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  Subjects -> ENGINEERING (Total: 2272 journals)
    - CHEMICAL ENGINEERING (190 journals)
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    - ENGINEERING (1204 journals)
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ENGINEERING (1204 journals)                  1 2 3 4 5 6 7 | Last

Showing 1 - 200 of 1205 Journals sorted alphabetically
3 Biotech     Open Access   (Followers: 7)
3D Research     Hybrid Journal   (Followers: 17)
AAPG Bulletin     Hybrid Journal   (Followers: 7)
AASRI Procedia     Open Access   (Followers: 15)
Abstract and Applied Analysis     Open Access   (Followers: 3)
Aceh International Journal of Science and Technology     Open Access   (Followers: 2)
ACS Nano     Full-text available via subscription   (Followers: 247)
Acta Geotechnica     Hybrid Journal   (Followers: 7)
Acta Metallurgica Sinica (English Letters)     Hybrid Journal   (Followers: 5)
Acta Polytechnica : Journal of Advanced Engineering     Open Access   (Followers: 2)
Acta Scientiarum. Technology     Open Access   (Followers: 3)
Acta Universitatis Cibiniensis. Technical Series     Open Access  
Active and Passive Electronic Components     Open Access   (Followers: 7)
Adaptive Behavior     Hybrid Journal   (Followers: 11)
Adıyaman Üniversitesi Mühendislik Bilimleri Dergisi     Open Access  
Adsorption     Hybrid Journal   (Followers: 4)
Advanced Engineering Forum     Full-text available via subscription   (Followers: 6)
Advanced Science     Open Access   (Followers: 5)
Advanced Science Focus     Free   (Followers: 3)
Advanced Science Letters     Full-text available via subscription   (Followers: 8)
Advanced Science, Engineering and Medicine     Partially Free   (Followers: 7)
Advanced Synthesis & Catalysis     Hybrid Journal   (Followers: 17)
Advances in Calculus of Variations     Hybrid Journal   (Followers: 2)
Advances in Catalysis     Full-text available via subscription   (Followers: 5)
Advances in Complex Systems     Hybrid Journal   (Followers: 7)
Advances in Engineering Software     Hybrid Journal   (Followers: 25)
Advances in Fuel Cells     Full-text available via subscription   (Followers: 16)
Advances in Fuzzy Systems     Open Access   (Followers: 5)
Advances in Geosciences (ADGEO)     Open Access   (Followers: 10)
Advances in Heat Transfer     Full-text available via subscription   (Followers: 22)
Advances in Human Factors/Ergonomics     Full-text available via subscription   (Followers: 26)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 9)
Advances in Natural Sciences: Nanoscience and Nanotechnology     Open Access   (Followers: 29)
Advances in Operations Research     Open Access   (Followers: 11)
Advances in OptoElectronics     Open Access   (Followers: 5)
Advances in Physics Theories and Applications     Open Access   (Followers: 12)
Advances in Polymer Science     Hybrid Journal   (Followers: 41)
Advances in Porous Media     Full-text available via subscription   (Followers: 4)
Advances in Remote Sensing     Open Access   (Followers: 37)
Advances in Science and Research (ASR)     Open Access   (Followers: 6)
Aerobiologia     Hybrid Journal   (Followers: 1)
African Journal of Science, Technology, Innovation and Development     Hybrid Journal   (Followers: 4)
AIChE Journal     Hybrid Journal   (Followers: 31)
Ain Shams Engineering Journal     Open Access   (Followers: 5)
Akademik Platform Mühendislik ve Fen Bilimleri Dergisi     Open Access  
Alexandria Engineering Journal     Open Access   (Followers: 1)
AMB Express     Open Access   (Followers: 1)
American Journal of Applied Sciences     Open Access   (Followers: 28)
American Journal of Engineering and Applied Sciences     Open Access   (Followers: 11)
American Journal of Engineering Education     Open Access   (Followers: 9)
American Journal of Environmental Engineering     Open Access   (Followers: 17)
American Journal of Industrial and Business Management     Open Access   (Followers: 23)
Analele Universitatii Ovidius Constanta - Seria Chimie     Open Access  
Annals of Combinatorics     Hybrid Journal   (Followers: 3)
Annals of Pure and Applied Logic     Open Access   (Followers: 2)
Annals of Regional Science     Hybrid Journal   (Followers: 7)
Annals of Science     Hybrid Journal   (Followers: 7)
Applicable Algebra in Engineering, Communication and Computing     Hybrid Journal   (Followers: 2)
Applicable Analysis: An International Journal     Hybrid Journal   (Followers: 1)
Applied Catalysis A: General     Hybrid Journal   (Followers: 6)
Applied Catalysis B: Environmental     Hybrid Journal   (Followers: 16)
Applied Clay Science     Hybrid Journal   (Followers: 5)
Applied Computational Intelligence and Soft Computing     Open Access   (Followers: 12)
Applied Magnetic Resonance     Hybrid Journal   (Followers: 4)
Applied Nanoscience     Open Access   (Followers: 8)
Applied Network Science     Open Access   (Followers: 1)
Applied Numerical Mathematics     Hybrid Journal   (Followers: 5)
Applied Physics Research     Open Access   (Followers: 3)
Applied Sciences     Open Access   (Followers: 2)
Applied Spatial Analysis and Policy     Hybrid Journal   (Followers: 4)
Arabian Journal for Science and Engineering     Hybrid Journal   (Followers: 5)
Archives of Computational Methods in Engineering     Hybrid Journal   (Followers: 4)
Archives of Foundry Engineering     Open Access  
Archives of Thermodynamics     Open Access   (Followers: 7)
Arkiv för Matematik     Hybrid Journal   (Followers: 1)
ASEE Prism     Full-text available via subscription   (Followers: 3)
Asia-Pacific Journal of Science and Technology     Open Access  
Asian Engineering Review     Open Access  
Asian Journal of Applied Science and Engineering     Open Access   (Followers: 1)
Asian Journal of Applied Sciences     Open Access   (Followers: 2)
Asian Journal of Biotechnology     Open Access   (Followers: 8)
Asian Journal of Control     Hybrid Journal  
Asian Journal of Current Engineering & Maths     Open Access  
Asian Journal of Technology Innovation     Hybrid Journal   (Followers: 8)
Assembly Automation     Hybrid Journal   (Followers: 2)
at - Automatisierungstechnik     Hybrid Journal   (Followers: 1)
ATZagenda     Hybrid Journal  
ATZextra worldwide     Hybrid Journal  
Australasian Physical & Engineering Sciences in Medicine     Hybrid Journal   (Followers: 1)
Australian Journal of Multi-Disciplinary Engineering     Full-text available via subscription   (Followers: 2)
Autonomous Mental Development, IEEE Transactions on     Hybrid Journal   (Followers: 8)
Avances en Ciencias e Ingeniería     Open Access  
Balkan Region Conference on Engineering and Business Education     Open Access   (Followers: 1)
Bangladesh Journal of Scientific and Industrial Research     Open Access  
Basin Research     Hybrid Journal   (Followers: 5)
Batteries     Open Access   (Followers: 5)
Bautechnik     Hybrid Journal   (Followers: 1)
Bell Labs Technical Journal     Hybrid Journal   (Followers: 23)
Beni-Suef University Journal of Basic and Applied Sciences     Open Access   (Followers: 4)
BER : Manufacturing Survey : Full Survey     Full-text available via subscription   (Followers: 2)
BER : Motor Trade Survey     Full-text available via subscription   (Followers: 1)
BER : Retail Sector Survey     Full-text available via subscription   (Followers: 2)
BER : Retail Survey : Full Survey     Full-text available via subscription   (Followers: 2)
BER : Survey of Business Conditions in Manufacturing : An Executive Summary     Full-text available via subscription   (Followers: 3)
BER : Survey of Business Conditions in Retail : An Executive Summary     Full-text available via subscription   (Followers: 3)
Bharatiya Vaigyanik evam Audyogik Anusandhan Patrika (BVAAP)     Open Access   (Followers: 1)
Biofuels Engineering     Open Access  
Biointerphases     Open Access   (Followers: 1)
Biomaterials Science     Full-text available via subscription   (Followers: 10)
Biomedical Engineering     Hybrid Journal   (Followers: 16)
Biomedical Engineering and Computational Biology     Open Access   (Followers: 13)
Biomedical Engineering Letters     Hybrid Journal   (Followers: 5)
Biomedical Engineering, IEEE Reviews in     Full-text available via subscription   (Followers: 17)
Biomedical Engineering, IEEE Transactions on     Hybrid Journal   (Followers: 32)
Biomedical Engineering: Applications, Basis and Communications     Hybrid Journal   (Followers: 5)
Biomedical Microdevices     Hybrid Journal   (Followers: 9)
Biomedical Science and Engineering     Open Access   (Followers: 4)
Biomedizinische Technik - Biomedical Engineering     Hybrid Journal  
Biomicrofluidics     Open Access   (Followers: 4)
BioNanoMaterials     Hybrid Journal   (Followers: 2)
Biotechnology Progress     Hybrid Journal   (Followers: 39)
Boletin Cientifico Tecnico INIMET     Open Access  
Botswana Journal of Technology     Full-text available via subscription   (Followers: 1)
Boundary Value Problems     Open Access   (Followers: 1)
Brazilian Journal of Science and Technology     Open Access   (Followers: 2)
Broadcasting, IEEE Transactions on     Hybrid Journal   (Followers: 10)
Bulletin of Canadian Petroleum Geology     Full-text available via subscription   (Followers: 14)
Bulletin of Engineering Geology and the Environment     Hybrid Journal   (Followers: 9)
Bulletin of the Crimean Astrophysical Observatory     Hybrid Journal  
Cahiers, Droit, Sciences et Technologies     Open Access  
Calphad     Hybrid Journal  
Canadian Geotechnical Journal     Hybrid Journal   (Followers: 22)
Canadian Journal of Remote Sensing     Full-text available via subscription   (Followers: 43)
Case Studies in Engineering Failure Analysis     Open Access   (Followers: 8)
Case Studies in Thermal Engineering     Open Access   (Followers: 4)
Catalysis Communications     Hybrid Journal   (Followers: 6)
Catalysis Letters     Hybrid Journal   (Followers: 2)
Catalysis Reviews: Science and Engineering     Hybrid Journal   (Followers: 8)
Catalysis Science and Technology     Free   (Followers: 7)
Catalysis Surveys from Asia     Hybrid Journal   (Followers: 3)
Catalysis Today     Hybrid Journal   (Followers: 8)
CEAS Space Journal     Hybrid Journal  
Cellular and Molecular Neurobiology     Hybrid Journal   (Followers: 3)
Central European Journal of Engineering     Hybrid Journal   (Followers: 1)
CFD Letters     Open Access   (Followers: 6)
Chaos : An Interdisciplinary Journal of Nonlinear Science     Hybrid Journal   (Followers: 2)
Chaos, Solitons & Fractals     Hybrid Journal   (Followers: 3)
Chinese Journal of Catalysis     Full-text available via subscription   (Followers: 2)
Chinese Journal of Engineering     Open Access   (Followers: 2)
Chinese Science Bulletin     Open Access   (Followers: 1)
Ciencia e Ingenieria Neogranadina     Open Access  
Ciencia en su PC     Open Access   (Followers: 1)
Ciencias Holguin     Open Access   (Followers: 1)
CienciaUAT     Open Access  
Cientifica     Open Access  
CIRP Annals - Manufacturing Technology     Full-text available via subscription   (Followers: 11)
CIRP Journal of Manufacturing Science and Technology     Full-text available via subscription   (Followers: 14)
City, Culture and Society     Hybrid Journal   (Followers: 22)
Clay Minerals     Full-text available via subscription   (Followers: 10)
Clean Air Journal     Full-text available via subscription   (Followers: 2)
Coal Science and Technology     Full-text available via subscription   (Followers: 3)
Coastal Engineering     Hybrid Journal   (Followers: 11)
Coastal Engineering Journal     Hybrid Journal   (Followers: 5)
Coatings     Open Access   (Followers: 4)
Cogent Engineering     Open Access   (Followers: 2)
Cognitive Computation     Hybrid Journal   (Followers: 4)
Color Research & Application     Hybrid Journal   (Followers: 1)
COMBINATORICA     Hybrid Journal  
Combustion Theory and Modelling     Hybrid Journal   (Followers: 13)
Combustion, Explosion, and Shock Waves     Hybrid Journal   (Followers: 13)
Communications Engineer     Hybrid Journal   (Followers: 1)
Communications in Numerical Methods in Engineering     Hybrid Journal   (Followers: 2)
Components, Packaging and Manufacturing Technology, IEEE Transactions on     Hybrid Journal   (Followers: 26)
Composite Interfaces     Hybrid Journal   (Followers: 6)
Composite Structures     Hybrid Journal   (Followers: 267)
Composites Part A : Applied Science and Manufacturing     Hybrid Journal   (Followers: 187)
Composites Part B : Engineering     Hybrid Journal   (Followers: 283)
Composites Science and Technology     Hybrid Journal   (Followers: 179)
Comptes Rendus Mécanique     Full-text available via subscription   (Followers: 2)
Computation     Open Access  
Computational Geosciences     Hybrid Journal   (Followers: 14)
Computational Optimization and Applications     Hybrid Journal   (Followers: 7)
Computational Science and Discovery     Full-text available via subscription   (Followers: 2)
Computer Applications in Engineering Education     Hybrid Journal   (Followers: 7)
Computer Science and Engineering     Open Access   (Followers: 17)
Computers & Geosciences     Hybrid Journal   (Followers: 28)
Computers & Mathematics with Applications     Full-text available via subscription   (Followers: 5)
Computers and Electronics in Agriculture     Hybrid Journal   (Followers: 4)
Computers and Geotechnics     Hybrid Journal   (Followers: 10)
Computing and Visualization in Science     Hybrid Journal   (Followers: 5)
Computing in Science & Engineering     Full-text available via subscription   (Followers: 30)
Conciencia Tecnologica     Open Access  
Concurrent Engineering     Hybrid Journal   (Followers: 3)
Continuum Mechanics and Thermodynamics     Hybrid Journal   (Followers: 7)
Control and Dynamic Systems     Full-text available via subscription   (Followers: 9)
Control Engineering Practice     Hybrid Journal   (Followers: 42)
