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ENGINEERING (1201 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: 19)
AAPG Bulletin     Full-text available via subscription   (Followers: 5)
AASRI Procedia     Open Access   (Followers: 14)
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: 207)
Acta Geotechnica     Hybrid Journal   (Followers: 6)
Acta Metallurgica Sinica (English Letters)     Hybrid Journal   (Followers: 5)
Acta Polytechnica : Journal of Advanced Engineering     Open Access   (Followers: 1)
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: 10)
Adıyaman Üniversitesi Mühendislik Bilimleri Dergisi     Open Access  
Adsorption     Hybrid Journal   (Followers: 4)
Advanced Engineering Forum     Full-text available via subscription   (Followers: 4)
Advanced Science     Open Access   (Followers: 4)
Advanced Science Focus     Free   (Followers: 3)
Advanced Science Letters     Full-text available via subscription   (Followers: 4)
Advanced Science, Engineering and Medicine     Partially Free   (Followers: 6)
Advanced Synthesis & Catalysis     Hybrid Journal   (Followers: 18)
Advances in Artificial Neural Systems     Open Access   (Followers: 3)
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: 14)
Advances in Fuzzy Systems     Open Access   (Followers: 5)
Advances in Geosciences (ADGEO)     Open Access   (Followers: 9)
Advances in Heat Transfer     Full-text available via subscription   (Followers: 18)
Advances in Human Factors/Ergonomics     Full-text available via subscription   (Followers: 22)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 7)
Advances in Natural Sciences: Nanoscience and Nanotechnology     Open Access   (Followers: 28)
Advances in Operations Research     Open Access   (Followers: 11)
Advances in OptoElectronics     Open Access   (Followers: 5)
Advances in Physics Theories and Applications     Open Access   (Followers: 13)
Advances in Polymer Science     Hybrid Journal   (Followers: 40)
Advances in Porous Media     Full-text available via subscription   (Followers: 4)
Advances in Remote Sensing     Open Access   (Followers: 34)
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: 28)
Ain Shams Engineering Journal     Open Access   (Followers: 5)
Akademik Platform Mühendislik ve Fen Bilimleri Dergisi     Open Access  
Alexandria Engineering Journal     Open Access  
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: 16)
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: 5)
Applied Catalysis B: Environmental     Hybrid Journal   (Followers: 6)
Applied Clay Science     Hybrid Journal   (Followers: 4)
Applied Computational Intelligence and Soft Computing     Open Access   (Followers: 12)
Applied Magnetic Resonance     Hybrid Journal   (Followers: 3)
Applied Nanoscience     Open Access   (Followers: 8)
Applied Numerical Mathematics     Hybrid Journal   (Followers: 5)
Applied Physics Research     Open Access   (Followers: 4)
Applied Sciences     Open Access   (Followers: 3)
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: 8)
Arkiv för Matematik     Hybrid Journal  
ASEE Prism     Full-text available via subscription   (Followers: 2)
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: 7)
Asian Journal of Control     Hybrid Journal  
Asian Journal of Current Engineering & Maths     Open Access  
Asian Journal of Technology Innovation     Hybrid Journal   (Followers: 9)
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: 7)
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: 3)
Batteries     Open Access   (Followers: 3)
Bautechnik     Hybrid Journal   (Followers: 1)
Bell Labs Technical Journal     Hybrid Journal   (Followers: 24)
Beni-Suef University Journal of Basic and Applied Sciences     Open Access   (Followers: 3)
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: 8)
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: 16)
Biomedical Engineering, IEEE Transactions on     Hybrid Journal   (Followers: 31)
Biomedical Engineering: Applications, Basis and Communications     Hybrid Journal   (Followers: 5)
Biomedical Microdevices     Hybrid Journal   (Followers: 8)
Biomedical Science and Engineering     Open Access   (Followers: 4)
Biomedizinische Technik - Biomedical Engineering     Hybrid Journal  
Biomicrofluidics     Open Access   (Followers: 4)
BioNanoMaterials     Hybrid Journal   (Followers: 1)
Biotechnology Progress     Hybrid Journal   (Followers: 40)
Boletin Cientifico Tecnico INIMET     Open Access  
Botswana Journal of Technology     Full-text available via subscription  
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: 3)
Bulletin of the Crimean Astrophysical Observatory     Hybrid Journal  
Cahiers, Droit, Sciences et Technologies     Open Access  
Calphad     Hybrid Journal  
Canadian Geotechnical Journal     Full-text available via subscription   (Followers: 14)
Canadian Journal of Remote Sensing     Full-text available via subscription   (Followers: 40)
Case Studies in Engineering Failure Analysis     Open Access   (Followers: 7)
Case Studies in Thermal Engineering     Open Access   (Followers: 4)
Catalysis Communications     Hybrid Journal   (Followers: 6)
Catalysis Letters     Hybrid Journal   (Followers: 3)
Catalysis Reviews: Science and Engineering     Hybrid Journal   (Followers: 7)
Catalysis Science and Technology     Free   (Followers: 6)
Catalysis Surveys from Asia     Hybrid Journal   (Followers: 3)
Catalysis Today     Hybrid Journal   (Followers: 5)
CEAS Space Journal     Hybrid Journal  
Cellular and Molecular Neurobiology     Hybrid Journal   (Followers: 4)
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: 10)
CIRP Journal of Manufacturing Science and Technology     Full-text available via subscription   (Followers: 13)
City, Culture and Society     Hybrid Journal   (Followers: 20)
Clay Minerals     Full-text available via subscription   (Followers: 8)
Clean Air Journal     Full-text available via subscription   (Followers: 2)
Coal Science and Technology     Full-text available via subscription   (Followers: 4)
Coastal Engineering     Hybrid Journal   (Followers: 10)
Coastal Engineering Journal     Hybrid Journal   (Followers: 3)
Coatings     Open Access   (Followers: 2)
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: 23)
Composite Interfaces     Hybrid Journal   (Followers: 5)
Composite Structures     Hybrid Journal   (Followers: 242)
Composites Part A : Applied Science and Manufacturing     Hybrid Journal   (Followers: 175)
Composites Part B : Engineering     Hybrid Journal   (Followers: 215)
Composites Science and Technology     Hybrid Journal   (Followers: 159)
