title: Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools.
abstract: BACKGROUND
  Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.
  
  
  RESULTS
  Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.
  
  
  CONCLUSIONS
  It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität Braunschweig, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: David
authors.forename: Antje
authors.initials: A
authors.lastname: Berger
authors.forename: Robert
authors.initials: R
authors.lastname: Hänsch
authors.forename: Manfred
authors.initials: M
authors.lastname: Rohde
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Franco-Lara
mesh_terms: D001410:Bacillus megaterium / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D050296:Microbial Viability; D059010:Single-Cell Analysis / Q000379:methods*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D057127:Single-Chain Antibodies