Strategies for high titre plasmid DNA production in Escherichia coli DH5α

O’Mahony, Kevin; Freitag, Ruth; Hilbrig, Frank; Müller, Patrick; Schumacher, Ivo

doi:10.1016/j.procbio.2007.03.014

Cited by 21 publications

(13 citation statements)

References 27 publications

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“…With the aim of building a robust model able to accurately predict a high range of plasmid bioprocesses from NIR spectra, five distinct E. coli cultures set up with different E. coli strains, medium composition and culture strategies, and producing maximum plasmid yields per biomass from 4 to 11 mg g −1 DCW (Table ), were monitored using a NIR transflectance fiber optic probe. Maximum plasmid titers from 28 to 142 mg L −1 were achieved, which are in the range of what is typically obtained in laboratory‐scale cultivation . The five cultures were divided into three groups to be investigated regarding three distinct goals.…”

Section: Resultsmentioning

confidence: 99%

In situNIR spectroscopy monitoring of plasmid production processes: effect of producing strain, medium composition and the cultivation strategy

Lopes

Gonçalves

Felício-Silva

et al. 2014

J of Chemical Tech & Biotech

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BACKGROUND While the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real‐time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTS Partial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed‐batch) and E. coli strain used, three strategies were adopted, using: (i) E. coli DH5α cultures conducted under different media compositions and culture strategies (batch and fed‐batch); (ii) engineered E. coli strains, MG1655ΔendAΔrecAΔpgi and MG1655ΔendAΔrecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed‐batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSION NIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination‐free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used. © 2014 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 99%

In situNIR spectroscopy monitoring of plasmid production processes: effect of producing strain, medium composition and the cultivation strategy

Lopes

Gonçalves

Felício-Silva

et al. 2014

J of Chemical Tech & Biotech

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“…Some studies clearly discuss the influence of oxygen limiting conditions on plasmid productivity in miniaturized E. coli cell cultures [31, 32], even though they just present plasmid yields in cultures carried out under anaerobic, oxygen limited or aerobic conditions. Oxygen limitations neither seems to have a direct negative influence on plasmid stability nor the plasmid copy number, however, the growth of E. coli to high cell densities is negatively influenced under oxygen limitation by the low biomass yield per glucose and the acidification of the culture medium by the mixed acids fermentation.…”

Section: Resultsmentioning

confidence: 99%

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier

Pilarek

Brand

Hillig

et al. 2012

Bioprocess Biosyst Eng

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A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase®) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 μL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The μL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.

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“…Consistent with this, a recent report compared 35°C versus 37°C growth during the batch phase, with subsequent fed-batch production at 37°C under restricted growth rate. Production yields with 35°C batch phase growth were dramatically improved (250 mg/L versus 50 mg/L; OMahony et al, 2007). Interestingly, creation of seed stocks from transformations performed at 30°C, rather than 37°C, also dramatically increased plasmid yield, productivity and seed stock viability with traditionally low yield plasmids (Williams et al, 2008; Carnes et al, 2008) indicating a general benefit on plasmid productivity of reduced temperature growth in all steps prior to the growth controlled restricted fed-batch stage.…”

Section: Metabolic Burden Limits Plasmid Productivitymentioning

confidence: 99%

Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production

Williams

Carnes

Hodgson

2009

Biotechnology Advances

155

131

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Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance's are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for followon products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.

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Strategies for high titre plasmid DNA production in Escherichia coli DH5α

Cited by 21 publications

References 27 publications

In situNIR spectroscopy monitoring of plasmid production processes: effect of producing strain, medium composition and the cultivation strategy

In situNIR spectroscopy monitoring of plasmid production processes: effect of producing strain, medium composition and the cultivation strategy

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier

Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production

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