| 1. |
- Ajjan, Fátima, et al.
(författare)
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Enhancing Energy Storage Devices with Biomacromolecules in Hybrid Electrodes
- 2019
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Ingår i: Biotechnology Journal. - : WILEY-V C H VERLAG GMBH. - 1860-6768 .- 1860-7314. ; 14:12
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Forskningsöversikt (refereegranskat)abstract
- The development of energy storage devices with higher energy and power outputs, and long cycling stability is urgently required in the pursuit of the expanding challenges of electrical energy storage. The utilization of biologically renewable redox compounds holds a great potential in designing sustainable energy storage systems and contributes in reducing the dependence on fossil fuels for energy materials. Quinones are the principal redox centers in natural organic materials and play a key role as charge storage electrode materials because of their abundance, multiple forms and integration into the materials flow through the biosphere. Electrical energy storage devices and systems can be significantly improved by the combination of scalable quinone-based biomaterials with good electronic conductors. This review uses recent examples to show how biopolymers are providing new directions in the development of renewable biohybrid electrodes for energy storage devices.
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| 2. |
- Alinaghi, Masoumeh, et al.
(författare)
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Hierarchical time-series analysis of dynamic bioprocess systems
- 2022
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Ingår i: Biotechnology Journal. - : John Wiley & Sons. - 1860-6768 .- 1860-7314. ; 17:12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Monoclonal antibodies (mAbs) are leading types of ‘blockbuster’ biotherapeutics worldwide; they have been successfully used to treat various cancers and chronic inflammatory and autoimmune diseases. Biotherapeutics process development and manufacturing are complicated due to lack of understanding the factors that impact cell productivity and product quality attributes. Understanding complex interactions between cells, media, and process parameters on the molecular level is essential to bring biomanufacturing to the next level. This can be achieved by analyzing cell culture metabolic levels connected to vital process parameters like viable cell density (VCD). However, VCD and metabolic profiles are dynamic parameters and inherently correlated with time, leading to a significant correlation without actual causality. Many time-series methods deal with such issues. However, with metabolic profiling, the number of measured variables vastly exceeds the number of experiments, making most of existing methods ill-suited and hard to interpret. Methods and MajorResults: Here we propose an alternative workflow using hierarchical dimension reduction to visualize and interpret the relation between evolution of metabolic profiles and dynamic process parameters. The first step of proposed method is focused on finding predictive relation between metabolic profiles and process parameter at all time points using OPLS regression. For each time point, the p(corr) obtained from OPLS model is considered as a differential metabogram and is further assessed using principal components analysis (PCA).Conclusions: Compared to traditional batch modeling, applying proposed methodology on metabolic data from Chinese Hamster Ovary (CHO) antibody production characterized the dynamic relation between metabolic profiles and critical process parameters.
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| 3. |
- Alm, Tove, et al.
(författare)
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High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
- 2007
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 2, s. 709-716
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Tidskriftsartikel (refereegranskat)abstract
- A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.
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| 4. |
- Andersson, Ken G., et al.
(författare)
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Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli
- 2019
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Ingår i: Biotechnology Journal. - : WILEY-V C H VERLAG GMBH. - 1860-6768 .- 1860-7314. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.
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| 5. |
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| 6. |
- Borodina, I., et al.
(författare)
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Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals
- 2014
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 9:5, s. 609-620
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Forskningsöversikt (refereegranskat)abstract
- Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast-based processes for chemicals production.
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| 7. |
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| 8. |
- Braissant, Olivier, et al.
