title: Multi-parameter flow cytometry as a process analytical technology (PAT) approach for the assessment of bacterial ghost production.
abstract: Flow cytometry (FCM) is a tool for the analysis of single-cell properties in a cell suspension. In this contribution, we present an improved FCM method for the assessment of E-lysis in Enterobacteriaceae. The result of the E-lysis process is empty bacterial envelopes-called bacterial ghosts (BGs)-that constitute potential products in the pharmaceutical field. BGs have reduced light scattering properties when compared with intact cells. In combination with viability information obtained from staining samples with the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylarbituric acid) trimethine oxonol (DiBAC4(3)), the presented method allows to differentiate between populations of viable cells, dead cells, and BGs. Using a second fluorescent dye RH414 as a membrane marker, non-cellular background was excluded from the data which greatly improved the quality of the results. Using true volumetric absolute counting, the FCM data correlated well with cell count data obtained from colony-forming units (CFU) for viable populations. Applicability of the method to several Enterobacteriaceae (different Escherichia coli strains, Salmonella typhimurium, Shigella flexneri 2a) could be shown. The method was validated as a resilient process analytical technology (PAT) tool for the assessment of E-lysis and for particle counting during 20-l batch processes for the production of Escherichia coli Nissle 1917 BGs.
authors.affiliation: BIRD-C GmbH & Co KG, Dr.-Bohr-Gasse 2-8, A-1030, Vienna, Austria.|RCPE-Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, A-8010, Graz, Austria.
authors.forename: Timo
authors.initials: T
authors.lastname: Langemann
authors.affiliation: BIRD-C GmbH & Co KG, Dr.-Bohr-Gasse 2-8, A-1030, Vienna, Austria.
authors.forename: Ulrike Beate
authors.initials: UB
authors.lastname: Mayr
authors.affiliation: RCPE-Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, A-8010, Graz, Austria.
authors.forename: Andrea
authors.initials: A
authors.lastname: Meitz
authors.affiliation: BIRD-C GmbH & Co KG, Dr.-Bohr-Gasse 2-8, A-1030, Vienna, Austria.|Center of Molecular Biology, University of Vienna, Dr.-Bohr-Gasse 9, A-1030, Vienna, Austria.
authors.forename: Werner
authors.initials: W
authors.lastname: Lubitz
authors.affiliation: Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1A 166/4, A-1060, Vienna, Austria. christoph.herwig@tuwien.ac.at.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
mesh_terms: D004755:Enterobacteriaceae / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes
keywords: Bacterial ghosts; E-lysis; Flow cytometry; Process analytical technology