(389d) Integrated Development of High-Throughput Phage Purification Strategies for Use in Human Phage Therapy

Bacteriophages are a promising antimicrobial alternative to modern antibiotics as they can rapidly overcome bacterial resistance. However, the propagation of therapeutic phages in gram-negative bacteria results in proinflammatory contaminants, notably endotoxins (lipopolysaccharides). To meet the strict regulations (0.5 EU/mL or 5 EU/kg/h for intravenous applications) regarding endotoxin levels, the widely accepted method for phage purification is based on polyethylene glycol precipitation followed by cesium chloride density gradient ultracentrifugation. However, this method is very labour-intensive while only producing small quantities of purified phage product. There have been a handful of studies on using membrane adsorbers in this application, however those studies have focused on only removing endotoxins or only recovering phages and thus there is a strong need for further study of removing endotoxins from bacteriophage preparations.

Two phages were isolated, sequenced, and propagated using E. coli plate cultures generating bacteriophage feedstocks with titers of 10⁹ - 10¹⁰ PFU/mL containing endotoxin concentrations in the range of 3 000 – 30 000 EU/mL. In some early purification tests with commercial anion exchange membrane absorbers, Mustang E (Pall) and Natrix Q (Millipore), in the standard syringe filter format, we discovered that the separation of phage from endotoxins was very poor. Recognizing the limitations of this sequential approach and the need to study a wide variety of solution conditions (e.g., complexation of endotoxins into various aggregate forms is determined by presence of detergents/ions), we have since pivoted to using a high-throughput screening approach recently developed at McMaster University. Also, we are using helium ion microscopy to directly visualize phage binding onto the membrane adsorbers. We anticipate that this approach will elucidate the governing factors for phage binding onto membranes which will be used to develop a universal method of generating clinical-grade phage preparations.