(författare)
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Isothermal microcalorimetry accurately detects bacteria, tumorous microtissues, and parasitic worms in a label-free well-plate assay
- 2015
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:3, s. 460-468
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Tidskriftsartikel (refereegranskat)abstract
- Isothermal microcalorimetry is a label-free assay that allows monitoring of enzymatic and metabolic activities. The technique has strengths, but most instruments have a low throughput, which has limited their use for bioassays. Here, an isothermal microcalorimeter, equipped with a vessel holder similar to a 48-well plate, was used. The increased throughput of this microcalorimeter makes it valuable for biomedical and pharmaceutical applications. Our results show that the sensitivity of the instrument allows the detection of 3 x 10(4) bacteria per vial. Growth of P. mirabilis in Luria Broth medium was detected between 2 and 9 h with decreasing inoculum. The culture released 2.1J with a maximum thermal power of 76 W. The growth rate calculated using calorimetric and spectrophotometric data were 0.60 and 0.57 h(-1), respectively. Additional insight on protease activities of P. mirabilis matching the last peak in heat production could be gathered as well. Growth of tumor microtissues releasing a maximum thermal power of 2.1 W was also monitored and corresponds to a diameter increase of the microtissues from ca. 100 to 428 m. This opens new research avenues in cancer research, diagnostics, and development of new antitumor drugs. For parasitic worms, the technique allows assessment of parasite survival using motor and metabolic activities even with a single worm.
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| 9. |
- Campbell, Kate, 1987, et al.
(författare)
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Cell-to-cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community
- 2016
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 11:9, s. 1169-1178
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Tidskriftsartikel (refereegranskat)abstract
- Cells that grow together respond heterogeneously to stress even when they are genetically similar. Metabolism, a key determinant of cellular stress tolerance, may be one source of this phenotypic heterogeneity, however, this relationship is largely unclear. We used self-establishing metabolically cooperating (SeMeCo) yeast communities, in which metabolic cooperation can be followed on the basis of genotype, as a model to dissect the role of metabolic cooperation in single-cell heterogeneity. Cells within SeMeCo communities showed to be highly heterogeneous in their stress tolerance, while the survival of each cell under heat or oxidative stress, was strongly determined by its metabolic specialization. This heterogeneity emerged for all metabolite exchange interactions studied (histidine, leucine, uracil, and methionine) as well as oxidant (H2O2, diamide) and heat stress treatments. In contrast, the SeMeCo community collectively showed to be similarly tolerant to stress as wild-type populations. Moreover, stress heterogeneity did not establish as sole consequence of metabolic genotype (auxotrophic background) of the single cell, but was observed only for cells that cooperated according to their metabolic capacity. We therefore conclude that phenotypic heterogeneity and cell to cell differences in stress tolerance are emergent properties when cells cooperate in metabolism.
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| 10. |
- Camsund, Daniel, et al.
(författare)
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Genetically engineered light sensors for control of bacterial gene expression
- 2011
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:7, s. 826-836
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Forskningsöversikt (refereegranskat)abstract
- Light of different wavelengths can serve as a transient, noninvasive means of regulating gene expression for biotechnological purposes. Implementation of advanced gene regulatory circuits will require orthogonal transcriptional systems that can be simultaneously controlled and that can produce several different control states. Fully genetically encoded light sensors take advantage of the favorable characteristics of light, do not need the supplementation of any chemical inducers or co-factors, and have been demonstrated to control gene expression in Escherichia coli. Herein, we review engineered light-sensor systems with potential for in vivo regulation of gene expression in bacteria, and highlight different means of extending the range of available light input and transcriptional output signals. Furthermore, we discuss advances in multiplexing different light sensors for achieving multichromatic control of gene expression and indicate developments that could facilitate the construction of efficient systems for light-regulated, multistate control of gene expression.
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| 11. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels
- 2013
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 8:5, s. 534-544
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Forskningsöversikt (refereegranskat)abstract
- Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.
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| 12. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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One-step enzyme extraction and immobilization for biocatalysis applications
- 2011
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:4, s. 463-469
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Tidskriftsartikel (refereegranskat)abstract
- An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.
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| 13. |
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| 14. |
- Chumnanpuen, Pramote, 1983, et al.