Control Theory and Informatics     Open Access   (Followers: 8)
Corrosion Science     Hybrid Journal   (Followers: 25)
CT&F Ciencia, Tecnologia y Futuro     Open Access   (Followers: 1)
CTheory     Open Access  

        1 2 3 4 5 6 7 | Last

Journal Cover Biotechnology Progress
  [SJR: 0.736]   [H-I: 101]   [39 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 8756-7938 - ISSN (Online) 1520-6033
   Published by John Wiley and Sons Homepage  [1579 journals]
  • Alternating flow filtration as an alternative to internal spin filter
           based perfusion process: Impact on productivity and product quality
    • Authors: María Bosco; Cristian Paillet, Laura Mauro, Ignacio Amadeo, Eduardo Orti, Guillermina Forno
      PubDate: 2017-04-26T11:20:47.133676-05:
      DOI: 10.1002/btpr.2487
  • Growth engineering of Synechococcus elongatus PCC 7942 for mixotrophy
           under natural light conditions for improved feedstock production
    • Authors: Aditya Sarnaik; Reena Pandit, Arvind Lali
      Abstract: Synechococcus elongatus PCC 7942 has been widely explored as cyanobacterial cell factory through genetic modifications for production of various value-added compounds. However, successful industrial scale-ups have not been reported for the system predominantly due to its obligate photoautotrophic metabolism and use of artificial light in photobioreactors. Hence, engineering the organism to perform mixotrophy under natural light could serve as an effective solution. Thus, we applied a genetically engineered strain of Synechococcus elongatus PCC 7942 expressing heterologous hexose transporter gene (galP) to perform mixotrophy under natural light in a temperature controlled environmental chamber (EC). We systematically studied the comparative performances of these transformants using autotrophy and mixotrophy, which showed 3.4 times increase in biomass productivity of mixotrophically grown transformants over autotrophs in EC. Chlorophyll-a yield was found to have decreased in mixotrophic conditions, possibly indicating reduced dependency on light for energy metabolism. Although pigment yield decreases under mixotrophy, titer was found to have improved due to increased biomass productivity. Carotenoid analysis showed that zeaxanthin is the major carotenoid produced by the species which is essential for photoprotection. Our work thus demonstrates that mixotrophy under temperature controlled natural light can serve as the viable solution to improve biomass productivity of Synechococcus elongatus PCC 7942 and for commercial production of natural or engineered value added compounds from the system. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-26T11:00:46.958939-05:
      DOI: 10.1002/btpr.2490
  • Identification of a novel miRNA that increases transient protein
           expression in combination with valproic acid
    • Authors: Hermann-Josef Meyer; Dorothea Reilly, Scott E. Martin, Athena W. Wong
      Abstract: Transient gene expression in mammalian cells is an efficient process to produce recombinant proteins for various research applications and large molecule therapeutics development. For the first time, we report a screen to identify human microRNAs (miRNAs) that increase titers after polyethylenimine (PEI) mediated transient transfection of a HEK293 cell line. From a library of 875 miRNAs, we identified 2 miRNAs, miR-26a-5p and miR-337-5p, that increased human IgG1 (huIgG1) yields by 50% and 25%, respectively. The titer increase was achievable by expressing miR-26a-5p from oligonucleotides or a plasmid. Furthermore, combining miR-26a-5p with valproic acid (VPA) treatment doubled huIgG1 titers. Assessment of miR-26a-5p and VPA treatment across a panel of 32 human and murine antibodies demonstrates that the level of yield enhancement was molecule-dependent, with most exhibiting a range of 50 – 100% titer increase. These findings exemplify that combining genetic and chemical manipulation can be an effective strategy to enhance transient transfection productivity. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-26T11:00:42.676952-05:
      DOI: 10.1002/btpr.2488
  • A practical approach in bioreactor scale-up and process transfer using a
           combination of constant P/V and vvm as the criterion
    • Authors: Sen Xu; Linda Hoshan, Rubin Jiang, Balrina Gupta, Eric Brodean, Kristin O'Neill, T. Craig Seamans, John Bowers, Hao Chen
      Abstract: Bioreactor scale-up is a critical step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). With the scale-up criterion such as similar power input per volume or O2 volumetric mass transfer coefficient (kLa), adequate oxygen supply and cell growth can be largely achieved. However, CO2 stripping in the growth phase is often inadequate. This could cascade down to increased base addition and osmolality, as well as residual lactate increase and compromised production and product quality. Here we described a practical approach in bioreactor scale-up and process transfer, where bioreactor information may be limited. We evaluated the sparger kLa and kLaCO2 (CO2 volumetric mass transfer coefficient) from a range of bioreactor scales (3 L to 2,000 L) with different spargers. Results demonstrated that kLa for oxygen is not an issue when scaling from small-scale to large-scale bioreactors at the same gas flow rate per reactor volume (vvm). Results also showed that sparging CO2 stripping, kLaCO2, is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N2 flow with a similar specific power was used as the general scale-up criterion. An equation was developed to determine the minimum vvm required for removing CO2 produced from cell respiration. We demonstrated the effectiveness of using such scale-up criterion with five MAb projects exhibiting different cell growth and metabolic characteristics, scaled from 3 L to 2,000 L bioreactors across four sites. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-25T03:51:02.626103-05:
      DOI: 10.1002/btpr.2489
  • A new approach to obtain cellulose nanocrystals and ethanol from
           eucalyptus cellulose pulp via the biochemical pathway
    • Authors: Thalita J. Bondancia; Luiz Henrique C. Mattoso, José M. Marconcini, Cristiane S. Farinas
      Abstract: The feasibility of integration of cellulosic ethanol production with the manufacture of cellulose nanofibers and cellulose nanocrystals was evaluated using eucalyptus cellulose pulp as feedstock and employing the biochemical route alone. For the enzymatic hydrolysis step, experimental central composite design (CCD) methodology was used as a tool to evaluate the effects of solids loading (SL) and enzymatic loading (EL) on glucose release and cellulose conversion. Glucose concentrations from 45 to 125 g/L were obtained after 24 h, with cellulose conversions from 35 to 96%. Validation of the statistical model was performed at SL of 20% and EL of 10 mg protein/g, which was defined by the desirability function as the optimum condition. The sugars released were used for the production of ethanol by Saccharomyces cerevisiae, resulting in 62.1 g/L ethanol after 8 h (yield of 95.5%). For all the CCD experimental conditions, the residual solids presented cellulose nanofiber (CNF) characteristics. Moreover, the use of a new strategy with temperature reduction from 50 to 35°C after 24 h of enzymatic hydrolysis enabled cellulose nanocrystals (CNC) to be obtained after 144 h. The CNC showed a crystallinity index of 83%, length of 260 nm, diameter of 15 nm, and aspect ratio (L/D) of 15. These characteristics are suitable for many applications, such as reinforcement in polymeric materials and other lower volume higher value bio-based products. The findings indicate the viability of obtaining ethanol and CNC using the biochemical route exclusively, potentially contributing to the future implementation of forest biorefineries. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-25T03:50:57.329954-05:
      DOI: 10.1002/btpr.2486
  • Production of itaconic acid from pentose sugars by Aspergillus terreus
    • Authors: Badal C. Saha; Gregory J. Kennedy, Nasib Qureshi, Michael J. Bowman
      Abstract: Itaconic acid (IA), an unsaturated 5-carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, 6 strains (NRRL strains 1960, 1961, 1962, 1972, 66125 and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8±0.3, 38.9±0.8, 34.8±0.9 and 33.2±2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose and arabinose by A. terreus. The results presented in the paper will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-25T03:50:34.505558-05:
      DOI: 10.1002/btpr.2485
  • The CRISPR/Cas9 system: their delivery, in vivo and ex vivo applications
           and clinical development by startups
    • Authors: Minjung Song
      Abstract: The CRISPR/Cas9 gene editing system was originally derived from the prokaryotic adaptive immune system mediated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas). The system has been successfully applied to genome editing in eukaryotes and has contributed to remarkable advances in the life sciences, in areas ranging from agriculture to genetic disease therapies. For efficient editing and extending the influence of this system, proper delivery of its components is crucial. Both viral and non-viral delivery methods are reviewed here, along with the advantages and disadvantages of each. In addition, we review ex vivo and in vivo CRISPR/Cas9 applications for disease therapies. Related remarkable studies are highlighted and relevant startup companies and their drug development pipelines are described. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-25T03:50:17.882866-05:
      DOI: 10.1002/btpr.2484
  • Advanced model-based control strategies for the intensification of
           upstream and downstream processing in mAb production
    • Authors: Maria M. Papathanasiou; Ana L. Quiroga-Campano, Fabian Steinebach, Montaña Elviro, Athanasios Mantalaris, Efstratios N. Pistikopoulos
      Abstract: Current industrial trends encourage the development of sustainable, environmentally friendly processes with minimal energy and material consumption. In particular, the increasing market demand in biopharmaceutical industry, as well as the tight regulations in product quality, necessitate efficient operating procedures that guarantee products of high purity. In this direction, process intensification via continuous operation paves the way for the development of novel, eco-friendly processes, characterized by higher productivity and lower production costs. This work focuses on the development of advanced control strategies for: (i) a cell culture system in a bioreactor and (ii) a semi-continuous purification process. More specifically, we consider a fed-batch culture of GS-NS0 cells and the semi-continuous Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) for the purification process. The controllers are designed following the PAROC framework/software platform and their capabilities are assessed in-silico, against the process models. It is demonstrated that the proposed controllers efficiently manage to increase the system productivity, returning strategies that can lead to continuous, stable process operation. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-12T03:42:01.929696-05:
      DOI: 10.1002/btpr.2483
  • Optimisation-based Framework for Resin Selection Strategies in
           Biopharmaceutical Purification Process Development
    • Authors: Songsong Liu; Spyridon Gerontas, David Gruber, Richard Turner, Nigel J. Titchener-Hooker, Lazaros G. Papageorgiou
      Abstract: This work addresses rapid resin selection for integrated chromatographic separations when conducted as part of a high-throughput screening (HTS) exercise during the early stages of purification process development. An optimisation-based decision support framework is proposed to process the data generated from microscale experiments in order to identify the best resins to maximise key performance metrics for a biopharmaceutical manufacturing process, such as yield and purity. A multiobjective mixed integer nonlinear programming (MINLP) model is developed and solved using the ε-constraint method. Dinkelbach's algorithm is used to solve the resulting mixed integer linear fractional programming (MILFP) model. The proposed framework is successfully applied to an industrial case study of a process to purify recombinant Fc Fusion protein from low molecular weight and high molecular weight product related impurities, involving two chromatographic steps with 8 and 3 candidate resins for each step, respectively. The computational results show the advantage of the proposed framework in terms of computational efficiency and flexibility. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:45:35.829808-05:
      DOI: 10.1002/btpr.2479
  • Characterization of Mammalian Cell Culture Raw Materials by Combining
           Spectroscopy and Chemometrics
    • Authors: Nicholas Trunfio; Haewoo Lee, Jason Starkey, Cyrus Agarabi, Jay Liu, Seongkyu Yoon