Comptes Rendus Mécanique     Full-text available via subscription   (Followers: 2)
Computation     Open Access  
Computational Geosciences     Hybrid Journal   (Followers: 12)
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: 6)
Computer Science and Engineering     Open Access   (Followers: 17)
Computers & Geosciences     Hybrid Journal   (Followers: 25)
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: 8)
Computing and Visualization in Science     Hybrid Journal   (Followers: 6)
Computing in Science & Engineering     Full-text available via subscription   (Followers: 25)
Conciencia Tecnologica     Open Access  
Concurrent Engineering     Hybrid Journal   (Followers: 3)
Continuum Mechanics and Thermodynamics     Hybrid Journal   (Followers: 6)
Control and Dynamic Systems     Full-text available via subscription   (Followers: 7)
Control Engineering Practice     Hybrid Journal   (Followers: 40)
Control Theory and Informatics     Open Access   (Followers: 7)
Corrosion Science     Hybrid Journal   (Followers: 24)
CT&F Ciencia, Tecnologia y Futuro     Open Access  
CTheory     Open Access  
Current Applied Physics     Full-text available via subscription   (Followers: 4)

        1 2 3 4 5 6 7 | Last

Journal Cover Biotechnology Progress
  [SJR: 0.736]   [H-I: 101]   [40 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  [1616 journals]
  • 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
    • Pages: 1 - 4
      PubDate: 2017-02-17T02:23:48.007247-05:
      DOI: 10.1002/btpr.2352
  • An In-situ glucose-stimulated insulin secretion assay under perfusion
           bioreactor conditions
    • Authors: Jamie Sharp; Patrick Vermette
      Abstract: Perfusion bioreactors, unlike traditional in vitro cell culture systems, offer stringent control of physiological parameters such as pH, flow, temperature, and dissolved oxygen concentration which have been shown to have an impact on cellular behaviour and viability. Due to the relative infancy and the growing interest in these in vitro culture systems, detection methods to monitor cell function under dynamic perfusion bioreactor conditions remains one of the main challenges. In this study, INS-1 cells, a cell line which exhibit glucose-stimulated insulin secretion, were embedded in fibrin and cultured under perfusion bioreactor conditions for 48 h and then exposed to either a high-, or low-glucose concentration for 24 h. These cultures were compared to non-bioreacted controls. Bioreacted cultures exposed to a high-glucose concentration showed the highest glucose-stimulated insulin secretion when compared to those in a low-glucose environment. The stimulation index, a marker for insulin secretion functionality, increased over time. A lower incidence of apoptotic cells was observed in the bioreacted cultures when compared to non-bioreacted ones, as evaluated by a TUNEL assay. Immunofluorescence staining of Ki67 and insulin was performed and showed no differences in the incidence of proliferative cells between conditions (bioreacted and non-bioreacted), where all cells stained positive for insulin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-12-16T23:36:26.968394-05:
      DOI: 10.1002/btpr.2407
  • Six-membered cyclic carbonates from trimethylolpropane: Lipase-mediated
           synthesis in a flow reactor and in silico evaluation of the reaction
    • Authors: Amin Bornadel; Mohamed Ismail, Mahmoud Sayed, Rajni Hatti-Kaul, Sang-Hyun Pyo
      Abstract: Six-membered cyclic carbonates with hydroxyl and methoxycarbonyloxy functional groups were prepared by transesterification of trimethylolpropane (TMP) with dimethylcarbonate (DMC) by solvent-free lipase-mediated flow reaction followed by thermal cyclization. The flow reaction efficiency was evaluated using different configurations of reactor consisting of packed beds of Novozym®435 (immobilized Candida antarctica lipase B—CalB—a.k.a. N435) and molecular sieves, flowrate, and biocatalyst loads. The mixed column of the biocatalyst and molecular sieves, allowing rapid and efficient removal of the by-product—methanol—was the most efficient setup. Higher conversion (81.6%) in the flow reaction compared to batch process (72%) was obtained using same amount of N435 (20% (w/w) N435:TMP) at 12 h, and the undesirable dimer and oligomer formation were suppressed. Moreover, the product was recovered easily without extra separation steps, and the biocatalyst and the molecular sieves remained intact for subsequent regeneration and recycling. The reaction of CalB with DMC and the primary transesterification product, monocarbonated TMP, respectively, as acyl donors was evaluated by in silico modeling and empirically to determine the role of the enzyme in the formation of cyclic carbonates and other side products. DMC was shown to be the preferred acyl donor, suggesting that TMP and its carbonated derivatives serve only as acyl acceptors in the lipase-catalyzed reaction. Subsequent cyclization to cyclic carbonate is catalyzed at increased temperature and not by the enzyme. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-12-01T04:25:32.191263-05:
      DOI: 10.1002/btpr.2406
  • Virus-like particle of Macrobrachium rosenbergii nodavirus produced in
           Spodoptera frugiperda (Sf9) cells is distinctive from that produced in
           Escherichia coli
    • Authors: Chare Li Kueh; Chean Yeah Yong, Seyedehsara Masoomi Dezfooli, Subha Bhassu, Soon Guan Tan, Wen Siang Tan
      Abstract: Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2–12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host–pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-11-29T04:01:14.591264-05:
      DOI: 10.1002/btpr.2409
  • Heterologous expression and purification of active L-asparaginase I of
           Saccharomyces cerevisiae in Escherichia coli host
    • Authors: João H. P. M. Santos; Iris M. Costa, João V. D. Molino, Mariana S. M. Leite, Marcela V. Pimenta, João A. P. Coutinho, Adalberto Pessoa, Sónia P. M. Ventura, André M. Lopes, Gisele Monteiro
      Abstract: l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6-tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+-charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg−1) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-11-29T04:01:08.238707-05:
      DOI: 10.1002/btpr.2410
  • Impact of phosphate and other medium components on physiological
           regulation of bacterial laccase production
    • Authors: Kavleen Kaur; Gursharan Singh, Vijaya Gupta, Neena Capalash, Prince Sharma