(författare)
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Lipid biosynthesis monitored at the single-cell level in Saccharomyces cerevisiae
- 2012
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:5, s. 594-601
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Tidskriftsartikel (refereegranskat)abstract
- There is increasing interest in bioengineering of lipids for use in functional foods, pharmaceuticals, and biofuels. Saccharomyces cerevisiae is a widely utilized cell factory for biotechnological production, thus a tempting alternative. Herein, we show how its neutral lipid accumulation varies throughout metabolic phases under nutritional conditions relevant for large-scale fermentation. Population-averaged metabolic data were correlated with lipid storage at the single-cell level monitored at submicron resolution by label-free coherent anti-Stokes Raman scattering (CARS) microscopy. While lipid droplet sizes are fairly constant, the number of droplets is a dynamic parameter determined by glucose and ethanol levels. The lowest number of lipid droplets is observed in the transition phase between glucose and ethanol fermentation. It is followed by a buildup during the ethanol phase. The surplus of accumulated lipids is then mobilized at concurrent glucose and ethanol starvation in the subsequent stationary phase. Thus, the highest amount of lipids is found in the ethanol phase, which is about 0.3 fL/cell. Our results indicate that the budding yeast, S. cerevisiae, can be used for the biosynthesis of lipids and demonstrate the strength of CARS microscopy for monitoring the dynamics of lipid metabolism at the single-cell level of importance for optimized lipid production.
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| 15. |
- De Jonge, L.P., et al.
(författare)
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Flux response of glycolysis and storage metabolism during rapid feast/famine conditions in Penicillium chrysogenum using dynamic 13C labeling
- 2014
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 9:3, s. 372-385
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Tidskriftsartikel (refereegranskat)abstract
- The scale-up of fermentation processes frequently leads to a reduced productivity compared to small-scale screening experiments. Large-scale mixing limitations that lead to gradients in substrate and oxygen availability could influence the microorganism performance. Here, the impact of substrate gradients on a penicillin G producing Penicillium chrysogenum cultivation was analyzed using an intermittent glucose feeding regime. The intermittent feeding led to fluctuations in the extracellular glucose concentration between 400 μM down to 6.5 μM at the end of the cycle. The intracellular metabolite concentrations responded strongly and showed up to 100-fold changes. The intracellular flux changes were estimated on the basis of dynamic 13C mass isotopomer measurements during three cycles of feast and famine using a novel hybrid modeling approach. The flux estimations indicated a high turnover of internal and external storage metabolites in P. chrysogenum under feast/famine conditions. The synthesis and degradation of storage requires cellular energy (ATP and UTP) in competition with other cellular functions including product formation. Especially, 38% of the incoming glucose was recycled once in storage metabolism. This result indicated that storage turnover is increased under dynamic cultivation conditions and contributes to the observed decrease in productivity compared to reference steady-state conditions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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| 16. |
- Erlendsson, Lýður S, et al.
(författare)
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Barley as a green factory for the production of functional Flt3 ligand
- 2009
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Ingår i: Biotechnology Journal. - Weinheim : Wiley-VCH Verlag GmbH & Co. KGaA. - 1860-7314 .- 1860-6768. ; 5:2, s. 163-171
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Tidskriftsartikel (refereegranskat)abstract
- Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.
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| 17. |
- Falk, Ronny, et al.
(författare)
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Targeted protein pullout from human tissue samples using competitive elution
- 2011
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:1, s. 28-37
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Tidskriftsartikel (refereegranskat)abstract
- One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, I MAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.
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| 18. |
- Forster, Anthony C., et al.
(författare)
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Editorial : NextGen SynBio has arrived...
- 2012
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:7, s. 827-827
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 19. |
- Forster, Anthony C.
