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Biotechnology and Bioengineering

Stephen Goldrick, William Holmes, Nick Bond, Gareth Lewis, Marcel Kuiper, Richard Turner, Suzanne S Farid
Product quality heterogeneities, such as a trisulfide bond (TSB) formation, can be influenced by multiple interacting process parameters. Identifying their root cause is a major challenge in biopharmaceutical production. To address this issue, this paper describes the novel application of advanced multivariate data analysis (MVDA) techniques to identify the process parameters influencing TSB formation in a novel recombinant antibody-peptide fusion expressed in mammalian cell culture. The screening dataset was generated with a high-throughput (HT) micro-bioreactor system (Ambr(TM) 15) using a design of experiments (DoE) approach...
May 13, 2017: Biotechnology and Bioengineering
Eunbi Yi, Jinsoo Oh, Ngoc Quynh Giao, Soohwan Oh, Se-Ho Park
Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type...
May 12, 2017: Biotechnology and Bioengineering
Ying Hou, Gazi Sakir Hossain, Jianghua Li, Hyun-Dong Shin, Guocheng Du, Jian Chen, Long Liu
Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD-dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve α-keto acid production-from the corresponding L-amino acids-catalyzed by FAD-dependent L-amino acid deaminase (L-AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine-tuned for FAD synthesis...
May 12, 2017: Biotechnology and Bioengineering
José L Adrio
Oleaginous yeasts have a unique physiology that makes them the best suited hosts for the production of lipids, oleochemicals and diesel-like fuels. Their high lipogenesis, capability of growing on many different carbon sources (including lignocellulosic sugars), easy large-scale cultivation, and an increasing number of genetic tools are some of the advantages that have encouraged their use to develop sustainable processes. This mini-review summarizes the metabolic engineering strategies developed in oleaginous yeasts within the last two years to improve process metrics (titer, yield and productivity) for the production of lipids, free fatty acids, fatty acid-based chemicals (eg...
May 12, 2017: Biotechnology and Bioengineering
Xueping Ee, Ying Yan, Daniel A Hunter, Lauren Schellhardt, Shelly E Sakiyama-Elbert, Susan E Mackinnon, Matthew D Wood
Providing temporally regulated glial cell line-derived neurotrophic factor (GDNF) to injured nerve can promote robust axon regeneration. However, it is poorly understood why providing highly elevated levels of GDNF to nerve can lead to axon entrapment in the zone containing elevated GDNF. This limited understanding represents an obstacle to the translation of GDNF therapies to treat nerve injuries clinically. Here, we investigated how transgenic Schwann cells (SCs) overexpressing GDNF-IRES-DsRed impact nerve regeneration...
May 8, 2017: Biotechnology and Bioengineering
Daniel Nampe, Ronak Joshi, Kevin Keller, Nicole Zur Nieden, Hideaki Tsutsui
The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters...
May 8, 2017: Biotechnology and Bioengineering
Wendy R Zeng, Siew-Joo Beh, Robert J Bryson-Richardson, Pauline M Doran
The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts...
May 5, 2017: Biotechnology and Bioengineering
Anindya Basu, Fengxue Xin, Teck Kwang Lim, Qingsong Lin, Kun-Lin Yang, Jianzhong He
Development of sustainable biobutanol production platforms from lignocellulosic materials is impeded by inefficient five carbon sugar uptake by solventogenic bacteria. The recently isolated Clostridium sp. strain BOH3 is particularly advantaged in this regard as it serves as a model organism which can simultaneously utilize both glucose and xylose for high butanol (>15 g/L) production. Strain BOH3 was therefore investigated for its metabolic mechanisms for efficient five carbon sugar uptake using a quantitative proteomics based approach...
May 5, 2017: Biotechnology and Bioengineering
Ryan Marshall, Colin S Maxwell, Scott P Collins, Chase L Beisel, Vincent Noireaux
E. coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from Escherichia coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles...
May 5, 2017: Biotechnology and Bioengineering
Ryosuke Yamada, Kazuki Wakita, Ryosuke Mitsui, Hiroyasu Ogino
Utilization of renewable feedstocks for the production of bio-based chemicals such as d-lactic acid by engineering metabolic pathways in the yeast Saccharomyces cerevisiae has recently become an attractive option. In this study, to realize efficient d-lactic acid production by S. cerevisiae, the expression of 12 glycolysis-related genes and the Leuconostoc mesenteroides d-LDH gene was optimized using a previously developed global metabolic engineering strategy, and repeated batch fermentation was carried out using the resultant strain YPH499/dPdA3-34/DLDH/1-18...