      Abstract: Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used. Shift to phosphate-less M162 medium led to successful isolations. Seven bacterial isolates belonging to genera Bacillus, Lysinibacillus, Bhargavaea and Rheinheimera were used to study the effect of medium constituents on laccase production. Inorganic phosphate (≥50 mM) was found to regulate laccase synthesis negatively though no inhibitory effect of phosphate (10–500 mM) was seen on laccase activity. All isolates ceased laccase synthesis when grown in the presence of tryptone (0.2–1%), with R. tangshanensis as an exception, or yeast extract (1.5–2%) as the only C/N source in M162 medium. Supplementation upto 0.1% of glucose in basal M162 medium increased laccase production in five isolates but decreased at higher concentrations. The influence of medium components on laccase synthesis was further affirmed by zymographic studies. These observations offer possibilities of isolating promising laccase producers from diverse environmental sources. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-11-24T00:30:38.351587-05:
      DOI: 10.1002/btpr.2408
  • Two-dimensional fluorescence as soft sensor in the monitoring of
           biotransformation performed by yeast
    • Authors: Marcin Zabadaj; Karolina Chreptowicz, Jolanta Mierzejewska, Patrycja Ciosek
      Abstract: Soft sensors are powerful tools for bioprocess monitoring due to their ability to perform online, noninvasive measurement, and possibility of detection of multiple components in cultivation media, which in turn can provide tools for the quantification of more than one metabolite/substrate/product in real time. In this work, soft sensor based on excitation-emission fluorescence is for the first time applied for the monitoring of biotransformation production of 2-phenylethanol (2-PE) by yeast strains. Main process parameters—such as optical density, glucose, and 2-PE concentrations—were determined with high accuracy and precision by fluorescence fingerprinting coupled with partial least squares regression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-10-31T05:39:34.595089-05:
      DOI: 10.1002/btpr.2381
  • Metabolic profiling of recombinant Escherichia coli cultivations based on
           high-throughput FT-MIR spectroscopic analysis
    • Authors: Kevin C. Sales; Filipa Rosa, Bernardo R. Cunha, Pedro N. Sampaio, Marta B. Lopes, Cecília R. C. Calado
      Abstract: Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic profile of recombinant E. coli cultures producing the pVAX-lacZ plasmid model was analyzed by rapid, economic and high-throughput Fourier Transform Mid-Infrared (FT-MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C-sources consumption phases, direct FT-MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016
      PubDate: 2016-10-31T05:38:09.716814-05:
      DOI: 10.1002/btpr.2378
  • Application of flow cytometry for monitoring the production of
           poly(3-hydroxybutyrate) by Halomonas boliviensis
    • Authors: María García-Torreiro; María López-Abelairas, Thelmo A. Lu-Chau, Juan M. Lema
      Abstract: In this study, a flow cytometry (FC) protocol was implemented to measure poly(3-hydroxybutyrate) (PHB) content in a halophilic bacterium, to have a faster and easier control of the process. The halophilic bacterium Halomonas boliviensis was stained with BODIPY 493/503 and analyzed using FC. Bacterial polymer accumulation induced by two different nutrient limitations during the operation of a 2 L bioreactor was studied using traditional gas chromatography (GC) analysis and FC. The application of this rapid and straightforward method is useful to obtain complex and precise information about PHB accumulation that could be used for the monitoring, control and optimization of the production of PHB. A clear correlation between the PHB concentration determined by GC and the fluorescence signal obtained from stained bacteria by using FC was observed. Additionally, the heterogeneity of bacterial population as a function of PHB content was measured. © 2016 American Institute of Chemical Engineers Biotechnol. Prog, 2016
      PubDate: 2016-10-20T01:04:55.613584-05:
      DOI: 10.1002/btpr.2373
  • The use of electric, magnetic, and electromagnetic field for directed cell
           migration and adhesion in regenerative medicine
    • Authors: Christina L. Ross
      Pages: 5 - 16
      Abstract: Directed cell migration and adhesion is essential to embryonic development, tissue formation and wound healing. For decades it has been reported that electric field (EF), magnetic field (MF) and electromagnetic field (EMF) can play important roles in determining cell differentiation, migration, adhesion, and evenwound healing. Combinations of these techniques have revealed new and exciting explanations for how cells move and adhere to surfaces; how the migration of multiple cells are coordinated and regulated; how cellsinteract with neighboring cells, and also to changes in their microenvironment. In some cells, speed and direction are voltage dependent. Data suggests that the use of EF, MF and EMF could advance techniques in regenerative medicine, tissue engineering and wound healing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:5–16, 2017
      PubDate: 2016-10-31T05:37:58.524999-05:
      DOI: 10.1002/btpr.2371
  • Methionine sulfoximine supplementation enhances productivity in
           GS–CHOK1SV cell lines through glutathione biosynthesis
    • Authors: Marc Feary; Andrew J. Racher, Robert J. Young, C. Mark Smales
      Pages: 17 - 25
      Abstract: In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS–CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS–CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS–CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS–CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS–CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17–25, 2017
      PubDate: 2016-10-31T05:41:02.904372-05:
      DOI: 10.1002/btpr.2372
  • Identification of active constraints in dynamic flux balance analysis
    • Authors: Ali Nikdel; Hector Budman
      Pages: 26 - 36
      Abstract: This study deals with the calibration of dynamic metabolic flux models that are formulated as the maximization of an objective subject to constraints. Two approaches were applied for identifying the constraints from data. In the first approach a minimal active number of limiting constraints is found based on data that are assumed to be bounded within sets whereas, in the second approach, the limiting constraints are found based on parametric sensitivity analysis. The ability of these approaches to finding the active limiting constraints was verified through their application to two case studies: an in-silico (simulated) data-based study describing the growth of E. coli and an experimental data-based study for Bordetella pertussis (B. pertussis). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:26–36, 2017