      Abstract: Two of the primary issues with characterizing the variability of raw materials used in mammalian cell culture, such as wheat hydrolysates, is that the analyses of these materials can be time consuming, and the results of the analyses are not straightforward to interpret. To solve these issues, spectroscopy can be combined with chemometrics to provide a quick, robust and easy to understand methodology for the characterization of raw materials; which will improve cell culture performance by providing an assessment of the impact that a given raw material will have on final product quality. In this study, four spectroscopic technologies: Near-Infrared Spectroscopy, Middle-Infrared Spectroscopy, Raman Spectroscopy and Fluorescence Spectroscopy were used in conjunction with Principal Component Analysis to characterize the variability of wheat hydrolysates, and to provide evidence that the classification of good and bad lots of raw material is possible. Then, the same spectroscopic platforms are combined with Partial Least Squares regressions to quantitatively predict two cell culture Critical Quality Attributes (CQA): Integrated Viable Cell Density and IgG titer. The results showed that Near-Infrared (NIR) spectroscopy and Florescence spectroscopy are capable of characterizing the wheat hydrolysate's chemical structure, with NIR performing slightly better; and that they can be used to estimate the raw material's impact on the CQAs. These results were justified by demonstrating that of all the components present in the wheat hydrolysates, six amino acids: Arginine, Glycine, Phenylalanine, Tyrosine, Isoleucine and Threonine; and five trace elements: Copper, Phosphorous, Molybdenum, Arsenic and Aluminum, had a large, statistically significant effect on the CQAs, and that NIR and Fluorescence spectroscopy performed the best for characterizing the important amino acids. It was also found that the trace elements of interest were not characterized well by any of the spectral technologies used; however, the trace elements were also shown to have a less significant effect on the CQAs than the amino acids. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:40:46.435043-05:
      DOI: 10.1002/btpr.2480
  • Enhanced Direct Ethanol Production by Cofactor Optimization of Cell
           Surface-Displayed Xylose Isomerase in Yeast
    • Authors: Yusuke Sasaki; Toshiyuki Takagi, Keisuke Motone, Kouichi Kuroda, Mitsuyoshi Ueda
      Abstract: Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d-xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC-displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co2+), especially enhanced the activity by 46-fold. Co2+ also contributed to d-xylose fermentation, and ethanol yields and xylose consumption rates were improved by 6.0- and 2.7-fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC-displaying yeast showed the faster d-xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co-culture system. A xylan-degrading yeast strain was established by displaying two kinds of xylanases; endo-1,4-β-xylanase (Xyn11B) from Saccharophagus degradans, and β-xylosidase (XlnD) from Aspergillus niger. The yeast co-culture system enabled fine-tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)-displaying yeast and XylC-displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 1013: 1.39 × 1013: 2.78 × 1013 molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co-culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:40:41.716685-05:
      DOI: 10.1002/btpr.2478
  • Comparative Study of Therapeutic Antibody Candidates Derived from
           Mini-Pool and Clonal Cell Lines
    • Authors: Lianchun Fan; Giovanni Rizzi, Kathleen Bierilo, Jun Tian, Joon Chong Yee, Reb Russel, Tapan Das
      Abstract: The long journey of developing a drug from initial discovery target identification to regulatory approval often leaves many patients with missed window of opportunities. Both regulatory agencies and biopharmaceutical industry continue to develop creative approaches to shorten the time of new drug development in order to deliver life-saving medicine to patients. Historically, drug substance materials to support the toxicology and early phase clinical study can only be manufactured after creating the final Master Cell Bank (MCB) of the clonally-derived cell line, which normally takes 1-2 years. With recent advances in cell line development, cell culture process and analytical technologies, generating more homogeneous bulk/mini-pool population with higher productivity has become a norm, thereby making it possible to shorten the timeline to initiate First in Human (FIH) trial by using bulk/mini-pool generated materials to support toxicology and FIH studies. In this study, two monoclonal antibodies of different subclasses (IgG1 and IgG4) were expressed from the mini-pool cells as well as clonally-derived cell lines generated from the same mini-pool. Cell growth, productivity, and product quality were compared between the materials generated from the mini-pool and clonally-derived cell line. The results demonstrate the similarity of the antibody products generated from mini-pool cells and clonally-derived cell lines from the same mini-pool, and strongly support the concept and feasibility of using antibody materials produced from mini-pool cultures for toxicology and FIH studies. The strategy to potentially shorten the FIH timeline is discussed. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:35:43.248139-05:
      DOI: 10.1002/btpr.2477
  • Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from biodiesel
           by-product and propionic acid by mutant strains of Pandoraea sp
    • Authors: Fabrício C. de Paula; Jonas Contiero, Carolina B. C. de Paula, José Gregório C. Gomez, Alexander Steinbüchel
      Abstract: Pandoraea sp. MA03 wild type strain was subjected to UV mutation in order to obtain mutants unable to grow on propionic acid (PA) but still able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from glycerol and PA at high 3HV yields. In shake flask experiments, mutant prp25 was selected from 52 mutants affected in the propionate metabolism exhibiting a conversion rate of PA into 3HV units of 0.78 gg−1. The utilization of crude glycerol (CG) plus PA or valeric acid resulted in a copolymer with 3HV contents varying from 21.9 to 30 mol% and 22.2 to 36.7 mol%, respectively. Fed-batch fermentations were performed using CG and PA and reached a 3HV yield of 1.16 gg−1, which is 86% of the maximum theoretical yield. Nitrogen limitation was a key parameter for polymer accumulation reaching up to 63.7% content and 18.1 mol% of 3HV. Henceforth, mutant prp25 is revealed as an additional alternative to minimize costs and support the P(3HB-co-3HV) production from biodiesel by-products. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:35:42.288946-05:
      DOI: 10.1002/btpr.2481
  • Upgrading laccase production and biochemical properties: Strategies and
    • Authors: Brandt Bertrand; Fernando Martínez Morales, María R. Trejo-Hernández
      Abstract: Improving laccases continues to be crucial in novel biotechnological developments and industrial applications, where they are concerned. This review breaks down and explores the potential of the strategies (conventional and modern) that can be used for laccase enhancement (increased production and upgraded biochemical properties such as stability and catalytic efficiency). The challenges faced with these approaches are briefly discussed. We also shed light on how these strategies merge and give rise to new options and advances in this field of work. Additionally, this paper seeks to serve as a guide for students and academic researchers interested in laccases. This document not only gives basic information on laccases, but also provides updated information on the state of the art of various technologies that are used in this line of investigation. It also gives the readers an idea of the areas extensively studied and the areas where there is still much left to be done. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:35:40.975023-05:
      DOI: 10.1002/btpr.2482
  • A non-chromatographic process for purification of secretory
           immunoglobulins from caprine whey
    • Authors: Alexander Matlschweiger; Gottfried Himmler, Clemens Linhart, Michael Harasek, Rainer Hahn
      Abstract: Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non-chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30 – 40 fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re-dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans-membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by-product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra-/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-05T18:18:20.196506-05:
      DOI: 10.1002/btpr.2466
  • Influence of mixed electrolytes on the adsorption of lysozyme, PEG and
           PEGylated lysozyme on a hydrophobic interaction chromatography resin
    • Authors: Eva Hackemann; Albert Werner, Hans Hasse
      Abstract: In a recent work1 the influence of mixed electrolytes on the adsorption of the macromolecules lysozyme, PEG and di-PEGylated lysozyme on a hydrophobic resin has been studied, but only at one overall ionic strength (3000 mM). The present work, therefore, extends these studies to other ionic strengths (2400 mM and 2700 mM), and explores the application of a model to predict the entire data set. The adsorbent is Toyopearl PPG-600M. The solvent is a 25 mM aqueous sodium phosphate buffer at pH 7.0. The studied salts are sodium chloride, ammonium sulfate, sodium sulfate and ammonium chloride. Both pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied at 25 °C. The loading of the adsorbent increases with increasing salt concentration for all macromolecules. Synergetic effects of the mixed electrolytes are observed. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:55:30.33573-05:0
      DOI: 10.1002/btpr.2474
  • Evaluation of the bony repair in rat cranial defect using near infrared
           reflectance spectroscopy and discriminant analysis
    • Authors: Leomir A. S. de Lima; Lana S. de S. Oliveira, Aurigena A. Araújo, Raimundo Fernandes de Araújo Junior, Kássio M. G. Lima
      Abstract: We set out to determine whether near infrared reflectance spectroscopy (NIRS) combined with principal component analysis–linear discriminant analysis (PCA–LDA) or, variable selection techniques employing successive projection algorithm (SPA) or genetic algorithm (GA) could evaluate the bone repair in cranial critical-size (5mm) defect after stimulation with collagen sponge scaffold and/or infrared low-level laser therapy directly on the local. Forty-five Winstar rats were divided into nine groups of five each, namely: group H – healthy, n=5 (without treatment and without cranial critical-size defect), (GI positive control – n=5, 21 days or n=5, 30 days) without treatment and with cranial critical-size defect; (GII-n=5, 21 days or n=5, 30 days) cranial critical-size defect filled with collagen sponge scaffold; (GIII–n=5, 21 days or n=5, 30 days) cranial critical-size defect submitted to low-level laser therapy; (GIV–n=5, 21 days or n=5, 30 days) cranial critical-size defect submitted to combined collagen sponge scaffold + low-level laser therapy treatment. In relation to the histological analysis, the collagen sponge scaffold + low-level laser therapy treatment group (GIV) 30 days showed the best result with the presence of secondary bone, immature bone (osteoid) and newly formed connective tissue (periosteum). GA–LDA model also successfully classified control class of the others classes. Thus, the results provided by the good-quality classification model revealed the feasibility of NIRS for application to evaluation of the wound healing in rat cranial defect, thanks to the short analysis time of a few seconds and non-destructive advantages of NIRS as an alternative approach for bone repair purposes. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:55:25.084268-05:
      DOI: 10.1002/btpr.2476
  • Evaluation of green solvents: Oil extraction from oleaginous yeast
           Lipomyces starkeyi using cyclopentyl methyl ether (CPME)
    • Authors: Kyle V. Probst; Michael D. Wales, Mary E. Rezac, Praveen V. Vadlani
      Abstract: Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1-propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1-propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:55:22.60629-05:0
      DOI: 10.1002/btpr.2473
  • Principles and approach to developing mammalian cell culture media for
           high cell density perfusion process leveraging established fed-batch media
    • Authors: Henry Lin; Robert Woodrow Leighty, Scott Godfrey, Samantha Boran Wang
      Abstract: Perfusion medium was successfully developed based on our fed-batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed-batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2x to increase medium depth. The medium osmolality was also optimized where we found ∼360 mOsm/kg was desirable resulting in a residual culture osmolality of ∼300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N and D were rebalanced to reduce lactate and ammonium levels, and increase the cell specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell-day yielding 1.2 g/L-day at steady-state. This perfusion process was then successfully scaled up to a 100L single-use bioreactor with an ATF6 demonstrating similar performance as a 2L bioreactor with an ATF2. Large volume handling challenges in our fed-batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug-and-play use with the bioreactor. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:50:34.963027-05:
      DOI: 10.1002/btpr.2472
  • A Strategy to Accelerate Protein Production from a Pool of Clones in
           Chinese Hamster Ovary Cells for Toxicology Studies
    • Authors: Zhilan Hu; Wendy Hsu, Abby Pynn, Domingos Ng, Donna Quicho, Yilma Adem, Zephie Kwong, Brad Mauger, John Joly, Bradley Snedecor, Michael W. Laird, Dana C. Andersen, Amy Shen
      Abstract: In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for Investigational New Drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies ("Pool for Tox strategy") during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately four months at Genentech), leading to a potential acceleration of four months for the IND submission. We explored the feasibility of the Pool for Tox (PFT) strategy with three antibodies, mAb1, mAb2, and mAb3 at the 2L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally we used this approach for two antibody molecules, mAb4 and mAb5 at Tox and GMP production. The materials from the Tox batch at 400L scale and three GMP batches at 2000L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally-derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:50:33.85476-05:0