(författare)
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Synthetic biology challenges long-held hypotheses in translation, codon bias and transcription
- 2012
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:7, s. 835-845
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Forskningsöversikt (refereegranskat)abstract
- Synthetic biology is a powerful experimental approach, not only for developing new biotechnology applications, but also for testing hypotheses in basic biological science. Here, examples from our research using the best model system, Escherichia coli, are reviewed. New evidence drawn from synthetic biology has overturned several long-standing hypotheses regarding the mechanisms of transcription and translation: (i) all native aminoacyl-tRNAs are not equally efficient in translation at equivalent concentrations; (ii) accommodation is not always rate limiting in translation, and may not be for any aminoacyl-tRNA; (iii) proline is the only N-alkyl-amino acid in the genetic code not because of special suitability for protein structure, but because of its comparatively high nucleophilicity; (iv) the usages of most sense codons in E. coli do not correlate with cognate tRNA abundances and (v) class II transcriptional pausing and termination by T7 RNA polymerase cannot be assumed to occur in vivo based on in vitro data. Implications of these conclusions for the biotechnology field are discussed.
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| 20. |
- Gernaey, Krist V, et al.
(författare)
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Monitoring and control of microbioreactors: An expert opinion on development needs
- 2012
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlag Berlin. - 1860-6768 .- 1860-7314. ; 7:10, s. 1308-1314
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Tidskriftsartikel (refereegranskat)abstract
- This perspective article is based on an expert panel review on microbioreactor applications in biochemical and biomedical engineering that was organized by the M3C (measurement, monitoring, modelling and control) Working Group of the European Section of Biochemical Engineering Science (ESBES) in the European Federation of Biotechnology (EFB). The aim of the panel was to provide an updated view on the present status of the subject and to identify critical needs and issues for furthering the successful development of microbioreactor monitoring and control. This will benefit future bioprocess development and in vitro toxicity testing. The article concludes with a set of recommendations for extended use and further development of microbioreactors.
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| 21. |
- Gong, Z. W., et al.
(författare)
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Engineering Robustness of Microbial Cell Factories
- 2017
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 12:10
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Forskningsöversikt (refereegranskat)abstract
- Metabolic engineering and synthetic biology offer great prospects in developing microbial cell factories capable of converting renewable feedstocks into fuels, chemicals, food ingredients, and pharmaceuticals. However, prohibitively low production rate and mass concentration remain the major hurdles in industrial processes even though the biosynthetic pathways are comprehensively optimized. These limitations are caused by a variety of factors unamenable for host cell survival, such as harsh industrial conditions, fermentation inhibitors from biomass hydrolysates, and toxic compounds including metabolic intermediates and valuable target products. Therefore, engineered microbes with robust phenotypes is essential for achieving higher yield and productivity. In this review, the recent advances in engineering robustness and tolerance of cell factories is described to cope with these issues and briefly introduce novel strategies with great potential to enhance the robustness of cell factories, including metabolic pathway balancing, transporter engineering, and adaptive laboratory evolution. This review also highlights the integration of advanced systems and synthetic biology principles toward engineering the harmony of overall cell function, more than the specific pathways or enzymes.
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| 22. |
- Grönwall, Caroline, et al.
(författare)
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Affibody-mediated transferrin depletion for proteomics applications
- 2007
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 2:11, s. 1389-1398
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Tidskriftsartikel (refereegranskat)abstract
- An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.
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| 23. |
- Hedhammar, My, et al.
(författare)
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Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag
- 2006
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 1, s. 187-196
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Tidskriftsartikel (refereegranskat)abstract
- A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.
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| 24. |
- Hober, Sophia
(författare)
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Biotech in the post genomic era
- 2009
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 4, s. 1631-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 25. |
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| 26. |
- Hober, Sophia
(författare)
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Biotech reviews on plants, lignocellulose, sequencing, genome engineering and Aspergilli
- 2012
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:9, s. 1057-1057
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cover illustration Special issue: Biotech Reviews Biofuels, Protein engineering, Plant biotechnology. This issue provides an excellent overview of the latest developments in biotechnology with its very broad topics. You will find articles on genome engineering of mammalian cells, engineering of plant cells for glycosylation and expression of proteins in different organelles, cellular responses to ethanol stress, neuronal stem cells and more. Cover image by Elena du Guerny (8 years old).
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| 27. |
- Hober, Sophia
(författare)
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Global biotech challenges
- 2010
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 5
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 28. |
- Ihling, Nina, et al.
