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Biotechnology and Bioengineering

Narendran Sekar, Chang-Hao Wu, Michael W W Adams, Ramaraja P Ramasamy
Hyperthermophiles are microorganisms that thrive in extremely hot environments with temperatures near and even above 100 (°) C. They are the most deeply rooted microorganisms on phylogenetic trees suggesting they may have evolved to survive in the early hostile earth. The simple respiratory systems of some of these hyperthermophiles made them potential candidates to develop microbial fuel cells (MFC) that can generate power at temperatures approaching the boiling point. We explored extracellular electron transfer ability of a hyperthermophilic archaeon Pyrococcus furiosus (Pf) by studying its ability to generate electricity in a two-chamber MFC...
February 20, 2017: Biotechnology and Bioengineering
Liang Zhao, Hsu-Yuan Fu, Ravali Raju, Nandita Vishwanathan, Wei-Shou Hu
In the past few years, transcriptome analysis has been increasingly employed to better understand the physiology of Chinese hamster ovary (CHO) cells at a global level. As more transcriptome data accumulated, meta-analysis on data sets collected from various sources can potentially provide better insights on common properties of those cells. Here, we performed meta-analysis on transcriptome data of different CHO cell lines obtained using NimbleGen or Affymetrix microarray platforms. Hierarchical clustering, non-negative matrix factorization (NMF) analysis, and principal component analysis (PCA) accordantly showed the samples were clustered into two groups: one consists of adherent cells in serum-containing medium, and the other suspension cells in serum-free medium...
February 20, 2017: Biotechnology and Bioengineering
Xuan Droz, Niamh Harraghy, Etienne Lançon, Valérie Le Fourn, David Calabrese, Thierry Colombet, Pascal Liechti, Amar Rida, Pierre-Alain Girod, Nicolas Mermod
We developed a method for the fast sorting and selection of mammalian cells expressing and secreting a protein at high levels. This procedure relies on cell capture using an automated microfluidic device handling antibody-coupled magnetic microparticles and on a timed release of the cells from the microparticles after capture. Using clinically compatible materials and procedures, we show that this approach is able to discriminate between cells that truly secrete high amounts of a protein from those that just display it at high levels on their surface without properly releasing it...
February 18, 2017: Biotechnology and Bioengineering
David Brühlmann, Michael Sokolov, Alessandro Butté, Markus Sauer, Jürgen Hemberger, Jonathan Souquet, Hervé Broly, Martin Jordan
Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action...
February 15, 2017: Biotechnology and Bioengineering
Asbjørn Toftgaard Pedersen, Teresa Melo de Carvalho, Euan Sutherland, Gustav Rehn, Robert Ashe, John M Woodley
Biocatalytic oxidation reactions employing molecular oxygen as the electron acceptor are difficult to conduct in a continuous flow reactor because of the requirement for high oxygen transfer rates. In this paper, the oxidation of glucose to glucono-1,5-lactone by glucose oxidase was used as a model reaction to study a novel continuous agitated cell reactor (ACR). The ACR consists of ten cells interconnected by small channels. An agitator is placed in each cell, which mixes the content of the cell when the reactor body is shaken by lateral movement...
February 10, 2017: Biotechnology and Bioengineering
Mara C Inniss, Kalpanie Bandara, Barbara Jusiak, Tim K Lu, Ron Weiss, Liliana Wroblewska, Lin Zhang
As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system...
February 10, 2017: Biotechnology and Bioengineering
Wai Keen Chung, Brian Russell, Yanhong Yang, Michael Handlogten, Suzanne Hudak, Mingyan Cao, Jihong Wang, David Robbins, Sanjeev Ahuja, Min Zhu
Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG2 mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process...
February 10, 2017: Biotechnology and Bioengineering
Karina Bora de Oliveira, David Spencer, Christopher Barton, Nitin Agarwal
Glycosylation often plays a key role in the safety and efficacy of therapeutic proteins to patients, thus underlying the need for consistent control of this important post-translational modification during biologics production. In this study, we profiled the site-specific evolution of N-glycans on a CTLA4-Fc-fusion protein, from the intracellular secretory pathway to the conditioned medium (CM) in fed-batch cell culture. For this, we developed an approach that combined sub-cellular fractionation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses...
February 10, 2017: Biotechnology and Bioengineering
Alberto Rodriguez, Juan A Martínez, Pierre Millard, Guillermo Gosset, Jean-Charles Portais, Fabien Létisse, Francisco Bolivar
Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high yield but that is also expected to impose a significant cellular burden...