      DOI: 10.1002/btpr.2467
  • Differential Scanning Calorimetry and Fluorimetry Measurements of
           Monoclonal Antibodies and Reference Proteins: Effect of Scanning Rate and
           Dye Selection
    • Authors: Brian E. Lang; Kenneth D. Cole
      Abstract: Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) were used to measure the transition temperatures of four proteins: RNase A, invertase, rituximab, and the NISTmAb (NIST Reference Material, RM 8671). The proteins were combined with several different fluorescent dyes for the DSF measurements. This study compares the results of DSC and DSF measurements of transition temperatures with different types of proteins, dye combinations, and thermal scan rates. Since protein unfolding is often influenced by kinetic effects, we measured the transition temperatures of the proteins using DSC over a range of temperature scan rates and compared them to the data obtained from DSF over comparable temperature scan rates. The results when the proteins were combined with Sypro Orange® and bis-ANS for the DSF measurements had the best correlations with the transition temperatures determined by calorimetry. The scan rate was found to be an important variable when comparing results between DSC and DSF. The van't Hoff enthalpy changes for the transitions were calculated from the DSC data by using a non-two state model and from the DSF values using a two-state model. The calculated van't Hoff enthalpy changes did not show a good correlation between the two methods. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:50:32.902742-05:
      DOI: 10.1002/btpr.2464
  • miR-143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype
           to enhance production of difficult-to-express proteins
    • Authors: Melanie Schoellhorn; Simon Fischer, Andreas Wagner, René Handrick, Kerstin Otte
      Abstract: In recent years, the number of complex but clinically effective biologicals such as multi-specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult-to-express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. In order to provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next-generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult-to-express proteins. In a previous study we identified miR-143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR-143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR-143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen-activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR-143-3p in CHO cells. Finally, siRNA-mediated knock-down of MAPK7 could be demonstrated to phenocopy pro-productive effects of miR-143. In summary, our data suggest that miR-143 might represent a novel genetic element to enhance production of difficult-to-express proteins in CHO cells which may be partly mediated by down-regulation of MAPK7. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:50:28.32717-05:0
      DOI: 10.1002/btpr.2475
  • Epitopes rationally selected through computational analyses induce T-cell
           proliferation in mice and are recognized by serum from individuals
           infected with Schistosoma mansoni
    • Authors: Marcelo D. Lopes; Flávio M. Oliveira, Ivan E. V. Coelho, Maria J. F. Passos, Clarice C. Alves, Alex G. Taranto, Moacyr C. Júnior, Luciana L. Santos, Cristina T. Fonseca, José A. F. P. Villar, Débora O. Lopes
      Abstract: Schistosomiasis is the second leading cause of death due to parasitic diseases in the world. Seeking an alternative for the control of disease, the World Health Organization funded the genome sequencing of the major species related to schistosomiasis to identify potential vaccines and therapeutic targets. Therefore, the aim of this work was to select T and B-cell epitopes from Schistosoma mansoni through computational analyses and evaluate the immunological potential of epitopes in vitro. Extracellular regions of membrane proteins from the Schistosoma mansoni were used to predict promiscuous epitopes with affinity to different human Major Histocompatibility Class II (MHCII) molecules by bioinformatics analysis. The three-dimensional structure of selected epitopes was constructed and used in molecular docking to verify the interaction with murine MHCII H2-IAb. In this process, four epitopes were selected and synthesized to assess their ability to stimulate proliferation of CD4+ T lymphocytes in mice splenocyte cultures. The results showed that Sm041370 and Sm168240 epitopes induced significant cell proliferation. Additionally, the four epitopes were used as antigens in the Indirect Enzyme-Linked Immunosorbent Assay (ELISA) to assess the recognition by serum from individuals infected with Schistosoma mansoni. Sm140560, Sm168240 and Sm041370 epitopes were recognized by infected individuals IgG antibodies. Therefore, Sm041370 and Sm168240 epitopes that stood out in in silico and in vitro analyses could be promising antigens in schistosomiasis vaccine development or diagnostic kits. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:50:26.289459-05:
      DOI: 10.1002/btpr.2463
  • Microbial cell disruption for improving lipid recovery using pressurized
           CO2: Role of CO2 solubility in cell suspension, sugar broth and spent
    • Authors: Md Shamim Howlader; William Todd French, Sara A. Shields-Menard, Marta Amirsadeghi, Magan Green, Neeraj Rai
      Abstract: The study of in situ gas explosion to lyse the triglyceride-rich cells involves the solubilization of gas (e.g. carbon dioxide, CO2) in lipid rich cells under pressure followed by a rapid decompression, which allows the gas inside the cell to rapidly expand and rupture the cell from inside out. The aim of this study was to perform the cell disruption using pressurized CO2 as well as to determine the solubility of CO2 in Rhodotorula glutinis cell suspension, sugar broth media, and spent media. Cell disruption of R. glutinis was performed at two pressures of 2000 and 3500 kPa, respectively at 295.2 K, and it was found from both scanning electron microscopy (SEM) and plate count that a substantial amount of R. glutinis was disrupted due to the pressurized CO2. We also found a considerable portion of lipid present in the aqueous phase after the disruption at P = 3500 kPa compared to control (no pressure) and P = 2000 kPa, which implied that more intracellular lipid was released due to the pressurized CO2. Solubility of CO2 in R. glutinis cell suspension was found to be higher than the solubility of CO2 in both sugar broth media and spent media. Experimental solubility was correlated using the extended Henry's law, which showed a good agreement with the experimental data. Enthalpy and entropy of dissolution of CO2 were found to -14.22 kJ.mol−1 and 48.10 kJ.mol−1.K−1, 9.64 kJ.mol−1 and 32.52 kJ.mol−1.K−1, and 7.50 kJ.mol−1 and 25.22 kJ.mol−1.K−1 in R. glutinis, spent media and sugar broth media, respectively. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:50:24.356167-05:
      DOI: 10.1002/btpr.2471
  • Selection of best conditions of inoculum preparation for optimum
           performance of the pigment production process by Talaromyces spp. using
           the Taguchi method
    • Authors: L. Morales-Oyervides; J.C. Oliveira, M.J. Sousa-Gallagher, A. Méndez-Zavala, J.C. Montañez
      Abstract: Process optimisation techniques increasingly need to be used early on in Research and Development of processes for new ingredients. There are different approaches and this paper illustrates the main issues at stake with a method that is an industry best practice, the Taguchi method, suggesting a procedure to assess the potential impact of its drawbacks. The Taguchi method has been widely used in various industrial sectors because it minimises the experimental requirements to define an optimum region of operation, which is particularly relevant when minimising variability is a target. However, it also has drawbacks, especially the intricate confoundings generated by the experimental designs used.This work reports a process optimisation of the synthesis of red pigments by a fungal strain, Talaromyces spp. using the Taguchi methodology and proposes an approach to assess from validation trials whether the conclusions can be accepted with confidence.The work focused on optimising the inoculum characteristics, the studied factors were spore age and concentration, agitation speed and incubation time. It was concluded that spore age was the most important factor for both responses, with optimum results at 5 days old, with the best other conditions being spores concentration, 100,000 (spores/mL); agitation 200 rpm, and incubation time 84 h. The interactive effects can be considered negligible and therefore this is an example where a simple experimental design approach was successful in speedily indicating conditions able to increase pigment production by 63% compared to an average choice of settings. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:45:33.724107-05:
      DOI: 10.1002/btpr.2470
  • Nanoparticles for Bone Tissue Engineering
    • Authors: Sílvia Vieira; Stephanie Vial, Rui L. Reis, J. Miguel Oliveira
      Abstract: Tissue Engineering (TE) envisions the creation of functional substitutes for damaged tissues through integrated solutions, where medical, biological and engineering principles are combined. Bone regeneration is one of the areas in which designing a model that mimics all tissue properties is still a challenge. The hierarchical structure and high vascularization of bone hampers a TE approach, especially in large bone defects. Nanotechnology can open-up a new era for TE, allowing the creation of nanostructures that are comparable in size to those appearing in natural bone. Therefore, nanoengineered systems are now able to more closely mimic the structures observed in naturally occurring systems, and it is also possible to combine several approaches, such drug delivery and cell labeling, within a single system. This review aims to cover the most recent developments on the use of different nanoparticles for bone TE, with emphasis on their application for scaffolds improvement; drug and gene delivery carriers, and labeling techniques. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:45:26.805635-05:
      DOI: 10.1002/btpr.2469
  • Low glucose concentrations within typical industrial operating conditions
           have minimal effect on the transcriptome of recombinant CHO cells
    • Authors: Yogender Kumar Gowtham; Christopher A. Saski, Sarah W. Harcum
      Abstract: Typically, mammalian cell culture medium contains high glucose concentrations that are analogous to diabetic levels in humans, suggesting that mammalian cells are cultivated in excessive glucose. Using RNA-Seq, this study characterized the Chinese hamster ovary (CHO) cell transcriptome under two glucose concentrations to assess the genetic effects associated with metabolic pathways, in addition to other global responses. The initial extracellular glucose concentrations used represented high (30 mM) and low (10 mM) glucose conditions, where at the time the transcriptomes were compared, the glucose concentrations were approximately 24 and 4.4 mM for the mid-exponential cultures, where 4.4 mM represents a common target concentration in the biopharmaceutical industry for controlled fed-batch cultures. A recombinant CHO cell line producing a monoclonal antibody was used, such that the impact on glycosylation genes could be evaluated. Relatively few genes were identified as being significantly different (FDR ≤ 0.01) between the high and low glucose conditions, e.g., only 575 genes, and only 40 of these genes had 2-fold or greater differences. Gene expression differences for glycolysis, TCA cycle, and glycosylation-related reactions were minimal and unlikely to have biological significance. This transcriptome study indicates that low glucose concentrations in the culture medium are unlikely to cause any biologically significant or detrimental changes to CHO cells at the transcriptome level. Furthermore, it is well-known that maintaining low glucose concentrations in fed-batch cultures can reduce lactate production, which in turn improves process outcomes. Taken together, the transcriptome data supports the continued development of low glucose-based processes to control lactate. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:40:30.433745-05:
      DOI: 10.1002/btpr.2462
  • Enrichment of apoplastic fluid with therapeutic recombinant protein for
           efficient biofarming
    • Authors: Aparajita Chatterjee; Narayan C. Das, Ankita Shrestha, Ahamed Khan, Sefali Acharya, Sumita Raha, Indu B. Maiti, Nrisingha Dey
      Abstract: Objective: For efficient biofarming we attempted to enrich plant interstitial fluid (IF)/apoplastic fluid with targeted recombinant therapeutic protein. We employed a synthetic human Glucocerebrosidase (GCB), a model biopharmaceutical protein gene in this study.Results: Twenty one Nicotiana varieties, species and hybrids were initially screened for individual IF recovery and based on the findings, we selected Nicotiana tabacum NN (S-9-6), Nicotiana tabacum nn (S-9-7) and Nicotiana benthamiana (S-6-6) as model plants for raising transgenic expressing GCB via Agrobacterium mediated transformation under the control of M24 promoter; GCB specific activity in each transgenic lines were analyzed and we observed higher concentration of recombinant GCB in IF of these transgenic lines (S-9-6, S-9-7, and S-6-6) in comparison to their concentration in crude leaf extracts.Conclusion: Recovery of valuable therapeutics in plant IF as shown in the present study holds great promise for promoting plant based biofarming. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:40:26.360106-05:
