Bacteriophage purification using CIMmultus monolithic OH-column chromatography for therapeutic purposes

Bacteriophages, viruses that selectively infect bacteria, are increasingly explored as potential therapies to combat antibiotic-resistant infections. Effective phage purification is vital for therapeutic use, particularly to reduce endotoxin (lipopolysaccharide, LPS) and host protein contamination from production processes in gram-negative bacterial lysates. This study investigates the purification efficiency of CIMmultus OH-monolithic chromatography for seven similarly sized tailed phages (Myoviridae and Siphoviridae families) infecting Pseudomonas aeruginosa. Using preferential exclusion chromatography with a potassium phosphate gradient, we achieved significant reductions in host proteins and endotoxins, yielding therapeutically compliant samples suitable for human intravenous injection. We observed recovery rates of 85-95% for smaller phages (e.g., PAML-31-1, LPS5), with lower recoveries for larger "giant" phages (e.g., OMKO1, PhiKZ). All purified samples met endotoxin limits, with average reductions of 98.5% for proteins and up to 6.30 log reductions for endotoxins. Despite increased DNA content post-purification in most samples, the approach shows promise for clinical-grade phage purification. This chromatographic strategy is scalable, reproducible, and free from hazardous chemicals, making it suitable for industrial phage production.