Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

Kopp, Julian; Kittler, Stefan; Slouka, Christoph; Herwig, Christoph; Spadiut, Oliver; Wurm, David Johannes

doi:10.3389/fbioe.2020.573607

Cited by 11 publications

(6 citation statements)

References 64 publications

(125 reference statements)

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“…In the cultures induced in 18 h (Figure 2B and 2D), the biomass at the time of induction was 23.22 g/L and 22.58 g/ L (OD 600 nm 50.0 and 46.0), respectively. In these cultures, it was possible to observe greater cell growth, in which the maximum biomass values reached were 31.15 g/L and 27.88 g/L, with values up to 1.4-fold larger than those obtained in cultures induced in 12 h. This behavior is due to the addition of IPTG to the cultures, since the inducer can be toxic to the cells, 34,35 resulting in a decrease in biomass concentration as was observed in the induction conditions at 12 h. The highest rASNase yield values were found when induction was carried out at 18 h of cultivation, in both feeding strategies (DO-stat and linear) (Figure 2B and 2D), as well as total protein quantification (data not shown). The highest enzymatic activity obtained was approximately 98,000 U/L in the DO-stat controlled cultures (Figure 2B).…”

Section: Resultsmentioning

confidence: 92%

Production Process Optimization of Recombinant Erwinia carotovora l-Asparaginase II in Escherichia coli Fed-Batch Cultures and Analysis of Antileukemic Potential

de Andrade,

Renard,

Gennari

et al. 2024

ACS Omega

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The aims of this work were to optimize the production of Erwinia carotovora L-asparaginase II enzyme in Escherichia coli by different fed-batch cultivation strategies using a benchtop bioreactor and to evaluate the therapeutic potential of the recombinant enzyme against different acute lymphoblastic leukemia cell lines. The highest enzyme activities (∼98,000 U/L) were obtained in cultures using the DO-stat feeding strategy with induction in 18 h of culture. Under these experimental conditions, the maximum values for recombinant L-asparaginase II (rASNase) yield per substrate, rASNase yield per biomass, and productivity were approximately 1204 U/g glucose , 3660 U/g cells , and 3260 U/(L• h), respectively. This condition was efficient for achieving high yields of the recombinant enzyme, which was purified and used in in vitro antileukemic potential tests. Of all the leukemic cell lines tested, RS4;11 showed the highest sensitivity to rASNase, with an IC 50 value of approximately 0.0006 U/mL and more than 70% apoptotic cells. The study demonstrated that the cultivation strategies used were efficient for obtaining high yield and productivity of rASNase with therapeutic potential inasmuch as cytotoxic activity and induction of apoptosis were demonstrated for this protein.

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Section: Resultsmentioning

confidence: 92%

Production Process Optimization of Recombinant Erwinia carotovora l-Asparaginase II in Escherichia coli Fed-Batch Cultures and Analysis of Antileukemic Potential

de Andrade,

Renard,

Gennari

et al. 2024

ACS Omega

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“…HRP was produced in E. coli BL21 (DE3) (Life Technologies, Carlsbad, CA, USA) with details stated in previous publications [37,38]. After a successful HRP expression [37,39,40], the cell broth was harvested via centrifugation and the biomass was stored at −20 • C.…”

Section: Production and Isolation Of Hrp Ibmentioning

confidence: 99%

At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization

et al. 2021

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Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.

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“…Rapid advances in the use of machine learning for optimizing bioprocesses is one of the drivers for high-throughput small volume cultivation [3], [4] and most systems suffer from substantial sampling fraction. Even reactors with larger operating volumes can be subject to substantial errors when significant samples are withdrawn to prevent reactor overflow, extend cultivation time and reduce time between fermentations [5], [6], particularly in the case where reactors are operated as cyclic or repetitive fed-batch modes.…”

Section: Introductionmentioning

confidence: 99%

Pseudo batch transformation: A novel method to correct for mass removal through sample withdrawal of fed-batch fermentations

Hesselberg-Thomsen,

Groves,

McCubbin

et al. 2024

Preprint

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SummaryWe present a novel “pseudo batch” transformation algorithm that maps analytical data obtained for fed-batch bioreactor cultivations onto a constant volume batch process, significantly decreasing the complexity of characterizing the fed-batch process.Availability and implementationOur method is implemented in both Excel and Python and is available with tutorials and example data fromhttps://github.com/biosustain/pseudobatch. The Python package is also available on PYPI under the name “pseudobatch”.ContactLars Keld Nielsen, e-mail:lars.nielsen@uq.edu.auSupplementary informationA comprehensive explanation of the simulated fed-batch, parameter estimation procedures, and the Bayesian model can be found in supplementary information (S1-S5).

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Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

Cited by 11 publications

References 64 publications

Production Process Optimization of Recombinant Erwinia carotovora l-Asparaginase II in Escherichia coli Fed-Batch Cultures and Analysis of Antileukemic Potential

Production Process Optimization of Recombinant Erwinia carotovora l-Asparaginase II in Escherichia coli Fed-Batch Cultures and Analysis of Antileukemic Potential

At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization

Pseudo batch transformation: A novel method to correct for mass removal through sample withdrawal of fed-batch fermentations

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