      DOI: 10.1002/btpr.2461
  • Evaluation of several protein A resins for application to multi-column
           chromatography for the rapid purification of fed-batch bioreactors
    • Authors: Nicolas-Julian Hilbold; Xavier Le Saoût, Eric Valery, Laurence Muhr, Jonathan Souquet, Alain Lamproye, Hervé Broly
      Abstract: Most of the existing production capacity is based on fed batch bioreactors. Thanks to the development of more efficient cell lines and the development of high performance culture media, cell productivity dramatically increased. In a manufacturing perspective, it is necessary to clear as quickly as possible the protein A capture step in order to respect the manufacturing agenda. This paper describes the methodology applied for the design of a multi-column chromatography process with the objective of purifying as quickly as possible 1,000 and 15,000L fed-batch bioreactors. Several recent and reference protein A resins are compared based on characteristic values obtained from breakthrough curves. The importance and relevance of resin parameters are explained, and purposely simple indicators are proposed to quickly evaluate the potential of each candidate. Based on simulation data, the optimum BioSC systems associated with each resin are then compared. The quality of the elution delivered by each resin is also compared to complete the assessment. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-03T11:40:23.086904-05:
      DOI: 10.1002/btpr.2465
  • Design, construction, and optimization of a novel, modular, and scalable
           incubation chamber for continuous viral inactivation
    • Authors: Raquel Orozco; Scott Godfrey, Jon Coffman, Linus Amarikwa, Stephanie Parker, Lindsay Hernandez, Chinenye Wachuku, Ben Mai, Brian Song, Shashidhar Hoskatti, Jinkeng Asong, Parviz Shamlou, Cameron Bardliving, Marcus Fiadeiro
      Abstract: We designed, built or 3D printed, and screened tubular reactors that minimize axial dispersion to serve as incubation chambers for continuous virus inactivation of biological products. Empirical residence time distribution data were used to derive each tubular design's volume equivalent to a theoretical plate (VETP) values at a various process flow rates. One design, the Jig in a Box (JIB), yielded the lowest VETP, indicating optimal radial mixing and minimal axial dispersion. A minimum residence time (MRT) approach was employed, where the MRT is the minimum time the product spends in the tubular reactor. This incubation time is typically 60 minutes in a batch process. We provide recommendations for combinations of flow rates and device dimensions for operation of the JIB connected in series that will meet a 60-min MRT. The results show that under a wide range of flow rates and corresponding volumes, it takes 75 ± 3 min for 99% of the product to exit the reactor while meeting the 60-min MRT criterion and fulfilling the constraint of keeping a differential pressure drop under 5 psi. Under these conditions, the VETP increases slightly from 3 to 5 mL though the number of theoretical plates stays constant at about 1326 ± 88. We also demonstrated that the final design volume was only 6% ± 1% larger than the ideal plug flow volume. Using such a device would enable continuous viral inactivation in a truly continuous process or in the effluent of a batch chromatography column. Viral inactivation studies would be required to validate such a design. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-03-17T02:20:39.661555-05:
      DOI: 10.1002/btpr.2442
  • Fine-tuning sortase-mediated immobilization of protein layers on surfaces
           using sequential deprotection and coupling
    • Authors: Maryam Raeeszadeh-Sarmazdeh; Ranganath Parthasarathy, Eric T. Boder
      Abstract: Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer-by-layer protein attachment. Here, we report a technique for controlling enzyme-mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase-cleavable peptide sequence was inserted at the N-terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A-mediated protein immobilization on the surface layer-by-layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N-terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-03-11T00:05:41.122316-05:
      DOI: 10.1002/btpr.2449
  • CHO Cells adapted to inorganic phosphate limitation show higher growth and
           higher pyruvate carboxylase flux in phosphate replete conditions
    • Authors: Vishwanathgouda Maralingannavar; Dharmeshkumar Parmar, Chetan Gadgil, Venkateswarlu Panchagnula, Mugdha Gadgil, Tejal Pant
      Abstract: Inorganic phosphate (Pi) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to Pi limitation have been reported in E. coli. Given the essential role of Pi, adaptation to Pi limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting Pi conditions for 75 days. Not surprisingly, adapted cells showed better survival under Pi limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U-13C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to Pi limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-03-11T00:05:29.720427-05:
      DOI: 10.1002/btpr.2450
  • Applied in situ product recovery in ABE fermentation
    • Authors: Victoria Outram; Carl-Axel Lalander, Jonathan G. M. Lee, E. Timothy Davies, Adam P. Harvey
      Abstract: The production of biobutanol is hindered by the product's toxicity to the bacteria, which limits the productivity of the process. In situ product recovery of butanol can improve the productivity by removing the source of inhibition. This paper reviews in situ product recovery techniques applied to the acetone butanol ethanol fermentation in a stirred tank reactor. Methods of in situ recovery include gas stripping, vacuum fermentation, pervaporation, liquid–liquid extraction, perstraction, and adsorption, all of which have been investigated for the acetone, butanol, and ethanol fermentation. All techniques have shown an improvement in substrate utilization, yield, productivity or both. Different fermentation modes favored different techniques. For batch processing gas stripping and pervaporation were most favorable, but in fed-batch fermentations gas stripping and adsorption were most promising. During continuous processing perstraction appeared to offer the best improvement. The use of hybrid techniques can increase the final product concentration beyond that of single-stage techniques. Therefore, the selection of an in situ product recovery technique would require comparable information on the energy demand and economics of the process. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-03-10T06:29:04.776911-05:
      DOI: 10.1002/btpr.2446
  • Comparison of Spectroscopy Technologies for Improved Monitoring of Cell
           Culture Processes in Miniature Bioreactors
    • Authors: Ruth C. Rowland-Jones; Frans van den Berg, Andrew J. Racher, Elaine B. Martin, Colin Jaques
      Abstract: Cell culture process development requires the screening of large numbers of cell lines and process conditions. The development of miniature bioreactor systems has increased the through-put of such studies; however, there are limitations with their use. One important constraint is the limited number of offline samples that can be taken compared to those taken for monitoring cultures in larger-scale bioreactors. The small volume of miniature bioreactor cultures (15mL) is incompatible with the large sample volume (600µL) required for bioanalysers routinely used. Spectroscopy technologies may be used to resolve this limitation. The purpose of this study was to compare the use of NIR, Raman and 2D-fluorescence to measure multiple analytes simultaneously in volumes suitable for daily monitoring of a miniature bioreactor system. A novel design-of-experiment approach is described that utilizes previously analysed cell culture supernatant to assess metabolite concentrations under various conditions whilst providing optimal coverage of the desired design space. Multivariate data analysis techniques were used to develop predictive models. Model performance was compared to determine which technology is more suitable for this application. 2D-fluorescence could more accurately measure ammonium concentration (RMSECV 0.031g.L−1) than Raman and NIR. Raman spectroscopy however, was more robust at measuring lactate and glucose concentrations (RMSECV 1.11 and 0.92g.L−1 respectively) than the other two techniques. The findings suggest that Raman spectroscopy is more suited for this application than NIR and 2D-fluorescence. The implementation of Raman spectroscopy increases at-line measuring capabilities, enabling daily monitoring of key cell culture components within miniature bioreactor cultures. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-08T03:40:25.594031-05:
      DOI: 10.1002/btpr.2459
  • A novel cholesterol/lipid delivery system for murine myeloma cell lines
    • Authors: Gaurav Chauhan; Albert E. Schmelzer
      Abstract: Murine myeloma NS0 cells are cholesterol-dependent auxotrophs and require externally provided cholesterol for sustained growth. Traditionally, cholesterol is provided to these cells by supplementing cell culture media with a concentrated solution of cholesterol and other water insoluble components dissolved in 200-proof ethanol. However, the solubility of cholesterol in ethanol is limited, and for processes requiring large amounts of cholesterol, the consequential increase in added ethanol may negatively impact cell growth. Additionally, the flammability of 200-proof ethanol may restrict the preparation scale and storage volumes at a large-scale facility, thus resulting in a more complex preparation procedure due to safety guidelines. This study proposes 1-propanol as an alternative solvent, which can dissolve up to 40 g L−1 of cholesterol along with other water insoluble components, as compared to ethanol, which can dissolve up to 10 g L−1 of the same. A concentrated formulation simplifies the preparation method and ameliorates the procedural and operational challenges, as well as reduces the total amount of alcohol added to a cell culture by ∼80% when compared to the ethanolic solution, to deliver the same amount of cholesterol, thereby significantly minimizing alcohol exposure to the cells and mitigating the fire hazards at a large-scale facility. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-03-07T23:45:37.54935-05:0
      DOI: 10.1002/btpr.2441
  • Bioreactor scale up and protein product quality characterization of
           piggyBac transposon derived CHO pools
    • Authors: Yashas Rajendra; Sowmya Balasubramanian, Robert B. Peery, James R. Swartling, Neil A. McCracken, Dawn L. Norris, Christopher C. Frye, Gavin C. Barnard
      Abstract: Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2–10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-03-07T23:45:25.864712-05:
      DOI: 10.1002/btpr.2447
  • Large-scale synthesis of tert-butyl
           (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by a stereoselective carbonyl
           reductase with high substrate concentration and product yield
    • Authors: Zhi-Qiang Liu; Zhong-Liang Hu, Xiao-Jian Zhang, Xiao-Ling Tang, Feng Cheng, Ya-Ping Xue, Ya-Jun Wang, Lin Wu, Dan-Kai Yao, Yi-Teng Zhou, Yu-Guo Zheng
      Abstract: To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient for (3R,5S)-CDHH production. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mmol L/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-07T19:11:22.29895-05:0
      DOI: 10.1002/btpr.2460
  • Antioxidant effect of thiazolidine molecules in cell culture media
           improves stability and performance
    • Authors: Jennifer Kuschelewski; Alisa Schnellbaecher, Sascha Pering, Maria Wehsling, Aline Zimmer
      Abstract: The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies.Results presented in this study indicate that 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid and 2-(2-carboxyethyl)-1,3-thiazolidine-2,4-dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha-ketoglutarate respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, L-methionine (met) and L-tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed-batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti-oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant-free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-07T19:11:15.092568-05:
      DOI: 10.1002/btpr.2458
  • Permeability enhancement of Escherichia coli by single-walled carbon
           nanotube treatment
    • Authors: Abdollah Mosleh; Anna Heintz, Ki-Taek Lim, Jin-Woo Kim, Robert Beitle
      Abstract: This research investigated the use of single-walled carbon nanotubes (SWNTs) as an additive to increase the permeability of a bacterial cell wall. Recombinant Escherichia coli BL21 (DE3) that expressed β-lactamase were exposed to SWNTs under various levels of concentration and agitation. Activity of β-lactamase in the culture fluid and Transmission Electron Microscopy (TEM) were used to determine the amount of released protein, and visually examine the permeability enhancement of the cells. It was found that β-lactamase release in the culture fluid occurred in a dose dependent manner with treatment by SWNTs and was also dependent on agitation rate. Based on TEM, this treatment successfully caused an increase in permeability without significant damage to the cell wall. Consequently, SWNTs can be used as an enhancement agent to cause the release of intracellular proteins. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-06T11:05:25.603585-05:
      DOI: 10.1002/btpr.2443
  • Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent
           Metabolic Fingerprints in Perfusion Culture
    • Authors: Daniel J. Karst; Robert F. Steinhoff, Marie R. G. Kopp, Elisa Serra, Miroslav Soos, Renato Zenobi, Massimo Morbidelli
      Abstract: Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-03-03T00:00:26.208315-05:
      DOI: 10.1002/btpr.2421
  • Effects of lysosomal biotherapeutic recombinant protein expression on cell
           stress and protease and general host cell protein (HCP) release in Chinese
           hamster ovary cells
    • Authors: Damiano Migani; C. Mark Smales, Daniel G. Bracewell
      Abstract: Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterised by GAA deficiency in the cell lysosomes1. The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines [GAA, mAb and Null] capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co-eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-01T18:26:05.302358-05:
      DOI: 10.1002/btpr.2455
  • Antibody Purification from CHO Cell Supernatant Using New Multimodal
    • Authors: Juan Wang; Jinxiang Zhou, Yogender K. Gowtham, Sarah W. Harcum, Scott M. Husson
      Abstract: This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG1 following a size-exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two step MMM chromatography process achieved high selectivity for capturing hIgG1 from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was>98% and HCP levels were 
      PubDate: 2017-03-01T06:15:47.00014-05:0
      DOI: 10.1002/btpr.2454
  • A Novel Soft Sensor Approach for Estimating Individual Biomass in Mixed
    • Authors: Kyle A. Stone; Devarshi Shah, Min Hea Kim, Nathan R. M. Roberts, Q. Peter He, Jin Wang
      Abstract: Due to many advantages associated with mixed cultures, the application of mixed cultures in biotechnology has expanded rapidly in recent years. At the same time, many challenges remain for effective mixed culture applications. One obstacle is how to efficiently and accurately monitor the individual cell populations. The current approaches on individual cell mass quantification are suitable for off-line, infrequent characterization. In this work, we propose a fast and accurate ‘soft sensor' approach for estimating individual cell concentrations in mixed cultures. The proposed approach utilizes optical density scanning spectrum of a mixed culture sample measured by a spectrophotometer over a range of wavelengths. A multivariate linear regression method, partial least squares or PLS, is applied to correlate individual cell concentrations to the spectrum. Three experimental case studies are used to examine the performance of the proposed soft sensor approach. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-01T06:15:41.919127-05:
      DOI: 10.1002/btpr.2453
  • A novel extracellular low-temperature active phytase from Bacillus
           aryabhattai RS1 with potential application in plant growth
    • Authors: Authors Moushree Pal Roy; Subhabrata Datta, Shilpi Ghosh
      Abstract: Bacillus aryabhattai RS1 isolated from rhizosphere produced an extracellular, low temperature active phytase. The cultural conditions for enzyme production were optimized to obtain 35 U ml−1 of activity. Purified phytase had specific activity and molecular weight of 72.97 U mg−1 and ∼40 kDa, respectively. The enzyme was optimally active at pH 6.5 and 40°C and was highly specific to phytate. It exhibited higher catalytic activity at low temperature, retaining over 40% activity at 10°C. Phytase was more thermostable in presence of Ca2+ ion and retained 100% residual activity on pre-incubation at 20-50°C for 30 min. Partial phytase encoding gene, phyB (816 bp) was cloned and sequenced. The encoded amino acid sequence (272 aa) contained two conserved motifs, DA[A/T/E]DDPA[I/L/V]W and NN[V/I]D[I/L/V]R[Y/D/Q] of β-propellar phytase (BPP) and had lower sequence homology with other Bacillus phytases, indicating its novelty. Phytase and the bacterial inoculum were effective in improving germination and growth of chickpea seedlings under phosphate limiting condition. Moreover, the potential applications of the enzyme with relatively high activity at lower temperatures (20-30°C) could also be extended to aquaculture and food processing. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-01T06:15:34.168313-05:
      DOI: 10.1002/btpr.2452
  • Effects of ferrous iron on the performance and microbial community in
           aerobic granular sludge in relation to nutrient removal
    • Authors: Gulsum Yilmaz; Ender Cetin, Umit Bozkurt, Karin Aleksanyan Magden
      Abstract: Lab-scale experiments were conducted to investigate the effects of ferrous iron on nutrient removal performance and variations in the microbial community inside aerobic granular sludge for 408 days. Two reactors were simultaneously operated, one without added ferrous iron (SBR1), and one with 10 mg Fe2+ L−1 of added ferrous iron (SBR2). 1 mg Fe2+ L−1 of added ferrous iron was applied to SBR1 starting from the 191st day to observe the resulting variations in the nutrient removal performance and the microbial community. The results show that ammonia-oxidizing bacteria (AOB) could not oxidize ammonia due to a lack of iron compounds, but they could survive in the aerobic granular sludge. Limited ferrous iron addition encouraged nitrification. Enhanced biological phosphorus removal (EBPR) from both reactors could not be maintained regardless of the amount of ferrous iron that was applied. EBPR was established in both reactors when the concentration of mixed liquor suspended solid (MLSS) and the percentage of Accumulibacteria increased. 10 mg Fe2+ L−1 of added ferrous iron had a relatively adverse effect on the growth of AOB species compared to 1 mg L−1 of added ferrous iron, but it encouraged the growth of Nitrospira sp. and Accumulibacteria, which requires further study. It could be said that the compact and stable structure of aerobic granular sludge preserved AOB and NOB from Fe-deficient conditions, and wash-out during the disintegration period. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-01T06:15:33.218398-05:
      DOI: 10.1002/btpr.2456
  • Hydrogel-Based Three-Dimensional Cell Culture for Organ-on-a-Chip
    • Authors: Seung Hwan Lee; Kyu Young Shim, Bumsang Kim, Jong Hwan Sung
      Abstract: Recent studies have reported that three-dimensionally cultured cells have more physiologically relevant functions than two-dimensionally cultured cells. Cells are three-dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ-on-a-chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation-based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ-on-a-chip. Last, we also discuss the challenges of hydrogel-based 3D cell culture techniques and propose future directions. This article is protected by copyright. All rights reserved.
      PubDate: 2017-03-01T06:12:01.485026-05:
      DOI: 10.1002/btpr.2457
  • Shock treatment of corn stover
    • Authors: Austin Bond; Hema Rughoonundun, Eric Petersen, Carol Holtzapple, Mark Holtzapple
      Abstract: Corn stover digestibility was enhanced via shock treatment. A slurry of lime-treated corn stover was placed in a partially filled closed vessel. From the ullage space, either a shotgun shell was fired into the slurry, or a gas mixture was detonated. Various conditions were tested (i.e., pressures, depth, solids concentrations, gas mixtures). A high pressurization rate (108,000 MPa/s shotgun shells; 4,160,000 MPa/s hydrogen/oxygen detonation) was the only parameter that improved enzymatic digestibility. Stoichiometric propane/air deflagration had a low pressurization rate (37.2 MPa/s) and did not enhance enzymatic digestibility. Without shock, enzymatic conversion of lime-treated corn stover was 0.80 g glucan digested/g glucan fed with an enzyme loading of 46.7 mg protein/g glucan. With shock, the enzyme loading was reduced by ∼2× while maintaining the same conversion. Detonations are extraordinarily fast; rapidly cycling three small vessels (0.575 m3 each) every 7.5 s enables commercially relevant shock treatment (2,000 tone/day). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-28T01:16:14.549875-05:
      DOI: 10.1002/btpr.2437
  • A robust feeding strategy to maintain set-point glucose in mammalian
           fed-batch cultures when input parameters have a large error
    • Authors: Viktor Konakovsky; Christoph Clemens, Markus Michael Müller, Jan Bechmann, Christoph Herwig
      Abstract: Industrial CHO cell cultures run under fed-batch conditions are required to be controlled in particular ranges of glucose, while glucose is constantly consumed and must be replenished by a feed. The most appropriate feeding rate is ideally stoichiometric and adaptive in nature to balance the dynamically changing rate of glucose consumption. However, high errors in biomass and glucose estimation as well as limited knowledge of the true metabolic state challenge the control strategy. In this contribution, we take these errors into account and simulate the output with uncertainty trajectories in silico in order to control glucose concentration. Other than many control strategies, which require parameter estimation, our assumptions are founded on two pillars: (i) first principles and (ii) prior knowledge about the variability of fed-batch CHO cell culture. The algorithm was exposed to an in-silico Design of Experiments (DoE), in which variations of parameters were changed simultaneously, such as clone-specific behavior, precision of equipment and desired control range used. The results demonstrate that our method achieved the target of holding the glucose concentration within an acceptable range. A robust and sufficient level of control could be demonstrated even with high errors for biomass or metabolic state estimation. In a time where blockbuster drugs are queuing up for time slots of their production, this transferable control strategy that is independent of tedious establishment runs may be a decisive advantage for rapid implementation during technology transfer and scale up and decrease in campaign change over time. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-28T01:16:09.893101-05:
      DOI: 10.1002/btpr.2438
  • Towards the design of an optimal strategy for the production of ergosterol
           from Saccharomyces cerevisiae yeasts
    • Authors: Jan Náhlík; Pavel Hrnčiřík, Jan Mareš, Mojmír Rychtera, Christopher A. Kent
      Abstract: The total yield of ergosterol produced by the fermentation of the yeast Saccharomyces cerevisiae depends on the final amount of yeast biomass and the ergosterol content in the cells. At the same time ergosterol purity—defined as percentage of ergosterol in the total sterols in the yeast—is equally important for efficient downstream processing. This study investigated the development of both the ergosterol content and ergosterol purity in different physiological (metabolic) states of the microorganism S. cerevisiae with the aim of reaching maximal ergosterol productivity. To expose the yeast culture to different physiological states during fermentation an on-line inference of the current physiological state of the culture was used. The results achieved made it possible to design a new production strategy, which consists of two preferable metabolic states, oxidative-fermentative growth on glucose followed by oxidative growth on glucose and ethanol simultaneously. Experimental application of this strategy achieved a value of the total efficiency of ergosterol production (defined as product of ergosterol yield coefficient and volumetric productivity), 103.84 × 10−6 g L−1h−1, more than three times higher than with standard baker's yeast fed-batch cultivations, which attained in average 32.14 × 10−6 g L−1h−1. At the same time the final content of ergosterol in dry biomass was 2.43%, with a purity 86%. These results make the product obtained by the proposed control strategy suitable for effective down-stream processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-22T02:40:37.615369-05:
      DOI: 10.1002/btpr.2436
  • Maximizing the functional lifetime of Protein A resins
    • Authors: Jennifer Zhang; Sethu Siva, Ryan Caple, Rob Gronke, Sanchayita Ghose
      Abstract: Protein A chromatography is currently the industry gold-standard for monoclonal antibody and Fc-fusion protein purification. The high cost of Protein A however makes resin lifetime and resin reuse an important factor for process economics. Typical resin lifetime studies performed in the industry usually examine the effect of resin re-use on binding capacity, yield, and product quality without answering the fundamental question of what is causing the decrease in performance. A two part mechanistic study was conducted in an attempt to decouple the effect of the two possible factors (resin hydrolysis and/or degradation vs. resin fouling) on column performance over lifetime of the most commonly used base-stable Protein A resins (MabSelect SuRe and MabSelect SuRe LX). We examined the change in binding capacity as a function of sodium hydroxide concentration (rate of hydrolysis), temperature, and stabilizing additives. Additionally, resin extraction studies and product cycling studies were conducted to determine cleaning effectiveness (resin fouling) of various cleaning strategies. Sodium hydroxide based cleaning solutions were shown to be more effective at preventing resin fouling. On the other hand, cold temperature and the use of stabilizing additives in conjunction with sodium hydroxide were found to be beneficial in minimizing the rate of Protein A ligand hydrolysis. An effective and robust cleaning strategy is presented here to maximize resin lifetime and thereby the number of column cycles for future manufacturing processes. This article is protected by copyright. All rights reserved.
