Kinetic modeling of plasmid bioproduction in Escherichia coli DH5α cultures over different carbon-source compositions

Lopes, Marta B.; Martins, Gabriel R.; Calado, Cecı́lia R.C.

doi:10.1016/j.jbiotec.2014.06.022

Cited by 15 publications

(27 citation statements)

References 48 publications

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“…As expected, different expression systems (resulting from different plasmids and E. coli strains) or different culture environments can result in highly different biomass growth and plasmid production profiles and on highly different cellular metabolisms, as those obtained e.g. from E. coli engineered strains .…”

Section: Resultsmentioning

confidence: 63%

“…Glycerol minimizes the overflow metabolism, as a lower glycerol transport to the cell increases the energetic metabolism efficiency, reducing this way the acetate production . Furthermore, the lower glycerol transport into the cells also results in lower specific growth rates that may lead to an increased recombinant product synthesis per biomass . Indeed, during the glucose consumption of batch culture B, almost twice the specific growth rate (of 0.59 h −1 ) was achieved in relation to the one observed during glycerol consumption (of 0.31 h −1 ).…”

Section: Resultsmentioning

confidence: 98%

“…The Escherichia coli bacterium is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids, due to its high specific growth rates, its capacity to grow under a wide range of conditions, from economic rich complex organic media to salt‐based chemically defined media, as well as a high number of available plasmid expression systems, engineered strains and cultivation strategies . However, differences in cultivation strategies (e.g., batch and fed‐batch), environmental conditions and medium composition affect the cloned gene stability and expression . The plasmid and host cell characteristics, i.e., the cell expression system, are also important factors that should be carefully evaluated .…”

Section: Introductionmentioning

confidence: 99%

“…The characterization of the recombinant cultivation process is most often limited to the following variables: biomass growth, recombinant product production, C‐sources consumption and by‐products (e.g. acetic acid) production and consumption . However, to understand the complexity of the interrelationships between the genetic and physiological characteristics of the expression system and the cultivation general environment, cell metabolic information along the culture would be highly desired.…”

Section: Introductionmentioning

confidence: 99%

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Metabolic profiling of recombinant Escherichia coli cultivations based on high‐throughput FT‐MIR spectroscopic analysis

Sales

Rosa

Cunha

et al. 2016

Biotechnology Progress

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Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic profile of recombinant E. coli cultures producing the pVAX-lacZ plasmid model was analyzed by rapid, economic and high-throughput Fourier Transform Mid-Infrared (FT-MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C-sources consumption phases, direct FT-MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:285-298, 2017.

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Section: Resultsmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Metabolic profiling of recombinant Escherichia coli cultivations based on high‐throughput FT‐MIR spectroscopic analysis

Sales

Rosa

Cunha

et al. 2016

Biotechnology Progress

Self Cite

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show abstract

“…The diafiltration process is essential since there is evidence of production of growth‐inhibition impurities such as acetate in high density E. coli cultures even when using glycerol as sole carbon source, which can be related to the high plasmid specific yields …”

Section: Resultsmentioning

confidence: 99%

Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

García-Rendón

Munguía-Soto

Montesinos-Cisneros

et al. 2016

J of Chemical Tech & Biotech

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BACKGROUND The clinical application of plasmid DNA (pDNA) vaccines is progressing and it is essential to develop industrial bioprocesses to economically manufacture these macromolecules. In order to contribute to achieving this goal, in this work an exponential‐fed perfusion (EFP) culture for the production of pVAX1‐NH36 hosted in Escherichia coli DH5α for application as specific vaccine was investigated using a theoretical–experimental approach. RESULTS An experimental plasmid productivity of 3.0 mg L−1 h−1 was obtained, which represent a 1.5‐fold increase in this performance criteria. A mathematical model to investigate the system performance was built and validated. A novel aspect of the study is the analysis of the influence of individual model parameters on selected output variables and on scale‐up. CONCLUSIONS This work contributes to the design and optimization of EFP cultures for the efficient production of pDNA for therapeutic use. The higher productivity of the EFP bioprocess might reduce both capital investment and operational costs and as a result be economically attractive compared with conventional bioprocess. © 2016 Society of Chemical Industry

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Assessing plasmid bioprocess reproducibility and C‐source uptake stage through multivariate analysis of offline and online data

Lopes

Calado

2018

J of Chemical Tech & Biotech

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Background Considerable effort has been made by the biopharmaceutical industry to guarantee high‐quality products and patient safety. Ensuring the reproducibility of microbial cultures is essential to achieve such high standards. Reproducibility is usually assessed by offline, costly and time‐consuming analyses of the final product, disregarding the culture behavior over time, which affects the final product. Online data provided in real time by the bioreactor unit control (e.g. temperature, pH, dissolved oxygen concentration), unlike offline data acquired along the culture, implies no extra cost or contamination risks, thus holding the promise of playing an important role in assessing culture reproducibility. Results Eleven batch recombinant Escherichia coli DH5‐α cultures producing the plasmid model pVAX‐LacZ were analyzed by Multivariate Data Analysis (MVDA) of online and offline data. Similarities between the cultures were inspected by Principal Component Analysis (PCA) and the significance of the differences determined by Multivariate Analysis of Variance (MANOVA) based on offline data. Linear Discriminant Analysis (LDA) based on the online bioreactor data was also successful in predicting the cell growth stage at each time point regarding the C‐source under consumption. Conclusion Multivariate data analysis based on offline and online data has proved to be a valuable tool in assessing the cultures' reproducibility and the metabolic stage regarding the C‐source consumption phase at a given time. These results may certainly impact the biopharmaceutical industry, by drawing attention to the usefulness of combining MVDA and bioprocess data, particularly online data, never used for these purposes. © 2018 Society of Chemical Industry

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Kinetic modeling of plasmid bioproduction in Escherichia coli DH5α cultures over different carbon-source compositions

Cited by 15 publications

References 48 publications

Metabolic profiling of recombinant Escherichia coli cultivations based on high‐throughput FT‐MIR spectroscopic analysis

Metabolic profiling of recombinant Escherichia coli cultivations based on high‐throughput FT‐MIR spectroscopic analysis

Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

Assessing plasmid bioprocess reproducibility and C‐source uptake stage through multivariate analysis of offline and online data

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