      PubDate: 2016-11-22T06:55:43.556378-05:
      DOI: 10.1002/btpr.2388
  • A demonstration of athermal effects of continuous microwave irradiation on
           the growth and antibiotic sensitivity of Pseudomonas aeruginosa PAO1
    • Authors: Ismini Nakouti; Glyn Hobbs, Yothin Teethaisong, David Phipps
      Pages: 37 - 44
      Abstract: Stress, caused by exposure to microwaves (2.45 GHz) at constant temperature (37 ± 0.5°C), alters the growth profile of Pseudomonas aeruginosa PAO1. In the absence of microwave treatment a simple, highly reproducible growth curve was observed over 24 h or more. Microwave treatment caused no reduction in growth during the first 6 h, but at a later stage (>12 h) the growth was markedly different to the controls. Secondary growth, typical of the presence of persisters clearly became apparent, as judged by both the dissolved oxygen and the cell density profiles. These treated cells showed distinct morphological changes, but on regrowth these cells reverted to normal. The microwave induced persisters were subject to antibiotic challenge (tobramycin) and showed increased sensitivity when compared to the unstressed planktonic cells. This is in marked contrast to antibiotic induced persisters which show increased resistance. This provides evidence for both a nonthermal effect of microwaves and a previously undescribed route to a novel form of antibiotic susceptible persister cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:37–44, 2017
      PubDate: 2016-11-14T23:01:39.731905-05:
      DOI: 10.1002/btpr.2392
  • Evaluating Light-Induced Promoters for the Control of Heterologous Gene
           Expression in Synechocystis sp. PCC 6803
    • Authors: Stevan C. Albers; Christie A. M. Peebles
      Pages: 45 - 53
      Abstract: Cyanobacteria are enticing microbial factories, but little is understood how their gene control elements respond to the periodic availability to light. This research tested the capability of PpsbAII to control gene expression during light/dark conditions when moved to a neutral location within the Synechocystis sp. PCC 6803 genome. When the eYFP reporter gene was run by PpsbAII in the promoter's native genomic location, mutants exposed to 12-hour light conditions experienced a 15.8× increase in transcript abundance over that observed from the same construct exposed to 12-hour dark conditions. When this same construct was moved to the hypothetical coding region slr0168 in the genome, transcripts generated during 12 hour light conditions accumulated to 1.67X of the levels of transcripts generated by the same construct during 12 hour dark conditions. Three additional promoter constructs, PpsbAIII, PgroEL2, and PsigD were also tested for differential expression in light and dark conditions within the neutral region slr0168. While low amounts of transcript accumulation were observed from PgroEL2 and PsigD, the PpsbAIII construct accumulated 5.79× more transcripts when compared to transcript abundance during dark conditions, which highlights the potential of this promoter to control gene expression during diel-cycle light conditions. Additionally, nucleotide mutations were made to regions within PpsbAII. Mutations to the cis-acting hexo-nucleotide region increased expression 3.71× over that of the native promoter, while the addition of the “HLR” nucleotide region to the PpsbAII::ΔHex construct increased expression 2.76× over that of the native promoter. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:45–53, 2017
      PubDate: 2016-11-17T07:20:34.857737-05:
      DOI: 10.1002/btpr.2396
  • Heterologous expression of the plant cysteine protease bromelain and its
           inhibitor in Pichia pastoris
    • Authors: Nora Luniak; Peter Meiser, Sonja Burkart, Rolf Müller
      Pages: 54 - 65
      Abstract: Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54–65, 2017
      PubDate: 2016-11-29T11:05:25.88405-05:0
      DOI: 10.1002/btpr.2405
  • Still stable after 11 years: A Catharanthus roseus Hairy root line
           maintains inducible expression of anthranilate synthase
    • Authors: Jiayi Sun; Li Ma, Ka-Yiu San, Christie A. M. Peebles
      Pages: 66 - 69
      Abstract: Hairy root cultures generated using Agrobacterium rhizogenes are an extensively investigated system for the overproduction of various secondary metabolite based pharmaceuticals and chemicals. This study demonstrated a transgenic Catharanthus roseus hairy root line carrying a feedback-insensitive anthranilate synthase (AS) maintained chemical and genetic stability for 11 years. The AS gene was originally inserted in the hairy root genome under the control of a glucocorticoid inducible promoter. After 11 years continuous maintenance of this hairy root line, genomic PCR of the ASA gene showed the presence of ASA gene in the genome. The mRNA level of AS was induced to 52-fold after feeding the inducer as compared to the uninduced control. The AS enzyme activity was 18.4 nmol/(min*mg) in the induced roots as compared to 2.1 nmol/(min*mg) in the control. In addition, the changes in terpenoid indole alkaloid concentrations after overexpressing AS were tracked over 11 years. The major alkaloid levels in induced and control roots at 11 years are comparable with the metabolite levels at 5 years. This study demonstrates the long term genetic and biochemical stability of hairy root lines, which has important implications for industrial scale applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:66–69, 2017
      PubDate: 2016-11-21T04:20:52.229867-05:
      DOI: 10.1002/btpr.2403
  • Recombinant exochitinase of the thermophilic mould Myceliopthora
           thermophila BJA: Characteristics and utility in generating N-acetyl
           glucosamine and in biocontrol of phytopathogenic fungi
    • Authors: Ashima Dua; Swati Joshi, T. Satyanarayana
      Pages: 70 - 80
