Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

Gorodnichev RB1, Kornienko MA1, Kuptsov NS1, Efimov AD2, Bogdan VI2, Letarov AV2, Shitikov EA1, Ilina EN1
About authors

1 Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia

2 Federal Research Center of Biotechnology, Moscow, Russia

Correspondence should be addressed: Roman B. Gorodnichev
Malaya Pirogovskaya, 1a, Moscow, 119435; moc.liamg@b.r.vehcindorog

About paper

Funding: all study expenses were covered by the funds allocated for the State Assignment on the Development of a personalized approach to the therapy of infections using virulent bacteriophages (Code: Bacteriophage).

Author contribution: Gorodnichev RB — planned the study, conducted the experiments, and wrote the manuscript; Kornienko MA, Letarov AV, Shitikov EA — planned the study, analyzed its results, and wrote the manuscript; Kuptsov NS, Efimov AD, Bogdan VI — conducted the experiments; Ilina EN — planned the study and wrote the manuscript.

Compliance with ethical standards: the experiments were conducted in full compliance with Biosafety Guidelines for working with risk group III–IV pathogens (SP 1.3.2322-08), Amendment 1 to Biosafety Guidelines for working with risk group III–IV pathogens (SP 1.3.2518-09), medical waste regulations (SanPin, and Federal Clinical Guidelines on the rational use of bacteriophages for therapy and prevention of diseases.

Received: 2021-07-20 Accepted: 2021-08-25 Published online: 2021-09-22

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

Keywords: phage therapy, bacteriophage, microbiology, Klebsiella pneumoniae, purification methods, bacterial lysate, endotoxin