CIM monolithic chromatography as a useful tool for endotoxin reduction and purification of bacteriophage particles supported with PAT analytics.
In: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, Jg. 1217 (2023-02-15), S. 123606
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Zugriff:
Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants. In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
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CIM monolithic chromatography as a useful tool for endotoxin reduction and purification of bacteriophage particles supported with PAT analytics.
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Autor/in / Beteiligte Person: | Rebula, L ; Raspor, A ; Bavčar, M ; Štrancar, A ; Leskovec, M |
Zeitschrift: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, Jg. 1217 (2023-02-15), S. 123606 |
Veröffentlichung: | Amsterdam ; New York : Elsevier, c2002-, 2023 |
Medientyp: | academicJournal |
ISSN: | 1873-376X (electronic) |
DOI: | 10.1016/j.jchromb.2023.123606 |
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