(författare)
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Scale‐down of CHO cell cultivation from shake flasks based on oxygen mass transfer allows application of parallelized, non‐invasive, and time‐resolved monitoring of the oxygen transfer rate in 48‐well microtiter plates
- 2023
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 18:11
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Tidskriftsartikel (refereegranskat)abstract
- Cultivating Chinese hamster ovary (CHO) cells in microtiter plates (MTPs) with time-resolved monitoring of the oxygen transfer rate (OTR) is highly desirable to provide process insights at increased throughput. However, monitoring of the OTR in MTPs has not been demonstrated for CHO cells, yet. Hence, a CHO cultivation process was transferred from shake flasks to MTPs to enable monitoring of the OTR in each individual well of a 48-well MTP. For this, the cultivation of an industrially relevant, antibody-producing cell line was transferred from shake flask to MTP based on the volumetric oxygen mass transfer coefficient (kLa). Culture behavior was well comparable (deviation of the final IgG titer less than 10%). Monitoring of the OTR in 48-well MTPs was then used to derive the cytotoxicity of dimethyl sulfoxide (DMSO) based on a dose–response curve in a single experiment using a second CHO cell line. Logistic fitting of the dose–response curve determined after 100 h was used to determine the DMSO concentration that resulted in a cytotoxicity of 50% (IC50). A DMSO concentration of 2.70% ± 0.25% was determined, which agrees with the IC50 previously determined in shake flasks (2.39% ± 0.1%). Non-invasive, parallelized, and time-resolved monitoring of the OTR of CHO cells in MTPs was demonstrated and offers excellent potential to speed up process development and assess cytotoxicity.
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| 29. |
- J T Carrondo, Manuel J T, et al.
(författare)
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How can measurement, monitoring, modeling and control advance cell culture in industrial biotechnology?
- 2012
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlag Berlin. - 1860-6768 .- 1860-7314. ; 7:12, s. 1522-1529
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Tidskriftsartikel (refereegranskat)abstract
- This report highlights the potential of measurement, monitoring, modeling and control ((MC)-C-3) methodologies in animal and human cell culture technology. In particular, state-of-the-art of (MC)-C-3 technologies and their industrial relevance of existing technology are addressed. It is a summary of an expert panel discussion between biotechnologists and biochemical engineers with both academic and industrial backgrounds. The latest ascents in (MC)-C-3 are discussed from a cell culture perspective for industrial process development and production needs. The report concludes with a set of recommendations for targeting (MC)-C-3 research toward the industrial interests. These include issues of importance for biotherapeutics production, miniaturization of measurement techniques and modeling methods.
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| 30. |
- Jansson, Ronnie, et al.
(författare)
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Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris
- 2016
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:5, s. 687-699
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Tidskriftsartikel (refereegranskat)abstract
- Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.
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| 31. |
- Jessop-Fabre, Mathew M., et al.
(författare)
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EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9
- 2016
-
Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 11:8, s. 1110-1117
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.
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| 32. |
- Kanje, Sara, et al.
(författare)
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In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
- 2015
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:4, s. 564-574
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.
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| 33. |
- Kleinhans, C., et al.
(författare)
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A perfusion bioreactor system efficiently generates cell-loaded bone substitute materials for addressing critical size bone defects
- 2015
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 10:11, s. 1727-1738
-
Tidskriftsartikel (refereegranskat)abstract
- Critical size bone defects and non-union fractions are still challenging to treat. Cell-loaded bone substitutes have shown improved bone ingrowth and bone formation. However, a lack of methods for homogenously colonizing scaffolds limits the maximum volume of bone grafts. Additionally, therapy robustness is impaired by heterogeneous cell populations after graft generation. Our aim was to establish a technology for generating grafts with a size of 10.5 mm in diameter and 25 mm of height, and thus for grafts suited for treatment of critical size bone defects. Therefore, a novel tailor-made bioreactor system was developed, allowing standardized flow conditions in a porous poly(L-lactide-co-caprolactone) material. Scaffolds were seeded with primary human mesenchymal stem cells derived from four different donors. In contrast to static experimental conditions, homogenous cell distributions were accomplished under dynamic culture. Additionally, culture in the bioreactor system allowed the induction of osteogenic lineage commitment after one week of culture without addition of soluble factors. This was demonstrated by quantitative analysis of calcification and gene expression markers related to osteogenic lineage. In conclusion, the novel bioreactor technology allows efficient and standardized conditions for generating bone substitutes that are suitable for the treatment of critical size defects in humans.