      Abstract: Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. To produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween-20 supported two-fold higher rMtChit production (5.5 × 103 U L−1). The consensus sequences S(132)xG(133)G(134) and D(168)xxD(171)xD(173)xE(175) in the enzyme have been found to represent the substrate binding and catalytic sites, respectively. The rMtChit, purified to homogeneity by a two-step purification strategy, is a monomeric glycoprotein of ∼48 kDa, which is optimally active at 55°C and pH 5.0. The enzyme is thermostable with t1/2 values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters Km, Vmax, kcat, and kcat/Km of the enzyme are 4.655 mg mL−1, 34.246 nmol mg−1 s−1, 3.425 × 106 min−1, and 1.36 × 10−6 mg mL−1 min−1, respectively. rMtChit is an unique exochitinase, since its action on chitin liberates N-acetylglucosamine NAG. The enzyme inhibits the growth of phytopathogenic fungi like Fusarium oxysporum and Curvularia lunata, therefore, this finds application as biofungicide at high temperatures during summer in tropics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:70–80, 2017
      PubDate: 2016-10-31T05:40:06.666472-05:
      DOI: 10.1002/btpr.2370
  • Opossum peptide that can neutralize rattlesnake venom is expressed in
           Escherichia coli
    • Authors: Claire F. Komives; Elda E. Sanchez, Anurag S. Rathore, Brandon White, Michael Balderrama, Montamas Suntravat, Angela Cifelli, Varsha Joshi
      Pages: 81 - 86
      Abstract: An eleven amino acid ribosomal peptide was shown to completely neutralize Western Diamondback Rattlesnake (Crotalus atrox) venom in mice when a lethal dose of the venom was pre-incubated with the peptide prior to intravenous injection. We have expressed the peptide as a concatenated chain of peptides and cleaved them apart from an immobilized metal affinity column using a protease. After ultrafiltration steps, the mixture was shown to partially neutralize rattlesnake venom in mice. Preliminary experiments are described here that suggest a potential life-saving therapy could be developed. To date, no recombinant therapies targeting cytotoxic envenomation have been reported. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:81–86, 2017
      PubDate: 2016-12-01T04:25:34.780205-05:
      DOI: 10.1002/btpr.2386
  • Influence of the experimental setup on the determination of enzyme kinetic
    • Authors: Jan-Hendrik Grosch; David Wagner, Niklas Knaup, Timm Keil, Antje C. Spieß
      Pages: 87 - 95
      Abstract: For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29–118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87–95, 2017
      PubDate: 2016-11-14T23:01:33.238828-05:
      DOI: 10.1002/btpr.2390
  • Thermodynamic activity-based intrinsic enzyme kinetic sheds light on
           enzyme–solvent interactions
    • Authors: Jan-Hendrik Grosch; David Wagner, Vasilios Nistelkas, Antje C. Spieß
      Pages: 96 - 103
      Abstract: The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme–solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme–solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96–103, 2017
      PubDate: 2016-11-22T06:55:37.728502-05:
      DOI: 10.1002/btpr.2401
  • Recent advances in immobilization strategies for glycosidases
    • Authors: Sercan Karav; Joshua L. Cohen, Daniela Barile, Juliana Maria Leite Nobrega de Moura Bell
      Pages: 104 - 112
      Abstract: Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N′-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:104–112, 2017
      PubDate: 2016-10-31T05:39:39.565312-05:
      DOI: 10.1002/btpr.2385
  • High concentration biotherapeutic formulation and ultrafiltration: Part 1
           pressure limits
    • Authors: Herb Lutz; Joshua Arias, Yu Zou
      Pages: 113 - 124
      Abstract: High therapeutic dosage requirements and the desire for ease of administration drive the trend to subcutaneous administration using delivery systems such as subcutaneous pumps and prefilled syringes. Because of dosage volume limits, prefilled syringe administration requires higher concentration liquid formulations, limited to about 30 cP or roughly 100–300 g L−1 for mAb's. Ultrafiltration (UF) processes are routinely used to formulate biological therapeutics. This article considers pressure constraints on the UF process that may limit its ability to achieve high final product concentrations. A system hardware analysis shows that the ultrafiltration cassette pressure drop is the major factor limiting UF systems. Additional system design recommendations are also provided. The design and performance of a new cassette with a lower feed channel flow resistance is described along with 3D modeling of feed channel pressure drop. The implications of variations in cassette flow channel resistance for scaling up and setting specifications are considered. A recommendation for a maximum pressure specification is provided. A review of viscosity data and theory shows that molecular engineering, temperature, and the use of viscosity modifying excipients including pH adjustment can be used to achieve higher concentrations. The combined use of a low pressure drop cassette with excipients further increased final concentrations by 35%. Guidance is provided on system operation to control hydraulics during final concentration. These recommendations should allow one to design and operate systems to routinely achieve the 30 cP target final viscosity capable of delivery using a pre-filled syringe. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:113–124, 2017
      PubDate: 2016-10-15T09:15:33.049379-05:
      DOI: 10.1002/btpr.2334
  • Isolation and characterization of circulating tumor cells using a novel
           workflow combining the CellSearch® system and the CellCelector™
    • Authors: Martin Horst Dieter Neumann; Helen Schneck, Yvonne Decker, Susanne Schömer, André Franken, Volker Endris, Nicole Pfarr, Wilko Weichert, Dieter Niederacher, Tanja Fehm, Hans Neubauer
      Pages: 125 - 132
      Abstract: Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch® and the CellCelector™ micromanipulation system. CTCs were isolated from CellSearch® cartridges using the CellCelector™ system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector™ screen we reidentified 97% of CellSearch® SKBR-3 cells. Furthermore, we isolated 97% of CellSearch®-proven patient CTCs using the CellCelector™ system. Therein, we found an almost perfect correlation of R2 = 0.98 (Spearman's rho correlation, n = 20, p 
      PubDate: 2016-05-17T08:15:22.703932-05:
      DOI: 10.1002/btpr.2294
  • Recovery of functionally-active protein from inclusion bodies using a
           thermal-cycling method
    • Authors: Rahul Sadavarte; Carlos D. M. Filipe, Raja Ghosh
      Pages: 133 - 139
