Plasmid DNA production for pharmaceutical use: Role of specific growth rate and impact on process design

Bohle, Kathrin; Ross, Avena C.

doi:10.1002/bit.23138

Cited by 19 publications

(18 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The experimental plasmid yield coefficient increased linearly during the cultures from 0.44 ± 0.08 to a final value of 0.64 ± 0.16 mg/(L‐OD) (average of value ± standard error of mean). This coefficient depends on the specific growth rate of the culture, e.g. we have reported a 0.9 mg/(L‐OD 600 ) plasmid yield coefficient, under different growing conditions.…”

Section: Resultsmentioning

confidence: 87%

“…By applying the optimal growth rate during the whole cultivation process the maximum plasmid content would be achieved (0% batch phase) of course. Increasing the proportion of the batch phase would reduce the plasmid yield indeed, but also reduces the cultivation time or rather increases plasmid productivity . Therefore, it is necessary to analyze the plasmid productivity relationship with ratio of batch to EFP phase.…”

Section: Resultsmentioning

confidence: 99%

“…The perfusion culture consist of an initial batch phase (B) followed by the exponential‐fed perfusion phase (EFP). A defined medium for the batch cultures was used with 13 g L −1 of glycerol as limiting source, according to the methods described by Bohle and Ross . This type of medium permits better replication, control and modeling of the system, which is important in the bioprocess development.…”

Section: Methodsmentioning

confidence: 99%

“…The clinical application of pDNA vaccines is progressing and it is essential to devise industrial processes whereby pDNA can be economically manufactured . However, comprehensive process knowledge is essential and – in terms of Process Analytical Technologies – even requested by regulatory agencies …”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

View full text Add to dashboard Cite

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

show abstract

Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

View full text Add to dashboard Cite

show abstract

“…The pDNA vaccines are believed to be potentially useful in a wide range of applications including preventive and therapeutic vaccines for viral, bacterial, and parasitic diseases as well as cancer [2,3]. The production of pDNA follow the steps of fermentation, primary and intermediate recovery, and purification.…”

Section: Introductionmentioning

confidence: 99%

Efficient Disruption of Escherichia coli for Plasmid DNA Recovery in a Bead Mill

et al. 2017

View full text Add to dashboard Cite

Featured Application: In general, the alkaline lysis is more acceptable than the mechanical lysis for pDNA recovery due to the risk of product degradation when the latter is used. In this work, the experimental design allowed us to explore mechanical disruption conditions under which such an effect was minimized. The application of this technique is important for novel pDNA-vaccine process development, since bead milling is one of the most preferable mechanical cell lysis methods on an industrial scale due to its scalability, ease of operation, controllability, and ability to load concentrated cell slurry. Abstract:The release kinetics of pDNA in a bead mill was studied. Samples taken during the process were analyzed to determine total pDNA (pDNA(t)) and supercoiled pDNA (pDNA(sc)) concentration. In order to identify important variables of the process and to develop an empirical model for optimal pDNA(t) and pDNA(sc) release, a two level 2 3 factorial design was used with variables: mill frequency, cell concentration, and bead size. The results were analyzed by response surface methodology. The optimized conditions for pDNA(t) yield 13.26 mg/g dcw (93.41% recovery), with a mill frequency of 30 Hz, a bead size of 0.10-0.25 mm, and a cell concentration of 20 g wcw/L. However, the optimized conditions for pDNA(sc) yield 7.65 mg/g dcw (92.05% recovery), with a mill frequency of 15 Hz, a bead size of 0.10-0.25 mm, and a cell concentration of 10 g wcw/L. Cell disruption in a bead mill was proved efficient for the release of pDNA(t) and pDNA(sc) compared to the alkaline treatment. The results obtained suggest a compromise between pDNA(sc) purity and recuperation in the process development.

show abstract

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

Munguía-Soto

García-Rendón

Garibay‐Escobar³

et al. 2015

Biotech and App Biochem

View full text Add to dashboard Cite

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.

show abstract

Plasmid DNA production for pharmaceutical use: Role of specific growth rate and impact on process design

Cited by 19 publications

References 31 publications

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

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

Efficient Disruption of Escherichia coli for Plasmid DNA Recovery in a Bead Mill

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

Contact Info

Product

Resources

About