      PubDate: 2017-02-20T07:25:23.58947-05:0
      DOI: 10.1002/btpr.2448
  • Evaluation of Heavy Chain C-terminal Deletions on Productivity and Product
           Quality of Monoclonal Antibodies in Chinese Hamster Ovary (CHO) Cells
    • Authors: Zhilan Hu; Danming Tang, Shahram Misaghi, Guoying Jiang, Christopher Yu, Mandy Yim, David Shaw, Brad Snedecor, Michael Laird, Amy Shen
      Abstract: Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK). Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. This article is protected by copyright. All rights reserved.
      PubDate: 2017-02-11T03:40:44.541975-05:
      DOI: 10.1002/btpr.2444
  • Control of Chitin Nanofiber Production by the Lipid-Producing Diatom
           Cyclotella sp. through Fed-Batch Addition of Dissolved Silicon and Nitrate
           in a Bubble-Column Photobioreactor
    • Authors: Omar Chiriboga; Gregory L. Rorrer
      Abstract: Diatoms are single-celled algae that make cell walls of nanopatterned biogenic silica called frustules through metabolic uptake of dissolved silicon and its templated condensation into biosilica. The centric marine diatom Cyclotella sp. also produces intracellular lipids and the valued co-product chitin, an N-acetyl glucosamine biopolymer that is extruded from selected frustule pores as pure nanofibers. The goal of this study was to develop a nutrient feeding strategy to control the production of chitin nanofibers from Cyclotella with the co-production of biofuel lipids. A two-stage phototrophic cultivation process was developed where Stage I set the cell suspension to a silicon-starved state under batch operation, and Stage II continuously added silicon and nitrate to the silicon-starved cells to enable one more cell doubling to 4 ⋅ 106 cells/mL. The silicon delivery rate was set to enable a silicon-limited cell division rate under cumulative delivery of 0.8 mM Si and 1.2 mM nitrate (1.5:1 mol N/mol Si) over a 4-14 day addition period. In Stage II, both cell number and chitin production were linear with time. Cell number and the specific chitin production rate increased linearly with increasing silicon delivery rate to achieve cumulative product yields of 12 ± 1 mg chitin/109 cells and 33 ± 3 mg lipid/109 cells. Therefore, chitin production is controlled through cell division, which is externally controlled through silicon delivery. Lipid production was not linearly correlated to silicon delivery and occurred primarily during Stage I, just after the complete co-consumption of both dissolved silicon and nitrate. This article is protected by copyright. All rights reserved.
      PubDate: 2017-02-11T03:35:30.726789-05:
      DOI: 10.1002/btpr.2445
  • Cell-free translational screening of an expression sequence tag library of
           Clonorchis sinensis for novel antigen discovery
    • Authors: Devi Kasi; Christy Catherine, Seung-Won Lee, Kyung-Ho Lee, Yu Jung Kim, Myeong Ro Lee, Jung Won Ju, Dong-Myung Kim
      Abstract: The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-06T11:00:51.949431-05:
      DOI: 10.1002/btpr.2440
  • Taming hyperactive hDNase I: Stable inducible expression of a hyperactive
           salt- and actin-resistant variant of human deoxyribonuclease I in CHO
    • Authors: Cynthia Lam; Lydia Santell, Blair Wilson, Mandy Yim, Salina Louie, Danming Tang, David Shaw, Pamela Chan, Robert A. Lazarus, Brad Snedecor, Shahram Misaghi
      Abstract: While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt- and Actin-Resistant (HSAR-hDNase I) have been developed previously. However, constitutive expression of HSAR-hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR-hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR-hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR-hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR-hDNase I was both hyperactive and resistant to inhibition by salt and G-actin, resulting in an enzyme having ca. 10-fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-06T11:00:50.044939-05:
      DOI: 10.1002/btpr.2439
  • Characterization of TAP Ambr 250 disposable bioreactors, as a reliable
           scale-down model for biologics process development
    • Authors: Ping Xu; Colleen Clark, Todd Ryder, Colleen Sparks, Jiping Zhou, Michelle Wang, Reb Russell, Charo Scott
      Abstract: Demands for development of biological therapies is rapidly increasing, as is the drive to reduce time to patient. In order to speed up development, the disposable Automated Microscale Bioreactor (Ambr 250) system is increasingly gaining interest due to its advantages, including highly automated control, high throughput capacity, and short turnaround time. Traditional early stage upstream process development conducted in 2 - 5 L bench-top bioreactors requires high foot-print, and running cost. The establishment of the Ambr 250 as a scale-down model leads to many benefits in process development. In this study, a comprehensive characterization of mass transfer coefficient (kLa) in the Ambr 250 was conducted to define optimal operational conditions. Scale-down approaches, including dimensionless volumetric flow rate (vvm), power per unit volume (P/V) and kLa have been evaluated using different cell lines. This study demonstrates that the Ambr 250 generated comparable profiles of cell growth and protein production, as seen at 5-L and 1000-L bioreactor scales, when using kLa as a scale-down parameter. In addition to mimicking processes at large scales, the suitability of the Ambr 250 as a tool for clone selection, which is traditionally conducted in bench-top bioreactors, was investigated. Data show that cell growth, productivity, metabolite profiles, and product qualities of material generated using the Ambr 250 were comparable to those from 5-L bioreactors. Therefore, Ambr 250 can be used for clone selection and process development as a replacement for traditional bench-top bioreactors minimizing resource utilization during the early stages of development in the biopharmaceutical industry. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-02-06T11:00:47.17839-05:0
      DOI: 10.1002/btpr.2417
  • Screening different host cell lines for the dynamic production of measles
    • Authors: Tanja A. Grein; Felix Schwebel, Marco Kress, Daniel Loewe, Hauke Dieken, Denise Salzig, Tobias Weidner, Peter Czermak
      Abstract: Measles virus (MV) has a natural affinity for cancer cells and oncolytic MV preparations have therefore been investigated in several clinical trials as a potential treatment for cancer. The main bottleneck in the administration of oncolytic MV to cancer patients is the production process, because very large doses of virus particles are required for each treatment. Here, we investigated the productivity of different host cells and found that a high infection efficiency did not necessarily result in high virus yields because virus release is also dependent on the host cell. As well as producing large numbers of active MV particles, host cells must perform well in dynamic cultivation systems. In screening experiments, the highest productivity was achieved by Vero and BJAB cells, but only the Vero cells maintained their high virus productivity when transferred to a stirred tank reactor. We used dielectric spectroscopy as an online monitoring system to control the infection and harvest times, which are known to be critical process parameters. The precise control of these parameters allowed us to achieve higher virus titers with Vero cells in a stirred tank reactor than in a static cultivation system based on T-flasks, with maximum titers of up to 1011 TCID50 ml−1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-06T11:00:43.930692-05:
      DOI: 10.1002/btpr.2432
  • Effect of guanidine and arginine on protein–ligand interactions in
           multimodal cation-exchange chromatography
    • Authors: Siddharth Parimal; Shekhar Garde, Steven M. Cramer
      Abstract: The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse-grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse-grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-02-03T00:41:01.258087-05:
      DOI: 10.1002/btpr.2419
  • Optimization of biopharmaceutical downstream processes supported by
           mechanistic models and artificial neural networks
    • Authors: Silvia M. Pirrung; Luuk A. M. van der Wielen, Ruud F. W. C. van Beckhoven, Emile J. A. X. van de Sandt, Michel H. M. Eppink, Marcel Ottens
      Abstract: Downstream process development is a major area of importance within the field of bioengineering. During the design of such a downstream process, important decisions have to be made regarding the type of unit operations as well as their sequence and their operating conditions. Current computational approaches addressing these issues either show a high level of simplification or struggle with computational speed. Therefore, this article presents a new approach that combines detailed mechanistic models and speed-enhancing artificial neural networks. This approach was able to simultaneously optimize a process with three different chromatographic columns toward yield with a minimum purity of 99.9%. The addition of artificial neural networks greatly accelerated this optimization. Due to high computational speed, the approach is easily extendable to include more unit operations. Therefore, it can be of great help in the acceleration of downstream process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-03T00:40:52.756031-05:
      DOI: 10.1002/btpr.2435
  • Design and control of integrated chromatography column sequences
    • Authors: Niklas Andersson; Anton Löfgren, Marianne Olofsson, Anton Sellberg, Bernt Nilsson, Peter Tiainen
      Abstract: To increase the productivity in biopharmaceutical production, a natural step is to introduce integrated continuous biomanufacturing which leads to fewer buffer and storage tanks, smaller sizes of integrated unit operations, and full automation of the operation. The main contribution of this work is to illustrate a methodology for design and control of a downstream process based on integrated column sequences. For small scale production, for example, pre-clinical studies, integrated column sequences can be implemented on a single chromatography system. This makes for a very efficient drug development platform. The proposed methodology is composed of four steps and is governed by a set of tools, that is presented, that makes the transition from batch separations to a complete integrated separation sequence as easy as possible. This methodology, its associated tools and the physical implementation is presented and illustrated on a case study where the target protein is separated from impurities through an integrated four column sequence. This article shows that the design and control of an integrated column sequence was successfully implemented for a tertiary protein separation problem. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-03T00:40:45.729488-05:
      DOI: 10.1002/btpr.2434
  • Economic improvement of continuous pharmaceutical production via the
           optimal control of a multifeed bioreactor
    • Authors: Jonathan P. Raftery; Melanie R. DeSessa, M. Nazmul Karim
      Abstract: Projections on the profitability of the pharmaceutical industry predict a large amount of growth in the coming years. Stagnation over the last 20 years in product development has led to the search for new processing methods to improve profitability by reducing operating costs or improving process productivity. This work proposes a novel multifeed bioreactor system composed of independently controlled feeds for substrate(s) and media used that allows for the free manipulation of the bioreactor supply rate and substrate concentrations to maximize bioreactor productivity and substrate utilization while reducing operating costs. The optimal operation of the multiple feeds is determined a priori as the solution of a dynamic optimization problem using the kinetic models describing the time-variant bioreactor concentrations as constraints. This new bioreactor paradigm is exemplified through the intracellular production of beta-carotene using a three feed bioreactor consisting of separate glucose, ethanol and media feeds. The performance of a traditional bioreator with a single substrate feed is compared to that of a bioreactor with multiple feeds using glucose and/or ethanol as substrate options. Results show up to a 30% reduction in the productivity with the addition of multiple feeds, though all three systems show an improvement in productivity when compared to batch production. Additionally, the breakeven selling price of beta-carotene is shown to decrease by at least 30% for the multifeed bioreactor when compared to the single feed counterpart, demonstrating the ability of the multifeed reactor to reduce operating costs in bioreactor systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-02-03T00:40:41.471256-05:
      DOI: 10.1002/btpr.2433
  • Transient and stable CHO expression, purification and characterization of
           novel hetero-dimeric bispecific IgG antibodies
    • Authors: Yashas Rajendra; Robert B. Peery, Maria D. Hougland, Gavin C. Barnard, Xiufeng Wu, Jonathan R. Fitchett, Michael Bacica, Stephen J. Demarest
      Abstract: IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or “hetero-mAbs” using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-02-03T00:40:34.479241-05:
      DOI: 10.1002/btpr.2414
  • Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108
           for enhanced enzymatic hydrolysis and efficient ethanol production