      Abstract: Heterologous overexpression of genes in Escherichia coli has made it possible to obtain high titers of recombinant proteins. However, this can result in the formation of aggregated protein particles known as ‘inclusion bodies’. Protein sequestered as inclusion body is inactive and needs to be converted back to its functional form by refolding using appropriate techniques. In the current study inclusion bodies of the enzyme aminoglycoside nucleotidyl transferase (or ANT(2″)-Ia) were first solubilized in urea and subsequently subjected to thermal cycling under controlled conditions as part of the refolding strategy. Thermal cycling led to disaggregation of the individual protein chains and simultaneously refolding the released protein molecules to their native state. The optimum condition was identified as 10–80°C thermal cycling at 3°C s−1 for 2 h. Enzyme activity measurements showed that thermal cycling under optimized conditions resulted in 257% activity recovery when compared with nonrefolded protein. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:133–139, 2017
      PubDate: 2016-10-31T05:41:26.794206-05:
      DOI: 10.1002/btpr.2376
  • Identification of an IgG CDR sequence contributing to co-purification of
           the host cell protease cathepsin D
    • Authors: Jared S. Bee; LeeAnn M. Machiesky, Li Peng, Kristin C. Jusino, Matthew Dickson, Jeffrey Gill, Douglas Johnson, Hung-Yu Lin, Kenneth Miller, Jenny Heidbrink Thompson, Richard L. Remmele
      Pages: 140 - 145
      Abstract: Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb-1 and mAb-6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb-1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb-1 and cathepsin D was weaker than that of mAb-6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb-1 are replaced with neutral serine residues in mAb-6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140–145, 2017
      PubDate: 2016-11-14T23:01:30.46976-05:0
      DOI: 10.1002/btpr.2397
  • Biomass-water interactions correlate to recalcitrance and are intensified
           by pretreatment: An investigation of water constraint and retention in
           pretreated spruce using low field NMR and water retention value techniques
    • Authors: Noah D. Weiss; Lisbeth Garbrecht Thygesen, Claus Felby, Christian Roslander, Keith Gourlay
      Pages: 146 - 153
      Abstract: The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO2 pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship. The water retention value (WRV) of the pretreated materials was measured, and water constraint was assessed using time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry. WRV increased with pretreatment severity, correlating to reduced recalcitrance, as measured by hydrolysis of cellulose using commercial enzyme preparations. Water constraint increased with pretreatment severity, suggesting that a higher level of biomass-water interaction is indicative of reduced recalcitrance in pretreated materials. Both WRV and water constraint increased significantly with reductions in particle size when pretreated materials were further milled, suggesting that particle size plays an important role in biomass water interactions. It is suggested that WRV may be a simple and effective method for measuring and comparing biomass recalcitrance. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:146–153, 2017
      PubDate: 2016-11-17T07:20:39.69984-05:0
      DOI: 10.1002/btpr.2398
  • Insertion of single-chain variable fragment (scFv) peptide linker improves
           surface display of influenza hemagglutinin (HA1) on non-recombinant
           Lactococcus lactis
    • Authors: Pui-Fong Jee; Fez-Shin Chen, Meng-Hooi Shu, Won Fen Wong, Raha Abdul Rahim, Sazaly AbuBakar, Li-Yen Chang
      Pages: 154 - 162
      Abstract: Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA, and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7 ± 1.4% and 3,594.0 ± 675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1 ± 1.5% and 1,652.0 ± 34.1, respectively). Higher amount of HA1/L/AcmA (∼2.9 × 104 molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (∼1.1 × 104 molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:154–162, 2017
      PubDate: 2016-11-17T07:20:45.034017-05:
      DOI: 10.1002/btpr.2400
  • Exploring the linkage between cell culture process parameters and
           downstream processing utilizing a plackett-burman design for a model
           monoclonal antibody
    • Authors: Cyrus D. Agarabi; Brittany K. Chavez, Scott C. Lute, Erik K. Read, Sarah Rogstad, David Awotwe-Otoo, Matthew R. Brown, Michael T. Boyne, Kurt A. Brorson
      Pages: 163 - 170
      Abstract: Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017
      PubDate: 2016-11-21T04:20:47.556344-05:
      DOI: 10.1002/btpr.2402
  • Affinity chromatography matrices for depletion and purification of casein
           glycomacropeptide from bovine whey
    • Authors: María F. Baieli; Nicolás Urtasun, María J. Martinez, Daniela B. Hirsch, Ana M. R. Pilosof, María V. Miranda, Osvaldo Cascone, Federico J. Wolman
      Pages: 171 - 180
      Abstract: Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171–180, 2017
      PubDate: 2016-11-29T07:05:31.257676-05:
      DOI: 10.1002/btpr.2404
  • Robust factor selection in early cell culture process development for the
           production of a biosimilar monoclonal antibody
    • Authors: Michael Sokolov; Jonathan Ritscher, Nicola MacKinnon, Jean-Marc Bielser, David Brühlmann, Dominik Rothenhäusler, Gian Thanei, Miroslav Soos, Matthieu Stettler, Jonathan Souquet, Hervé Broly, Massimo Morbidelli, Alessandro Butté
      Pages: 181 - 191
      Abstract: This work presents a multivariate methodology combining principal component analysis, the Mahalanobis distance and decision trees for the selection of process factors and their levels in early process development of generic molecules. It is applied to a high throughput study testing more than 200 conditions for the production of a biosimilar monoclonal antibody at microliter scale. The methodology provides the most important selection criteria for the process design in order to improve product quality towards the quality attributes of the originator molecule. Robustness of the selections is ensured by cross-validation of each analysis step. The concluded selections are then successfully validated with an external data set. Finally, the results are compared to those obtained with a widely used software revealing similarities and clear advantages of the presented methodology. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:181–191, 2017
