Bacteriophage Particle Morphology Defines the Efficiency of Steric Exclusion Chromatography in Centrifuge-Free...

Bacteriophages have emerged as biological solution to control bacterial outbreaks in agriculture, food production, and healthcare; however, phage isolation and initial purification remain a costly and time-consuming process. In this study, the classic phage purification method containing two centrifugation steps was compared to the alternative, in which low-speed centrifugation for cell debris removal was replaced by microfiltration in combination with filter aid, and high-speed centrifugation for particle precipitation was substituted with steric exclusion chromatography. The efficiency of both approaches in phage recovery, concentration, and purification was assessed on bacteriophages of different morphology (tailed and non-tailed), accumulated on Pseudomonas syringae, Pseudomonas aeruginosa, and Serratia marcescens. The results revealed that the centrifuge-free approach shortened the purification procedure by more than twice and increased the final titer of non-tailed phages up to 100×, resulting in over 90% yield of suspension of sufficient quality for downstream characterization. In the case of tailed phages, only low-speed centrifugation step was successfully replaced, indicating the crucial impact of particle morphology on efficiency of purification by steric exclusion chromatography. The difference in efficiency between isometric and tailed particles highlights the need for tailored method development for the manufacturing of bacteriophage-based preparations.