    • Authors: Badal C. Saha; Gregory J. Kennedy, Nasib Qureshi, Michael A. Cotta
      Abstract: Biological pretreatment of lignocellulosic biomass by white-rot fungus can represent a low-cost and eco-friendly alternative to harsh physical, chemical, or physico-chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid-state cultivation of corn stover with Phlebia brevispora NRRL-13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed-batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed-batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL-13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-28T09:36:34.657044-05:
      DOI: 10.1002/btpr.2420
  • Spore-displayed enzyme cascade with tunable stoichiometry
    • Authors: Long Chen; Ashok Mulchandani, Xin Ge
      Abstract: Taking the advantages of inert and stable nature of endospores, we developed a biocatalysis platform for multiple enzyme immobilization on Bacillus subtilis spore surface. Among B. subtilis outer coat proteins, CotG mediated a high expression level of Clostridium thermocellum cohesin (CtCoh) with a functional display capability of ∼104 molecules per spore of xylose reductase-C. thermocellum dockerin fusion protein (XR-CtDoc). By co-immobilization of phosphite dehydrogenase (PTDH) on spore surface via Ruminococcus flavefaciens cohesin-dockerin modules, regeneration of NADPH was achieved. Both xylose reductase (XR) and PTDH exhibited enhanced stability upon spore surface display. More importantly, by altering the copy numbers of CtCoh and RfCoh fused with CotG, the molar ratio between immobilized enzymes was adjusted in a controllable manner. Optimization of spore-displayed XR/PTDH stoichiometry resulted in increased yields of xylitol. In conclusion, endospore surface display presents a novel approach for enzyme cascade immobilization with improved stability and tunable stoichiometry. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-28T09:36:32.653377-05:
      DOI: 10.1002/btpr.2416
  • Structural and performance characteristics of representative anion
           exchange resins used for weak partitioning chromatography
    • Authors: Shaojie Zhang; Tim Iskra, William Daniels, Jeffrey Salm, Christopher Gallo, Ranga Godavarti, Giorgio Carta
      Abstract: Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product Kp value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-28T09:36:30.774589-05:
      DOI: 10.1002/btpr.2412
  • Bioreactor productivity and media cost comparison for different
           intensified cell culture processes
    • Authors: Sen Xu; John Gavin, Rubin Jiang, Hao Chen
      Abstract: Process intensification in biomanufacturing has attracted a great deal of interest in recent years. Manufacturing platform improvements leading to higher cell density and bioreactor productivity have been pursued. Here we evaluated a variety of intensified mammalian cell culture processes for producing monoclonal antibodies. Cell culture operational modes including fed-batch (normal seeding density or high seeding density with N-1 perfusion), perfusion, and concentrated fed-batch (CFB) were assessed using the same media set with the same Chinese Hamster Ovary (CHO) cell line. Limited media modification was done to quickly fit the media set to different operational modes. Perfusion and CFB processes were developed using an alternating tangential flow filtration device. Independent of the operational modes, comparable cell specific productivity (fed-batch: 29.4 pg/cell/day; fed-batch with N-1 perfusion: 32.0 pg/cell/day; perfusion: 31.0 pg/cell/day; CFB: 20.1 – 45.1 pg/cell/day) was reached with similar media conditions. Continuous media exchange enabled much higher bioreactor productivity in the perfusion (up to 2.29 g/L/day) and CFB processes (up to 2.04 g/L/day), compared with that in the fed-batch processes (ranging from 0.39 to 0.49 g/L/day), largely due to the higher cell density maintained. Furthermore, media cost per gram of antibody produced from perfusion was found to be highly comparable with that from fed-batch; and the media cost for CFB was the highest due to the short batch duration. Our experimental data supports the argument that media cost for perfusion process could be even lower than that in a fed-batch process, as long as sufficient bioreactor productivity is achieved. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-28T09:36:24.163483-05:
      DOI: 10.1002/btpr.2415
  • Analysis of Tubespins as a suitable scale-down model of bioreactors for
           high cell density CHO cell culture
    • Authors: Natalia Gomez; Malhar Ambhaikar, Li Zhang, Chung-Jr Huang, Hedieh Barkhordarian, Jonathan Lull, Chris Gutierrez
      Abstract: High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed-batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab-scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2-week 50-mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell-specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C-terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS-simulated HCD cultures can be effectively used as a simple scale-down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-28T09:36:16.250424-05:
      DOI: 10.1002/btpr.2418
  • Integrated continuous processing of proteins expressed as inclusion
           bodies: GCSF as a case study
    • Authors: Nikhil Kateja; Harshit Agarwal, Vishwanath Hebbi, Anurag S. Rathore
      Abstract: Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (
      PubDate: 2017-01-28T09:35:57.679679-05:
      DOI: 10.1002/btpr.2413
  • Effect of copper variation in yeast hydrolysate on C-terminal lysine
           levels of a monoclonal antibody
    • Authors: Fernie G. Mitchelson; Jessica P. Mondia, Erik H. Hughes
      Abstract: The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C-terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large-scale production of a murine cell line exhibited variation in the C-terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small-scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C-terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C-terminal lysine levels of the batches in manufacturing. Small-scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C-terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast-hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-01-10T03:55:46.43279-05:0
      DOI: 10.1002/btpr.2411
  • Current state and perspectives in modeling and control of human
           pluripotent stem cell expansion processes in stirred-tank bioreactors
    • Authors: Vytautas Galvanauskas; Vykantas Grincas, Rimvydas Simutis, Yuki Kagawa, Masahiro Kino-oka
      Abstract: Implementation of model-based practices for process development, control, automation, standardization, and validation are important factors for therapeutic and industrial applications of human pluripotent stem cells. As robust cultivation strategies for pluripotent stem cell expansion and differentiation have yet to be determined, process development could be enhanced by application of mathematical models and advanced control systems to optimize growth conditions. Therefore, it is important to understand both the potential of possible applications and the apparent limitations of existing mathematical models to improve pluripotent stem cell cultivation technologies. In the present review, the authors focus on these issues as they apply to stem cell expansion processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-10T03:55:42.135768-05:
      DOI: 10.1002/btpr.2431
  • General solutions to decompose heterogeneous compositions using antibody
           afucosylation as a model system
    • Authors: John D. Chung; Peter L. Zhan
      Abstract: Methods involving the use of mathematical models of competitive ligand—receptor binding to characterize mixtures of ligands in terms of compositions and properties of the component ligands have been developed. The associated mathematical equations explicitly relate component ligand physical–chemical properties and mole fractions to measurable properties of the mixture including steady state binding activity, 1/Kd,apparent or equivalently 1/EC50, and kinetic rate constants kon,apparent and koff,apparent allowing: (1) component ligand physical property determination and (2) mixture property predictions. Additionally, mathematical equations accounting for combinatorial considerations associated with ligand assembly are used to compute ligand mole fractions. The utility of the methods developed is demonstrated using published experimental ligand–receptor binding data obtained from mixtures of afucosylated antibodies that bind FcγRIIIa (CD16a) to: (1) extract component ligand physical property information that has hitherto evaded researchers, (2) predict experimental observations, and (3) provide explanations for unresolved experimental observations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-01-10T03:55:36.88808-05:0
      DOI: 10.1002/btpr.2428
  • Optimization of enzymatic hydrolysis and fermentation conditions for
           improved bioethanol production from potato peel residues
    • Authors: Imen Ben Taher; Patrick Fickers, Sofien Chniti, Mnasser Hassouna
      Abstract: The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L−1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L−1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-01-10T03:55:30.605142-05:
      DOI: 10.1002/btpr.2427
  • Mammalian cell culture monitoring using in situ spectroscopy: Is your
           method really optimised'
    • Authors: Silvère André; Sylvain Lagresle, Zahia Hannas, Éric Calvosa, Ludovic Duponchel
      Abstract: In recent years, as a result of the process analytical technology initiative of the US Food and Drug Administration, many different works have been carried out on direct and in situ monitoring of critical parameters for mammalian cell cultures by Raman spectroscopy and multivariate regression techniques. However, despite interesting results, it cannot be said that the proposed monitoring strategies, which will reduce errors of the regression models and thus confidence limits of the predictions, are really optimized. Hence, the aim of this article is to optimize some critical steps of spectroscopic acquisition and data treatment in order to reach a higher level of accuracy and robustness of bioprocess monitoring. In this way, we propose first an original strategy to assess the most suited Raman acquisition time for the processes involved. In a second part, we demonstrate the importance of the interbatch variability on the accuracy of the predictive models with a particular focus on the optical probes adjustment. Finally, we propose a methodology for the optimization of the spectral variables selection in order to decrease prediction errors of multivariate regressions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-01-10T03:55:27.140765-05:
      DOI: 10.1002/btpr.2430
  • Electrodialytic separation of levulinic acid catalytically synthesized
           from woody biomass for use in microbial conversion
    • Authors: Hiroshi Habe; Susumu Kondo, Yuya Sato, Tomoyuki Hori, Manabu Kanno, Nobutada Kimura, Hideaki Koike, Kohtaro Kirimura
      Abstract: Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar-derived LA solution (40–160 g L−1) was demonstrated. When using reagent LA solution with pH5.0–6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg−1. With cedar-derived LA solution (pH6.0), good agreement in levulinate recovery (88–99%), and energy consumption (0.18–0.22 kWh kg−1) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar-derived LA. From 0.5 mol L−1 of the ED-concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L−1 LA solutions (40–160 g L−1). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-01-10T03:55:23.041709-05:
      DOI: 10.1002/btpr.2425
  • Production of 8,11-dihydroxy and 8-hydroxy unsaturated fatty acids from
           unsaturated fatty acids by recombinant Escherichia coli expressing
           8,11-linoleate diol synthase from Penicillium chrysogenum
    • Authors: Min-Ji Kim; Min-Ju Seo, Kyung-Chul Shin, Deok-Kun Oh
      Abstract: Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11-Linoleate diol synthase (8,11-LDS) catalyzes the conversion of unsaturated fatty acid to 8-hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11-dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11-LDS for the production of 8,11-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8,11-DiHODE), 8,11-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid (8,11-DiHOTrE), 8-hydroxy-9(Z)-hexadecenoic acid (8-HHME), and 8-hydroxy-9(Z)-octadecenoic acid (8-HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α-linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11-DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11-DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8-HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8-HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11-DiHODE, 8,11-DiHOTrE, 8-HHME, and 8-HOME. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2017-01-05T00:55:31.370313-05:
      DOI: 10.1002/btpr.2426
  • Issue Information
    • First page: 271
      PubDate: 2017-04-24T22:38:14.329357-05:
      DOI: 10.1002/btpr.2353
  • Welcome Brad Bundy and Caryn Heldt
    • First page: 275
      PubDate: 2017-04-24T22:38:14.449004-05:
      DOI: 10.1002/btpr.2451
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
Tel: +00 44 (0)131 4513762
Fax: +00 44 (0)131 4513327
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