      PubDate: 2016-10-31T05:38:22.565042-05:
      DOI: 10.1002/btpr.2374
  • Studies on fluid dynamics of the flow field and gas transfer in orbitally
           shaken tubes
    • Authors: Li-kuan Zhu; Bo-yan Song, Zhen-long Wang, Dominique T. Monteil, Xiao Shen, David L. Hacker, Maria De Jesus, Florian M. Wurm
      Pages: 192 - 200
      Abstract: Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50-mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high-throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three-dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (kLa) was determined experimentally and from simulations under various working conditions. We also determined the liquid-phase mass transfer coefficient (kL) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of kL. High oxygen transfer rates, sufficient for supporting the high-density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192–200, 2017
      PubDate: 2016-10-31T05:41:40.896999-05:
      DOI: 10.1002/btpr.2375
  • Combined effect of pulsed electromagnetic field and sound wave on In vitro
           and In vivo neural differentiation of human mesenchymal stem cells
    • Authors: Yun-Kyong Choi; Enerelt Urnukhsaikhan, Hee-Hoon Yoon, Young-Kwon Seo, Hyunjin Cho, Jong-Seob Jeong, Soo-Chan Kim, Jung-Keug Park
      Pages: 201 - 211
      Abstract: Biophysical wave stimulus has been used as an effective tool to promote cellular maturation and differentiation in the construction of engineered tissue. Pulsed electromagnetic fields (PEMFs) and sound waves have been selected as effective stimuli that can promote neural differentiation. The aim of this study was to investigate the synergistic effect of PEMFs and sound waves on the neural differentiation potential in vitro and in vivo using human bone marrow mesenchymal stem cells (hBM–MSCs). In vitro, neural-related genes in hBM–MSCs were accelerated by the combined exposure to both waves more than by individual exposure to PEMFs or sound waves. The combined wave also up-regulated the expression of neural and synaptic-related proteins in a three-dimensional (3-D) culture system through the phosphorylation of extracellular signal-related kinase. In a mouse model of photochemically induced ischemia, exposure to the combined wave reduced the infarction volume and improved post-injury behavioral activity. These results indicate that a combined stimulus of biophysical waves, PEMFs and sound can enhance and possibly affect the differentiation of MSCs into neural cells. Our study is meaningful for highlighting the potential of combined wave for neurogenic effects and providing new therapeutic approaches for neural cell therapy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:201–211, 2017
      PubDate: 2016-11-18T00:35:35.15672-05:0
      DOI: 10.1002/btpr.2389
  • Development of a high yielding E. coli periplasmic expression system for
           the production of humanized Fab' fragments
    • Authors: Mark Ellis; Pareshkumar Patel, Marjory Edon, Walter Ramage, Robert. Dickinson, David P. Humphreys
      Pages: 212 - 220
      Abstract: Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017
      PubDate: 2016-11-17T07:20:43.075503-05:
      DOI: 10.1002/btpr.2393
  • Modeling spatial distribution of oxygen in 3d culture of islet beta-cells
    • Authors: John McReynolds; Yu Wen, Xiaofei Li, Jianjun Guan, Sha Jin
      Pages: 221 - 228
      Abstract: Three-dimensional (3D) scaffold culture of pancreatic β-cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β-cells is that β-cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β-cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell-laden collagen culture on β-cell proliferation, a culture model with encapsulation of an oxygen-generator was established. The oxygen-generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β-cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial-temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation-aided 3D culture would augment the oxygen supply required for the β-cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell-laden scaffold cultures with an in situ oxygen supply significantly improved the β-cells' biological function. These β-cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β-cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221–228, 2017
      PubDate: 2016-11-21T04:20:55.380298-05:
      DOI: 10.1002/btpr.2395
  • Combining endocytic and freezing-induced trehalose uptake for
           cryopreservation of mammalian cells
    • Authors: Miao Zhang; Harriëtte Oldenhof, Harald Sieme, Willem F. Wolkers
      Pages: 229 - 235
      Abstract: Fibroblasts take up trehalose during freezing and thawing, which facilitates cryosurvival of the cells. The aim of this study was to investigate if trehalose uptake via fluid-phase endocytosis prefreeze increases cryosurvival. To determine endocytic trehalose uptake in attached as well as suspended fibroblasts, intracellular trehalose concentrations were determined during incubation at 37°C using an enzymatically based trehalose assay. In addition, freezing-induced trehalose uptake of extracellularly added trehalose was determined. Cryosurvival rates were determined via trypan blue staining. Intracellular trehalose contents of attached as well as suspended cells were found to increase linearly with time, consistent with fluid-phase endocytosis. Furthermore, the intracellular trehalose concentration increased with increasing extracellular trehalose concentration (0–100 mM) in a linear fashion. Prefreeze loading of cells with trehalose via fluid-phase endocytosis only showed increased cryosurvival rates at extracellular trehalose concentrations lower than 50 mM in the cryopreservation medium. To obtain satisfactory cryosurvival rates after endocytic preloading, extracellular trehalose is needed to prevent efflux of trehalose during freezing and thawing and for freezing-induced trehalose uptake. At trehalose concentrations greater than 100 mM, cryosurvival rates were similar or slightly higher if cells were not loaded with trehalose prefreeze. Cells that were grown in the presence of trehalose showed a tendency to aggregate after harvesting. It is concluded that it is particularly freezing-induced trehalose uptake that facilitates cryosurvival when trehalose is used as the sole cryoprotectant for cryopreservation of fibroblasts. Preloading with trehalose does not increase cryosurvival rates if trehalose is also added as extracellular protectant. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:229–230, 2017
