Arne Echterhof, Tejas Dharmaraj, Patrick Blankenberg, Maryam Hajfathalian, Francis G. Blankenberg, Jessica C. Sacher, Paul L. Bollyky
Bacteriophages are increasingly explored as antibacterial agents for therapeutic applications and pathogen biocontrol. Rigorous quality control and removal of microbial growth byproducts is essential for safety and compliance with regulatory standards for the use of phages; for phages isolated from Gram-negative bacterial lysates, this involves removal of endotoxin (lipopolysaccharide/LPS) and contaminating host proteins. Here, we describe the development of weakly hydrophobic hydroxyl-functional monolithic chromatography (CIMmultus-OH) as a single-step purification process for five tailed anti-pseudomonal phages, including myoviral and siphoviral morphologies, as well as extra-large bacteriophages so-called “jumbo” phages, yielding therapeutically compliant samples suitable for human intravenous administration. Using a salt-driven preferential exclusion chromatography with a potassium phosphate gradient, we achieved near-complete removal of endotoxin (99.98–≥99.99% depletion) and protein impurities (99.12–99.83% depletion). We found that phage recovery post-purification was inversely associated with tail length and effective particle size (hydrodynamic diameter), with shorter-tailed and smaller phages demonstrating greater recovery. We conclude that single-step CIMmultus OH-monolithic chromatography is scalable, reproducible, and free from hazardous chemicals, supporting its integration into industrial phage purification processes.