title: Segregated growth kinetics of Escherichia coli DH5α-NH36 in exponential-fed perfusion culture for pDNA vaccine production.
abstract: The clinical demand of plasmid DNA (pDNA) has been increasing constantly. An exponential-fed perfusion (EFP) culture is a new mode for plasmid production for clinical trials and commercialization. However, the culture conditions may lead to cell filamentation and growth cessation. In this study, the variation of the physiological state and the plasmid contents of Escherichia coli DH5α hosting pVAX1-NH36 in an EFP culture for application as a Leishmaniasis vaccine was investigated. The culture performance was monitored using flow cytometry (FC) and real-time quantitative PCR. The FC studies showed a high viability of cell population and a constant distribution of complexity and size. A high homogeneity of pDNA (>95 % of supercoiled) was obtained, which might be attributed to a better culture environment. The obtained plasmid specific and volumetric yields of 1.8 mg/g dcw and 36.5 mg/L represent typical values for laboratory-scale plasmid production in a defined medium. A segregated kinetic model of the perfusion system was developed and fitted to the experimental data (R(2) > 0.96). A practical conclusion of this work is that a space-time yield analysis of a bioprocess requires a viability evaluation. This new strategy of culture operation might help in the efficient production of pDNA for therapeutic use.
authors.affiliation: Posgrado en Ciencias de la Ingeniería, Universidad de Sonora, Hermosillo, Sonora, México.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Munguía-Soto
authors.affiliation: Posgrado en Ciencias de la Ingeniería, Universidad de Sonora, Hermosillo, Sonora, México.
authors.forename: Aurora
authors.initials: A
authors.lastname: García-Rendón
authors.affiliation: Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Hermosillo, Sonora, México.
authors.forename: Adriana
authors.initials: A
authors.lastname: Garibay-Escobar
authors.affiliation: Departamento de Ingeniería Química y Metalurgia, Universidad de Sonora, Hermosillo, Sonora, México.
authors.forename: Patricia
authors.initials: P
authors.lastname: Guerrero-Germán
authors.affiliation: Departamento de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Hermosillo, Sonora, México.
authors.forename: Armando
authors.initials: A
authors.lastname: Tejeda-Mansir
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002470:Cell Survival; D046508:Culture Techniques / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development*; D005285:Fermentation; D007700:Kinetics; D054332:Leishmaniasis Vaccines / Q000235:genetics; D008954:Models, Biological; D010477:Perfusion; D010957:Plasmids / Q000235:genetics*; D019444:Vaccines, DNA / Q000235:genetics*
chemical_list: D054332:Leishmaniasis Vaccines; D019444:Vaccines, DNA
keywords: exponential fed; growth kinetics; perfusion culture; plasmid DNA