Amplification and purification of T4-Like Escherichia coli phages for phage therapy: From laboratory to pilot scale

Abstract

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 109 to 1010 PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μmmembrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10mMmagnesium ions, phages showed no loss of titer over 1 month at 30C. © 2014, American Society for Microbiology.