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 10 12 -10 13 pfu/ml, led to the strongest reduction of endotoxins, withan 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.

Human neutrophil response to Pseudomonas bacteriophages

“…LPS) sensing Toll-like receptor 4 (1). Thus, trace amounts of LPS in the phage preparation might be responsible for IL-8 secretion in neutrophils (26). However, LPS tends to enhance neutrophil sensitivity to subsequent inflammatory stimuli (27), which was not observed after exposure to the phage PAK_P1 preparation.…”

Pharmacological limitations of phage therapy

“…Reducing the variation coming from differences in immune response would require that phages that are going to be used in clinical trials be produced meeting the requirements set by international regulatory bodies for medicines. There is, however, currently a lack of a common best practice and standardised purification methods, and the degree of purity varies between methods (76,77). The best purifying method would need to be scaled up for fast production of larger volumes, and the phages would in most cases need to be formulated into stable medical preparations without reducing phage titres or phage function.…”

Review article: bacteriophages in gastroenterology—from biology to clinical applications

“…Highly purified phage particles are essential in order to prevent unwanted side effects due to the presence of microbial contaminants or endotoxins in crude phage preparations. Phage purification strategies are already available and are deemed necessary . Theoretically, the use of lytic phages can induce a rapid bacterial lysis, which can lead to a release of endotoxin, possibly causing a Jarisch‐Herxheimer reaction or an immunologic reaction to the release of endotoxin during antibiotic treatment .…”

“…Phage purification strategies are already available and are deemed necessary. 50,51 Theoretically, the use of lytic phages can induce a rapid bacterial lysis, which can lead to a release of endotoxin, possibly causing a Jarisch-Herxheimer reaction or an immunologic reaction to the release of endotoxin during antibiotic treatment. 52 However, lytic phages induce less endotoxin release than B-lactam antibiotics.…”