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| 34. |
- Knuf, Christoph, 1984, et al.
(författare)
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Aspergilli: Systems biology and industrial applications
- 2012
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:9, s. 1147-1155
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Forskningsöversikt (refereegranskat)abstract
- Aspergilli are widely used as cell factories for the production of food ingredients, enzymes and antibiotics. Traditionally, improvement of these cell factories has been done using classical methods, that is, random mutagenesis and screening; however, advances in methods for performing directed genetic modifications has enabled the use of metabolic engineering strategies. Genome sequencing of Aspergilli was originally trailing behind developments in the field of bacteria and yeasts, but with the recent availability of genome sequences for several industrially relevant Aspergilli, it has become possible to implement systems biology tools to advance metabolic engineering. These tools include genome-wide transcription analysis and genome-scale metabolic models. Herein, we review achievements in the field and highlight the impact of Aspergillus systems biology on industrial biotechnology.
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| 35. |
- Koutsilieri, Stefania, et al.
(författare)
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Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models
- 2024
-
Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 19:3
-
Tidskriftsartikel (refereegranskat)abstract
- Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.
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| 36. |
- Lauschke, Volker M., et al.
(författare)
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3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity : Emerging Culture Paradigms and Applications
- 2019
-
Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 14:7
-
Forskningsöversikt (refereegranskat)abstract
- Recent research has shown that the maintenance of relevant liver functions ex vivo requires models in which the cells exhibit an in vivo-like phenotype, often achieved by reconstitution of appropriate cellular interactions. Multiple different models have been presented that differ in the cells utilized, media, and culture conditions. Furthermore, several technologically different approaches have been presented including bioreactors, chips, and plate-based systems in fluidic or static media constituting of chemically diverse materials. Using such models, the ability to predict drug metabolism, drug toxicity, and liver functionality have increased tremendously as compared to conventional in vitro models in which cells are cultured as 2D monolayers. Here, the authors highlight important considerations for microphysiological systems for primary hepatocyte culture, review current culture paradigms, and discuss their opportunities for studies of drug metabolism, hepatotoxicity, liver biology, and disease.
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| 37. |
- Lindberg, Hanna, et al.
(författare)
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A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
- 2015
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:11, s. 1707-1718
-
Tidskriftsartikel (refereegranskat)abstract
- The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.
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| 38. |
- Lindberg, Hanna, et al.
(författare)
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Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method
- 2017
-
Ingår i: Biotechnology Journal. - : John Wiley & Sons. - 1860-6768 .- 1860-7314. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.
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| 39. |
- Lindberg, Hanna, et al.
(författare)
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Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
- 2013
-
Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 8:1, s. 139-145
-
Tidskriftsartikel (refereegranskat)abstract
- We have previously generated an affibody molecule for the disease-associated amyloid beta (A beta) peptide, which has been shown to inhibit the formation of various A beta aggregates and revert the neurotoxicity of A beta in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the A beta-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and A beta binding. Overall, the detailed engineering and characterization of this promising A beta-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.
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| 40. |
- Luttmann, Reiner, et al.
