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https://www.readbyqxmd.com/read/28233468/an-automated-laboratory-scale-methodology-for-the-generation-of-sheared-mammalian-cell-culture-samples
#1
Adrian Joseph, Stephen Goldrick, Michael Mollet, Richard Turner, Jean Bender, David Gruber, Suzanne S Farid, Nigel Titchener-Hooker
Continuous disk-stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing-scale. The use of scale-down methods to characterise disk-stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot-scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines...
February 24, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28230321/disruption-of-genes-involved-in-corvet-complex-leads-to-enhanced-secretion-of-heterologous-carboxylesterase-only-in-protease-deficient-pichia-pastoris
#2
Lukas Marsalek, Clemens Gruber, Friedrich Altmann, Markus Aleschko, Diethard Mattanovich, Brigitte Gasser, Verena Puxbaum
The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work we have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET tethering complex at the early stages of endosomal sorting...
February 23, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28221723/animal-component-free-agrobacterium-tumefaciens-cultivation-media-for-better-gmp-compliance-increases-biomass-yield-and-pharmaceutical-protein-expression-in-nicotiana-benthamiana
#3
Marcel Houdelet, Anna Galinski, Tanja Holland, Kathrin Wenzel, Stefan Schillberg, Johannes Felix Buyel
Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP)...
February 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28220995/development-of-hydrogels-for-regenerative-engineering
#4
REVIEW
Xiaofei Guan, Meltem Avci-Adali, Emine Alarçin, Hao Cheng, Sara Saheb Kashaf, Yuxiao Li, Aditya Chawla, Hae Lin Jang, Ali Khademhosseini
The aim of regenerative engineering is to restore complex tissues and biological systems through convergence in the fields of advanced biomaterials, stem cell science, and developmental biology. Hydrogels are one of the most attractive biomaterials for regenerative engineering, since they can be engineered into tissue mimetic 3D scaffolds to support cell growth due to their similarity to native extracellular matrix. Advanced nano- and micro-technologies have dramatically increased the ability to control properties and functionalities of hydrogel materials by facilitating biomimetic fabrication of more sophisticated compositions and architectures, thus extending our understanding of cell-matrix interactions at the nanoscale...
February 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28217906/production-of-taxadiene-by-engineering-of-mevalonate-pathway-in-escherichia-coli-and-endophytic-fungus-alternaria-alternata-tpf6
#5
Guangkai Bian, Yujie Yuan, Hui Tao, Xiaofei Shi, Xiaofang Zhong, Yichao Han, Shuai Fu, Chengxiang Fang, Zixin Deng, Tiangang Liu
Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, we first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. We then developed an Agrobacterium tumefaciens-mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6...
February 20, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28177193/manufacturing-of-recombinant-adeno-associated-viruses-using-mammalian-expression-platforms
#6
REVIEW
Marc-André Robert, Parminder S Chahal, Alexandre Audy, Amine Kamen, Rénald Gilbert, Bruno Gaillet
Manufacturing practices for recombinant adeno-associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10(4) -10(5) vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used...
February 8, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28169491/fermentative-production-of-l-pipecolic-acid-from-glucose-and-alternative-carbon-sources
#7
Fernando Pérez-García, Joe Max Risse, Karl Friehs, Volker F Wendisch
Corynebacterium glutamicum is used for the million-ton scale production of amino acids and has recently been engineered for production of the cyclic non-proteinogenic amino acid L-pipecolic acid (L-PA). In this synthetic pathway L-lysine was converted to L-PA by oxidative deamination, dehydration and reduction by L-lysine 6-dehydrogenase (deaminating) from Silicibacter pomeroyi and pyrroline 5-carboxylate reductase from C. glutamicum. However, production of L-PA occurred as by-product of L-lysine production only...
February 7, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28166394/exosome-separation-using-microfluidic-systems-size-based-immunoaffinity-based-and-dynamic-methodologies
#8
REVIEW
Fang Yang, Xiangzhi Liao, Yuan Tian, Guiying Li
Exosomes, nanovesicles secreted by most types of cells, exist in virtually all bodily fluids. Their rich nucleic acid and protein content make them potentially valuable biomarkers for noninvasive molecular diagnostics. They also show promise, after further development, to serve as a drug delivery system. Unfortunately, existing exosome separation technologies, such as ultracentrifugation and methods incorporating magnetic beads, are time-consuming, laborious and separate only exosomes of low purity. Thus, a more effective separation method is highly desirable...
February 6, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28146316/mir-92a-enhances-recombinant-protein-productivity-in-cho-cells-by-increasing-intracellular-cholesterol-levels
#9
Wan Ping Loh, Yuansheng Yang, Kong Peng Lam
MicroRNAs (miRNAs) have emerged as promising targets for engineering of CHO cell factories to enhance recombinant protein productivity. Manipulation of miRNA levels in CHO cells have been shown to improve product yield by increasing proliferation and specific productivity (qP), resisting apoptosis and enhancing oxidative metabolism. We previously demonstrated that over-expressing miR-92a results in increases in qP and titer of CHO-IgG cells. However, the mechanisms by which miR-92a enhances qP in CHO cells are still uninvestigated...
