Monitoring and control of E. coli cell integrity

Kastenhofer, Jens; Rajamanickam, Vignesh; Libiseller-Egger, Julian; Spadiut, Oliver

doi:10.1016/j.jbiotec.2021.01.009

Cited by 16 publications

(11 citation statements)

References 163 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this respect it should be mentioned that it is relatively easy to isolate the periplasmic fraction of E. coli also at an industrial scale ( Mergulhao et al, 2005 ; Ellis and Humphreys, 2014 ). Furthermore, periplasmic protein production can also lead to the “spontaneous” release of the protein into the extracellular milieu ( Kastenhofer et al, 2021 ). The release of a protein into the extracellular milieu can also be promoted by actively making the outer membrane more permeable or by fusing the protein to a fusion partner that is naturally secreted into the extracellular milieu ( Hsiung et al, 1989 ; Zhang et al, 2006 ; Natarajan et al, 2017 ; Gao et al, 2018 )).…”

Section: Introductionmentioning

confidence: 99%

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Karyolaimos

Gier

2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

show abstract

Section: Introductionmentioning

confidence: 99%

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Karyolaimos

Gier

2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…Processes 2021, 9, x FOR PEER REVIEW 14 of 18 study could reduce the between-run variance, the within-run variance was still dominated by these correlated process dynamics. In addition to detection of the onset of leakiness and lysis, predicting the concentrations of target analytes, such as extracellular protein or DNA, by in-line spectroscopy can facilitate advanced, model-based process control [1]. PLSR is a well-established chemometric tool for MIR spectroscopy and has been frequently used for the quantification of small organic metabolites or inorganic medium components in bioprocesses [5][6][7][8][9].…”

Section: Discussionmentioning

confidence: 99%

“…Soluble expression of recombinant protein in Escherichia coli is often achieved by translocation of the product to the periplasm, the space between the inner membrane (IM) and outer membrane (OM). For successful bioprocessing aligned with Quality by Design and process analytical technology principles, the integrity of both membranes needs to be monitored in real-time to allow timely decision-making and control [1]. Protein leakage through the OM to the extracellular space has a strong impact on the downstream process, either as a way for selective product release and simplified primary recovery or as unwanted loss of product.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Monitoring E. coli Cell Integrity by ATR-FTIR Spectroscopy and Chemometrics: Opportunities and Caveats

et al. 2021

Self Cite

View full text Add to dashboard Cite

During recombinant protein production with E. coli, the integrity of the inner and outer membrane changes, which leads to product leakage (loss of outer membrane integrity) or lysis (loss of inner membrane integrity). Motivated by current Quality by Design guidelines, there is a need for monitoring tools to determine leakiness and lysis in real-time. In this work, we assessed a novel approach to monitoring E. coli cell integrity by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. Various preprocessing strategies were tested in combination with regression (partial least squares, random forest) or classification models (partial least squares discriminant analysis, linear discriminant analysis, random forest, artificial neural network). Models were validated using standard procedures, and well-performing methods were additionally scrutinized by removing putatively important features and assessing the decrease in performance. Whereas the prediction of target compound concentration via regression was unsuccessful, possibly due to a lack of samples and low sensitivity, random forest classifiers achieved prediction accuracies of over 90% within the datasets tested in this study. However, strong correlations with untargeted spectral regions were revealed by feature selection, thereby demonstrating the need to rigorously validate chemometric models for bioprocesses, including the evaluation of feature importance.

show abstract

“…Selecting the proper regeneration solution can be one of the most challenging steps in designing an SPR assay. This is a major shortcoming of affinity biosensors [132]. Using a lowaffinity ligand with a rapid dissociation rate has been suggested in the aim of performing continuous immunosensing [38] without the need for regeneration.…”

Section: Limitations Of Spr Biosensing In the Context Of Bioprocess Monitoringmentioning

confidence: 99%

On the Use of Surface Plasmon Resonance-Based Biosensors for Advanced Bioprocess Monitoring

et al. 2021

View full text Add to dashboard Cite

Biomanufacturers are being incited by regulatory agencies to transition from a quality by testing framework, where they extensively test their product after their production, to more of a quality by design or even quality by control framework. This requires powerful analytical tools and sensors enabling measurements of key process variables and/or product quality attributes during production, preferably in an online manner. As such, the demand for monitoring technologies is rapidly growing. In this context, we believe surface plasmon resonance (SPR)-based biosensors can play a role in enabling the development of improved bioprocess monitoring and control strategies. The SPR technique has been profusely used to probe the binding behavior of a solution species with a sensor surface-immobilized partner in an investigative context, but its ability to detect binding in real-time and without a label has been exploited for monitoring purposes and is promising for the near future. In this review, we examine applications of SPR that are or could be related to bioprocess monitoring in three spheres: biotherapeutics production monitoring, vaccine monitoring, and bacteria and contaminant detection. These applications mainly exploit SPR’s ability to measure solution species concentrations, but performing kinetic analyses is also possible and could prove useful for product quality assessments. We follow with a discussion on the limitations of SPR in a monitoring role and how recent advances in hardware and SPR response modeling could counter them. Mainly, throughput limitations can be addressed by multi-detection spot instruments, and nonspecific binding effects can be alleviated by new antifouling materials. A plethora of methods are available for cell growth and metabolism monitoring, but product monitoring is performed mainly a posteriori. SPR-based biosensors exhibit potential as product monitoring tools from early production to the end of downstream processing, paving the way for more efficient production control. However, more work needs to be done to facilitate or eliminate the need for sample preprocessing and to optimize the experimental protocols.

show abstract

Monitoring and control of E. coli cell integrity

Cited by 16 publications

References 163 publications

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Monitoring E. coli Cell Integrity by ATR-FTIR Spectroscopy and Chemometrics: Opportunities and Caveats

On the Use of Surface Plasmon Resonance-Based Biosensors for Advanced Bioprocess Monitoring

Contact Info

Product

Resources

About