TY - JOUR
T1 - A comparative study of different strategies for removal of endotoxins from bacteriophage preparations
AU - Van Belleghem, Jonas D.
AU - Merabishvili, Maya
AU - Vergauwen, Bjorn
AU - Lavigne, Rob
AU - Vaneechoutte, Mario
N1 - Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2017/1/1
Y1 - 2017/1/1
N2 - 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 1012–1013 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.
AB - 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 1012–1013 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.
KW - Bacteriophages
KW - Endotoxin quantification
KW - Endotoxin removal
KW - Endotoxins
KW - Lipopolysaccharides (LPS)
KW - Pseudomonas aeruginosa
KW - Staphylococcus aureus
UR - http://www.scopus.com/inward/record.url?scp=85006136660&partnerID=8YFLogxK
U2 - 10.1016/j.mimet.2016.11.020
DO - 10.1016/j.mimet.2016.11.020
M3 - Article
C2 - 27913133
AN - SCOPUS:85006136660
SN - 0167-7012
VL - 132
SP - 153
EP - 159
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
ER -