February 1, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28139886/genomic-integration-of-the-full-length-dystrophin-coding-sequence-in-duchenne-muscular-dystrophy-induced-pluripotent-stem-cells
#10
Alfonso P Farruggio, Mital S Bhakta, Haley du Bois, Julia Ma, Michele P Calos
We developed plasmid vectors that express the full-length human dystrophin coding sequence in human cells. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in E. coli. We expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E...
January 31, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28139884/streptomyces-venezuelae-tx-tl-a-next-generation-cell-free-synthetic-biology-tool
#11
Simon J Moore, Hung-En Lai, Hannah Needham, Karen M Polizzi, Paul S Freemont
Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S...
January 31, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28133942/microbiome-engineering-current-applications-and-its-future
#12
REVIEW
Jee Loon Foo, Hua Ling, Yung Seng Lee, Matthew Wook Chang
Microbiomes exist in all ecosystems and are composed of diverse microbial communities. Perturbation to microbiomes brings about undesirable phenotypes in the hosts, resulting in diseases and disorders, and disturbs the balance of the associated ecosystems. Engineering of microbiomes can be used to modify structures of the microbiota and restore ecological balance. Consequently, microbiome engineering has been employed for improving human health and agricultural productivity. The importance and current applications of microbiome engineering, particularly in humans, animals, plants and soil is reviewed...
January 30, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28120503/physiological-capacities-decline-during-induced-bioprocesses-leading-to-substrate-accumulation
#13
Wieland N Reichelt, Markus Brillmann, Peter Thurrold, Peter Keil, Jens Fricke, Christoph Herwig
During the cultivation of E. coli for recombinant protein production, substrate accumulation is often observed in induction phase. Uncontrolled substrate accumulation leads to difficulties in transferring or scaling processes and even to failed batches. The phenomenon of metabolite/substrate accumulation occurs as a result of exceeding the physiological capacity to metabolize substrate (qScrit ). In contrast to the common understanding of qScrit as "static" value, we hypothesize that qScrit essentially has a dynamic nature...
January 25, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28106351/real-time-ph-monitoring-of-industrially-relevant-enzymatic-reactions-in-a-microfluidic-side-entry-reactor-%C3%AE-ser-shows-potential-for-ph-control
#14
Pia Gruber, Marco P C Marques, Philipp Sulzer, Roland Wohlgemuth, Torsten Mayr, Frank Baganz, Nicolas Szita
Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (μSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control...
January 20, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28105684/bacterial-microcompartment-directed-polyphosphate-kinase-promotes-stable-polyphosphate-accumulation-in-e-coli
#15
Mingzhi Liang, Stefanie Frank, Heinrich Lünsdorf, Martin J Warren, Michael B Prentice
Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E...
January 20, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28098436/bringing-3d-tumor-models-to-the-clinic-predictive-value-for-personalized-medicine
#16
REVIEW
Kathrin Halfter, Barbara Mayer
Current decision-guiding algorithms in cancer drug treatment are based on decades of research and numerous clinical trials. For the majority of patients, this data is successfully applied for a systemic disease management. For a number of patients however, treatment stratification according to clinically based risk criteria will not be sufficient. The most effective treatment options are ideally identified prior to the start of clinical drug therapy. This review will discuss the implementation of three-dimensional (3D) cell culture models as a preclinical testing paradigm for the efficacy of clinical cancer treatment...
January 18, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28098428/the-self-sufficient-p450-rhf-expressed-in-a-whole-cell-system-selectively-catalyses-the-5-hydroxylation-of-diclofenac
#17
Jan M Klenk, Bernd A Nebel, Joanne L Porter, Justyna K Kulig, Shaneela A Hussain, Sven M Richter, Michele Tavanti, Nicholas J Turner, Martin A Hayes, Bernhard Hauer, Sabine L Flitsch
P450 monooxygenases are able to catalyse the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites...
January 18, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28078826/the-impact-of-ph-inhomogeneities-on-cho-cell-physiology-and-fed-batch-process-performance-two-compartment-scale-down-modelling-and-intracellular-ph-excursion
#18
Matthias Brunner, Philipp Braun, Philipp Doppler, Christoph Posch, Dirk Behrens, Christoph Herwig, Jens Fricke
Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m(3) stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH were hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9...
January 12, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28029739/drying-techniques-for-the-visualisation-of-agarose-based-chromatography-media-by-scanning-electron-microscopy
#19
Mauryn C Nweke, Mark Turmaine, R Graham McCartney, Daniel G Bracewell
The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub-micron level. Achieving suitable drying conditions is especially important with agarose-based chromatography resins, as over-drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under-drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect™/dw ≈85 µm and Capto™ Adhere/dw ≈75 µm) and provides a complete method for both...
December 28, 2016: Biotechnology Journal
https://www.readbyqxmd.com/read/28008726/multi-objective-global-optimization-mogo-algorithm-and-case-study-in-gradient-elution-chromatography
#20
Lars Freier, Eric von Lieres
Biotechnological separation processes are routinely designed and optimized using parallel high-throughput experiments and/or serial experiments. Well-characterized processes can further be optimized using mechanistic models. In all these cases - serial/parallel experiments and modeling - iterative strategies are customarily applied for planning novel experiments/simulations based on the previously acquired knowledge. Process optimization is typically complicated by conflicting design targets, such as productivity and yield...
December 23, 2016: Biotechnology Journal
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