(författare)
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Editorial Material: Soft sensors in bioprocessing: A status report and recommendations
- 2012
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlag Berlin. - 1860-6768 .- 1860-7314. ; 7:8, s. 1040-1048
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The following report with recommendations is the result of an expert panel meeting on soft sensor applications in bioprocess engineering that was organized by the Measurement, Monitoring, Modelling and Control (M3C) Working Group of the European Federation of Biotechnology - Section of Biochemical Engineering Science (ESBES). The aim of the panel was to provide an update on the present status of the subject and to identify critical needs and issues for the furthering of the successful development of soft sensor methods in bioprocess engineering research and for industrial applications, in particular with focus on biopharmaceutical applications. It concludes with a set of recommendations, which highlight current prospects for the extended use of soft sensors and those areas requiring development.
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| 41. |
- Löfblom, John
(författare)
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Bacterial display in combinatorial protein engineering
- 2011
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:9, s. 1115-1129
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Forskningsöversikt (refereegranskat)abstract
- Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.
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| 42. |
- Machleid, Rafael, et al.
(författare)
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Feasibility and performance of cross-clone Raman calibration models in CHO cultivation
- 2024
-
Ingår i: Biotechnology Journal. - : John Wiley & Sons. - 1860-6768 .- 1860-7314. ; 19:1
-
Tidskriftsartikel (refereegranskat)abstract
- Raman spectroscopy is widely used in monitoring and controlling cell cultivations for biopharmaceutical drug manufacturing. However, its implementation for culture monitoring in the cell line development stage has received little attention. Therefore, the impact of clonal differences, such as productivity and growth, on the prediction accuracy and transferability of Raman calibration models is not yet well described. Raman OPLS models were developed for predicting titer, glucose and lactate using eleven CHO clones from a single cell line. These clones exhibited diverse productivity and growth rates. The calibration models were evaluated for clone-related biases using clone-wise linear regression analysis on cross validated predictions. The results revealed that clonal differences did not affect the prediction of glucose and lactate, but titer models showed a significant clone-related bias, which remained even after applying variable selection methods. The bias was associated with clonal productivity and lead to increased prediction errors when titer models were transferred to cultivations with productivity levels outside the range of their training data. The findings demonstrate the feasibility of Raman-based monitoring of glucose and lactate in cell line development with high accuracy. However, accurate titer prediction requires careful consideration of clonal characteristics during model development.
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| 43. |
- Malm, Magdalena, et al.
(författare)
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Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension
- 2014
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 9:9, s. 1215-1222
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Tidskriftsartikel (refereegranskat)abstract
- Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.
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| 44. |
- Mandenius, Carl-Fredrik, et al.
(författare)
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Quality-by-design for biotechnology-related pharmaceuticals.
- 2009
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Ingår i: Biotechnology journal. - : Wiley. - 1860-7314 .- 1860-6768. ; 4:5, s. 600-9
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Tidskriftsartikel (refereegranskat)abstract
- The following article is a report from a workshop on Quality-by-Design (QbD) held at the 7th European Symposium on Biochemical Engineering Science (7 September 2008, Faro, Portugal).The aim of the workshop was to provide an update on the present status of using QbD in biotechnology-related applications in the pharmaceutical industry. The report summarizes the essential parts of the presentations and covers the industrial, academic, and regulatory aspects of QbD. It concludes with recommendations for further work and development.
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| 45. |
- Mandenius, Carl-Fredrik, et al.
(författare)
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Scale-up of cell culture bioreactors using biomechatronic design
- 2012
-
Ingår i: Biotechnology Journal. - : Wiley-VCH Verlag Berlin. - 1860-6768 .- 1860-7314. ; 7:8, s. 1026-1039
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Tidskriftsartikel (refereegranskat)abstract
- Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes.
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| 46. |
- Mardinoglu, Adil, 1982, et al.
(författare)
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Genome-scale modeling of human metabolism - a systems biology approach.
