - growth & development
Centrifugation
Cesium - chemistry
Chlorides - chemistry
Colony Count, Microbial
Detergents
Endotoxins - isolation & purification
Octoxynol - chemistry
Pseudomonas aeruginosa
Pseudomonas aeruginosa - virology
Solvents
Staphylococcus aureus
Staphylococcus aureus - virology
Virus Cultivation
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Summary: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 10 -10 pfu/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.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2016.11.020