Amplification, purification, and lyophilization of mycobacteriophages for therapeutic use
Bibliographic record
Abstract
ABSTRACT 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 Mycobacterium smegmatis 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. IMPORTANCE Bacteriophages specific for Mycobacterium hosts show promise as potential therapeutic agents for controlling nontuberculosis Mycobacterium 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".