- 2013
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 8:9, s. 985-996
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Forskningsöversikt (refereegranskat)abstract
- Altered metabolism is linked to the appearance of various human diseases and a better understanding of disease-associated metabolic changes may lead to the identification of novel prognostic biomarkers and the development of new therapies. Genome-scale metabolic models (GEMs) have been employed for studying human metabolism in a systematic manner, as well as for understanding complex human diseases. In the past decade, such metabolic models – one of the fundamental aspects of systems biology – have started contributing to the understanding of the mechanistic relationship between genotype and phenotype. In this review, we focus on the construction of the Human Metabolic Reaction database, the generation of healthy cell type- and cancer-specific GEMs using different procedures, and the potential applications of these developments in the study of human metabolism and in the identification of metabolic changes associated with various disorders. We further examine how in silico genome-scale reconstructions can be employed to simulate metabolic flux distributions and how high-throughput omics data can be analyzed in a context-dependent fashion. Insights yielded from this mechanistic modeling approach can be used for identifying new therapeutic agents and drug targets as well as for the discovery of novel biomarkers. Finally, recent advancements in genome-scale modeling and the future challenge of developing a model of whole-body metabolism are presented. The emergent contribution of GEMs to personalized and translational medicine is also discussed.
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| 47. |
- Meister, Sebastian, et al.
(författare)
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Engineering of TEV protease variants with redesigned substrate specificity
- 2023
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 18:11
-
Tidskriftsartikel (refereegranskat)abstract
- Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. Engineered proteases with redesigned substrate specificities have the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering-based screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of tobacco etch virus (TEV) protease. The target substrate EKLVFQA differs at three out of seven positions from the TEV consensus substrate sequence. Flow cytometric sorting of a semi-rational TEV protease library, consisting of focused mutations of the substrate binding pockets as well as random mutations throughout the enzyme, led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate.
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| 48. |
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| 49. |
- Mikkonen, Saara, et al.
(författare)
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Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation
- 2022
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314.
-
Tidskriftsartikel (refereegranskat)abstract
- The increased use of biopharmaceuticals calls for improved means of bioprocess monitoring. In this work, capillary electrophoresis (CE) and microchip electrophoresis (MCE) methods were developed and applied for the analysis of amino acids (AAs) in cell culture supernatant. In samples from different days of a Chinese hamster ovary cell cultivation process, all 19 proteinogenic AAs containing primary amine groups could be detected using CE, and 17 out of 19 AAs using MCE. The relative concentration changes in different samples agreed well with those measured by high-performance liquid chromatography (HPLC). Compared to the more commonly employed HPLC analysis, the CE and MCE methods resulted in faster analysis, while significantly lowering both the sample and reagent consumption, and the cost per analysis.
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| 50. |
- Mohammadi, Elyas, et al.
(författare)
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Improvement of the performance of anticancer peptides using a drug repositioning pipeline
- 2022
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 17:1, s. 2100417-
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Tidskriftsartikel (refereegranskat)abstract
- The use of anticancer peptides (ACPs) as an alternative/complementary strategy to conventional chemotherapy treatments has been shown to decrease drug resistance and/or severe side effects. However, the efficacy of the positively-charged ACP is inhibited by elevated levels of negatively-charged cell-surface components which trap the peptides and prevent their contact with the cell membrane. Consequently, this decreases ACP-mediated membrane pore formation and cell lysis. Negatively-charged heparan sulphate (HS) and chondroitin sulphate (CS) have been shown to inhibit the cytotoxic effect of ACPs. In this study, we propose a strategy to promote the broad utilization of ACPs. In this context, we developed a drug repositioning pipeline to analyse transcriptomics data generated for four different cancer cell lines (A549, HEPG2, HT29, and MCF7) treated with hundreds of drugs in the LINCS L1000 project. Based on previous studies identifying genes modulating levels of the glycosaminoglycans (GAGs) HS and CS at the cell surface, our analysis aimed at identifying drugs inhibiting genes correlated with high HS and CS levels. As a result, we identified six chemicals as likely repositionable drugs with the potential to enhance the performance of ACPs. The codes in R and Python programming languages are publicly available in https://github.com/ElyasMo/ACPs_HS_HSPGs_CS. As a conclusion, these six drugs are highlighted as excellent targets for synergistic studies with ACPs aimed at lowering the costs associated with ACP-treatment.
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