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The Removal of Endo- and Enterotoxins From Bacteriophage Preparations
Ville Hietala1, Jenni Horsma-Heikkinen1,2, Annelie Carron1
1Department of Bacteriology and Immunology, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
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_ApiM_fHyAci03, and Staphylococcus phage vB_SauM_fRuSau02. 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_fHoEco02 lysate from 3.5 × 104 Endotoxin Units (EU)/109 plaque forming units (PFU) to 0.09 EU/109 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/109 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/109 PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.
Insights
Phage purification for therapeutic use requires efficient endotoxin and toxin removal. Ultrafiltration combined with an EndoTrap HD affinity column proved most effective for endotoxin reduction, while ultrafiltration alone efficiently removed protein toxins.
Area of Science:
- Microbiology and Biotechnology
- Biopharmaceutical Manufacturing
- Purification Technologies
Background:
- Phage therapy requires scalable and efficient purification methods to remove harmful impurities like endotoxins and protein toxins.
- Endotoxins from gram-negative bacteria pose a significant safety risk, with strict limits for parenteral administration (5 EU/kg/h).
- Protein toxins from pathogenic bacteria also necessitate removal for safe clinical application of phage preparations.
Purpose of the Study:
- To evaluate the efficacy of various purification techniques for removing endotoxins and protein toxins from phage lysates.
- To identify optimal purification strategies for producing clinical-grade phage preparations.
- To assess the feasibility of different methods for endotoxin and toxin clearance in large-scale phage production.
Main Methods:
- Tested purification methods included polyethylene glycol precipitation, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, and specialized endotoxin removal columns.
- Assays involved three different phages: *Escherichia* phage vB_EcoM_fHoEco02, *Acinetobacter* phage vB_ApiM_fHyAci03, and *Staphylococcus* phage vB_SauM_fRuSau02.
- Efficiency was measured by phage titer and quantification of endotoxins or staphylococcal enterotoxins (SEA and SEC).
Main Results:
- The combination of ultrafiltration and an EndoTrap HD affinity column significantly reduced endotoxins in *E. coli* phage lysate (from 3.5 × 10^4 EU/10^9 PFU to 0.09 EU/10^9 PFU).
- Ultrafiltration coupled with anion exchange chromatography achieved an endotoxin ratio of 96 EU/10^9 PFU, further improved threefold with octanol extraction.
- Ultrafiltration (100,000 MWCO) effectively removed enterotoxins from *S. aureus* phage lysate (from 1.3 to 0.06 ng SEA/10^9 PFU).
Conclusions:
- Ultrafiltration combined with affinity chromatography (EndoTrap HD) is a highly effective method for endotoxin removal in phage purification.
- Ultrafiltration and anion exchange chromatography are suitable for reducing both endotoxins and protein toxins in phage preparations.
- These optimized purification strategies are crucial for the safe and scalable production of therapeutic phages.
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