Amplification, purification, and lyophilization of mycobacteriophages for therapeutic use

Infections with nontuberculous mycobacterium (NTM) strains are frequently nonresponsive to antibiotic regimens, even after multiple drugs are used for extensive periods of months or years. Therapy with bacteriophages specific for NTM clinical isolates shows considerable promise in compassionate-use single-patient cases but, like antibiotics, must be administered over long periods of time and typically intravenously. It is thus important that the phage preparations are highly purified and stable to avoid adverse reactions or decay of viability. Here, we describe methods for purification of mycobacteriophages including CsCl equilibrium gradient ultracentrifugation, desalting by either dialysis or column chromatography, and stabilization for storage by lyophilization. These phage preparations have low levels of contaminating <i>Mycobacterium smegmatis</i> host proteins and host genomic DNA, and the lack of host DNA strongly indicates that these phages are not competent for generalized transduction. Desalting by column chromatography is both rapid and efficient for removal of CsCl after equilibrium density ultracentrifugation, and the preparations maintain viability well in lyophilization using trehalose as an excipient. Once lyophilized, the phage preparations typically maintain viability over extended periods of time, although once reconstituted at working dose concentrations and distributed into syringes, they experience about a 10-fold loss in viability over a 2-week period.IMPORTANCEBacteriophages specific for <i>Mycobacterium</i> hosts show promise as potential therapeutic agents for controlling nontuberculosis <i>Mycobacterium</i> infections. Phage administration for compassionate-use cases frequently involves intravenous twice-daily doses, but over a period of many months or years; the biosafety profile is, therefore, of substantial importance. Here, we describe the detailed methods we have used to grow mycobacteriophages to high titers, to concentrate and purify them so that they are devoid of major contaminants, and methods for stable long-term storage.