February 10, 2017: Biotechnology and Bioengineering
Lisa A Pieper, Michaela Strotbek, Till Wenger, Monilola A Olayioye, Angelika Hausser
The dynamics of protein folding and secretion are key issues in improving the productivity and robustness of Chinese Hamster Ovary (CHO) producer cells. High recombinant protein secretion in CHO producer clones triggers the activation of the unfolded protein response (UPR), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). We previously reported that the human microRNA (miRNA) miR-1287 enhances productivity in IgG expressing CHO cells (CHO-IgG)...
February 6, 2017: Biotechnology and Bioengineering
Matthew D Ooms, Percival J Graham, Brian Nguyen, Edward H Sargent, David Sinton
The spectral distribution of light influences microalgae productivity; however, development of photobioreactors has proceeded largely without regard to spectral optimization. Here we use monochromatic light to quantify the joint influence of path length, culture density, light intensity and wavelength on productivity and efficiency in Synechococcus elongatus. The productivity of green light was ∼4 x that of red at the highest levels of culture density, depth and light intensity. This performance is attributed to the combination of increased dilution and penetration of this weakly absorbed wavelength over a larger volume fraction of the reactor...
February 6, 2017: Biotechnology and Bioengineering
Luke N Latimer, John E Dueber
A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities...
February 6, 2017: Biotechnology and Bioengineering
G Sandoval, D Espinoza, N Figueroa, J A Asenjo
In this work a biotechnological multiproduct batch plant that manufactures four different recombinant proteins for human application is described in some detail. This batch plant design is then optimized with regards to the size of equipment using a mixed-integer linear programming (MILP) formulation recently developed by us in order to find a hypothetical new biotechnological batch plant based on the information of real known processes for the production of the four recombinant protein products. The real plant was divided for practical purposes into 2 sub-processes or facilities: a fermentation facility and a purification facility...
February 1, 2017: Biotechnology and Bioengineering
Gregory W Hiller, Ana Maria Ovalle, Matthew P Gagnon, Meredith L Curran, Wenge Wang
A simple method originally designed to control lactate accumulation in fed-batch cultures of Chinese Hamster Ovary (CHO) cells has been modified and extended to allow cells in culture to control their own rate of perfusion to precisely deliver nutritional requirements. The method allows for very fast expansion of cells to high density while using a minimal volume of concentrated perfusion medium. When the short-duration cell-controlled perfusion is performed in the production bioreactor and is immediately followed by a conventional fed-batch culture using highly concentrated feeds, the overall productivity of the culture is approximately doubled when compared with a highly optimized state-of-the-art fed-batch process...
January 27, 2017: Biotechnology and Bioengineering
F Masri, K Lawrence, I Wall, M Hoare
Tools that allow cost-effective screening of the susceptibility of cell lines to operating conditions which may apply during full scale processing are central to the rapid development of robust processes for cell-based therapies. In this paper, an ultra scale-down (USD) device has been developed for the characterization of the response of a human cell line to membrane-based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. The cell line used to develop the measurements was a clinically relevant human fibroblast cell line...
January 23, 2017: Biotechnology and Bioengineering
Lisa Goers, Catherine Ainsworth, Cher Hui Goey, Cleo Kontoravdi, Paul S Freemont, Karen M Polizzi
Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimise the productivity of these cultures it is important to monitor cellular metabolism, for example the utilisation of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness...
January 23, 2017: Biotechnology and Bioengineering
Andrew S Thomson, Shing Mai, Michael P Byrne
Recombinant monoclonal antibody (mAb) products are widely produced in the pharmaceutical industry using Chinese hamster ovary (CHO) cells. Host cell proteins (HCPs) are one of many process-related impurities generated during the production of mAb products. The multi-analyte HCP enzyme linked immunosorbent assay (ELISA) is the industry standard accepted assay to measure clearance of HCPs from recombinant protein therapeutics. While similar platform processes are used for expression and purification, varying amounts of HCPs are found in final drug substances for different mAb products...
January 23, 2017: Biotechnology and Bioengineering
Jian Li, He Wang, Yong-Chan Kwon, Michael C Jewett
Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 µg/mL with batch reactions lasting up to 3 h...
January 23, 2017: Biotechnology and Bioengineering
Yosuke Fukutani, Jun Ishii, Akihiko Kondo, Takeaki Ozawa, Hiroaki Matsunami, Masafumi Yohda
The budding yeast S. cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and β-arrestin, which are independent of the endogenous S...
January 23, 2017: Biotechnology and Bioengineering
Christian Scherkus, Sandy Schmidt, Uwe T Bornscheuer, Harald Gröger, Selin Kara, Andreas Liese
A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO) and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach...
January 23, 2017: Biotechnology and Bioengineering
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