May 5, 2017: Biotechnology and Bioengineering
Haifeng Su, Jiafu Lin, Yuanhong Wang, Qiao Chen, Furong Tan
Biosynthesis of advanced biofuels by engineered non-natural microorganisms has been proposed to be the most promising approach for the replacement of dwindling fossil fuel resources. Brevibacterium flavum (B.f) is a model brevibacterium aerobe which lacks basic and applied research that could enable this species to produce any biofuels. There are also no reports regarding using this microorganism to produce advanced alcohols before. Here, for the first time, we developed the bacterium as a novel biosynthetic platform to produce advanced alcohols derived from 2-ketoacids metabolic pathways via the mutagenesis and engineering approachs...
May 2, 2017: Biotechnology and Bioengineering
Hyun Yong Shin, Jeroen G Nijland, Paul P de Waal, Arnold J M Driessen
Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% D-glucose and 4% D-xylose...
May 2, 2017: Biotechnology and Bioengineering
Youngbin Baek, Nripen Singh, Abhiram Arunkumar, Michael Borys, Zheng J Li, Andrew L Zydney
Ultrafiltration (UF) is used for the final concentration and formulation of essentially all antibody-based therapeutics including both monoclonal antibodies (mAbs) and Fc-fusion proteins. The objective of this study was to quantitatively compare the filtrate flux behavior for two highly purified mAbs and an Fc-fusion protein under identical flow and buffer conditions. Filtrate flux data were obtained using a Pellicon 3 tangential flow filtration cassette over a wide range of transmembrane pressures and bulk protein concentrations...
May 2, 2017: Biotechnology and Bioengineering
Yang Yang, Ajoy Velayudhan, Nina F Thornhill, Suzanne S Farid
The need for high-concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high-concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high-concentration mAb formulation conditions in terms of their ease of manufacture...
May 2, 2017: Biotechnology and Bioengineering
Scott C Pierobon, Jason Riordon, Brian Nguyen, Matthew D Ooms, David Sinton
High-density biomass production is currently only realized in biofilm-based photobioreactors. Harvest yields of whole biofilms are self-limited by daughter-upon-parent cell growth that hinders light and leads to respiratory biomass losses. In this work we demonstrate a sustainable multi-harvest approach for prolonged generation of high-density biomass. Calcium-alginate hydrogel cultures loaded with Synechococcus elongatus PCC 7942 achieved production densities comparable to that of biofilms (10(9) cells mL(-1) ) and optimal total productivity in harvest periods of two or three days that allowed high-density surface growth without self-limiting cell buildup or surface death...
May 2, 2017: Biotechnology and Bioengineering
Jianhua Yang, Anna Joëlle Ruff, Marcus Arlt, Ulrich Schwaneberg
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g. high temperature, extreme pH, ionic liquids or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance...
May 2, 2017: Biotechnology and Bioengineering
Janis Baeten, Mark van Loosdrecht, Eveline Volcke
Aquasim is public-domain and user-friendly software that is widely used for biofilm modeling. The program uses built-in process unit blocks, such as biofilm and mixed compartments. In the literature, often several of these compartments are linked with artificial advective flows to represent a single physical well-mixed system, such as a granular sludge reactor. This work shows that caution should be exercised with this popular approach because numerical errors occur when too high artificial advective flows are used...
May 2, 2017: Biotechnology and Bioengineering
Akinlolu Oyekunle Oluseun Odeleye, Sara Castillo-Avila, Mathew Boon, Haydn Martin, Karen Coopman
The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non-invasive and automated monitoring of cell number, confluence and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance...
May 2, 2017: Biotechnology and Bioengineering
Jeong H Lee, Jason Reier, Kelley M Heffner, Christopher Barton, David Spencer, Albert E Schmelzer, Raghavan Venkat
Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has ten sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation...
April 24, 2017: Biotechnology and Bioengineering
Camila A Orellana, Esteban Marcellin, Peter P Gray, Lars K Nielsen
For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm)...
April 24, 2017: Biotechnology and Bioengineering
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