Segregated growth kinetics of Escherichia coli DH5α-NH36 in exponential-fed perfusion culture for pDNA vaccine production

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...

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Veröffentlicht in:Biotechnology and applied biochemistry 2015-11, Vol.62 (6), p.795-805
Hauptverfasser: Munguía-Soto, Rodolfo, García-Rendón, Aurora, Garibay-Escobar, Adriana, Guerrero-Germán, Patricia, Tejeda-Mansir, Armando
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container_issue 6
container_start_page 795
container_title Biotechnology and applied biochemistry
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creator Munguía-Soto, Rodolfo
García-Rendón, Aurora
Garibay-Escobar, Adriana
Guerrero-Germán, Patricia
Tejeda-Mansir, Armando
description 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 (R2 > 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.
doi_str_mv 10.1002/bab.1339
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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 (&gt;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 (R2 &gt; 0.96). A practical conclusion of this work is that a space–time yield analysis of a bioprocess requires a viability evaluation. 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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 (&gt;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 (R2 &gt; 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.</abstract><cop>United States</cop><pub>Blackwell Publishing Ltd</pub><pmid>25556882</pmid><doi>10.1002/bab.1339</doi><tpages>11</tpages></addata></record>
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subjects Biotechnology
Biotechnology - methods
Cell culture
Cell Survival
Clinical trials
Commercialization
Constants
Culture
Culture Techniques - methods
Deoxyribonucleic acid
DNA
E coli
Escherichia coli
Escherichia coli - cytology
Escherichia coli - genetics
Escherichia coli - growth & development
exponential fed
Fermentation
Filamentation
Flow cytometry
Growth kinetics
Homogeneity
Kinetics
Leishmaniasis
Leishmaniasis Vaccines - genetics
Models, Biological
Perfusion
perfusion culture
plasmid DNA
Plasmids
Plasmids - genetics
Strategy
Vaccines
Vaccines, DNA - genetics
Vector-borne diseases
Viability
title Segregated growth kinetics of Escherichia coli DH5α-NH36 in exponential-fed perfusion culture for pDNA vaccine production
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