      PubDate: 2016-11-21T04:20:44.973875-05:
      DOI: 10.1002/btpr.2399
  • Immobilization of recombinant pectate lyase from Clostridium thermocellum
           ATCC-27405 on magnetic nanoparticles for bioscouring of cotton fabric
    • Authors: Soumyadeep Chakraborty; Tingirikari Jagan Mohan Rao, Arun Goyal
      Pages: 236 - 244
      Abstract: Recombinant pectate lyase from family 1 polysaccharide lyase (PL1B) was immobilized on synthesized magnetic nanoparticles (MNPs) after 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride activation. At 70 mg/mL MNPs 100% binding of 1 mg/mL PL1B was achieved. The immobilized PL1B-MNP displayed activity of 20.3 and 18.2 U/mg against polygalacturonic acid and citrus pectin, respectively, which was higher than the activity of free PL1B, on the same substrates of 17.8 and 16.2 U/mg. The immobilized PL1B-MNP showed 32 fold and 14 fold enhanced thermal stability at 80°C and 90°C, respectively as compared with free PL1B at same temperatures. At high temperature the immobilized PL1B-MNP retained its activity for a longer duration than free PL1B. The immobilized PL1B-MNP could be reused till five cycles and after that it retained 70% of initial activity. It could be easily recovered from the reaction mixture with the help of a magnet. Bioscouring of cotton fabric was carried out with immobilized PL1B-MNP which showed efficient removal of pectin from the fabric surface. The enhanced wettability of fabric resulted in the decrease of the water absorbing time period from 3 min taken by the free PL1B treated fabric to 15 s taken by the immobilized PL1B-MNP treated fabric. As per our knowledge this is the first attempt of bioscouring of coarse cotton fabric by pectinase immobilized on magnetic nanoparticles. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:231–244, 2017
      PubDate: 2016-10-31T05:39:54.75147-05:0
      DOI: 10.1002/btpr.2379
  • Impact of pectin esterification on the antimicrobial activity of
           nisin-loaded pectin particles
    • Authors: Tatjana Krivorotova; Ramune Staneviciene, Juliana Luksa, Elena Serviene, Jolanta Sereikaite
      Pages: 245 - 251
      Abstract: The relationship between pectin structure and the antimicrobial activity of nisin-loaded pectin particles was examined. The antimicrobial activity of five different nisin-loaded pectin particles, i.e., nisin-loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0–7.0 by agar-diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin-loaded pectin particles. Nisin-loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin-loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin-loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245–251, 2017
      PubDate: 2016-11-18T00:35:28.209368-05:
      DOI: 10.1002/btpr.2391
  • A PEGylated bovine hemoglobin as a potent hemoglobin-based oxygen carrier
    • Authors: Ying Wang; Linli Wang, Weili Yu, Dawei Gao, Guoxing You, Penglong Li, Shan Zhang, Jun Zhang, Tao Hu, Lian Zhao, Hong Zhou
      Pages: 252 - 260
      Abstract: Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been used as blood substitutes in surgery medicine and oxygen therapeutics for ischemic stroke. As a potent HBOC, the PEGylated Hb has received much attention for its oxygen delivery and plasma expanding ability. Two PEGylated Hbs, Euro-Hb, and MP4 have been developed for clinical trials, using human adult hemoglobin (HbA) as the original substrate. However, HbA was obtained from outdated human blood and its quantity available from this source may not be sufficient for mass production of PEGylated HbA. In contrast, bovine Hb (bHb) has no quantity constraints for its ample resource. Thus, bHb is of potential to function as an alternative substrate to obtain a PEGylated bHb (bHb-PEG). bHb-PEG was prepared under the same reaction condition as HbA-PEG, using maleimide chemistry. The structural, functional, solution and physiological properties of bHb-PEG were determined and compared with those of HbA-PEG. bHb-PEG showed higher hydrodynamic volume, colloidal osmotic pressure, viscosity and P50 than HbA-PEG. The high P50 of bHb can partially compensate the PEGylation-induced perturbation in the R to T state transition of HbA. bHb-PEG was non-vasoactive and could efficiently recover the mean arterial pressure of mice suffering from hemorrhagic shock. Thus, bHb-PEG is expected to function as a potent HBOC for its high oxygen delivery and strong plasma expanding ability. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:252–260, 2017
      PubDate: 2016-10-31T05:37:54.727226-05:
      DOI: 10.1002/btpr.2380
  • Bioprocess development workflow: Transferable physiological knowledge
           instead of technological correlations
    • Authors: Wieland N. Reichelt; Florian Haas, Patrick Sagmeister, Christoph Herwig
      Pages: 261 - 270
      Abstract: Microbial bioprocesses need to be designed to be transferable from lab scale to production scale as well as between setups. Although substantial effort is invested to control technological parameters, usually the only true constant parameter is the actual producer of the product: the cell. Hence, instead of solely controlling technological process parameters, the focus should be increasingly laid on physiological parameters. This contribution aims at illustrating a workflow of data life cycle management with special focus on physiology. Information processing condenses the data into physiological variables, while information mining condenses the variables further into physiological descriptors. This basis facilitates data analysis for a physiological explanation for observed phenomena in productivity. Targeting transferability, we demonstrate this workflow using an industrially relevant Escherichia coli process for recombinant protein production and substantiate the following three points: (1) The postinduction phase is independent in terms of productivity and physiology from the preinduction variables specific growth rate and biomass at induction. (2) The specific substrate uptake rate during induction phase was found to significantly impact the maximum specific product titer. (3) The time point of maximum specific titer can be predicted by an easy accessible physiological variable: while the maximum specific titers were reached at different time points (19.8 ± 7.6 h), those maxima were reached all within a very narrow window of cumulatively consumed substrate dSn (3.1 ± 0.3 g/g). Concluding, this contribution provides a workflow on how to gain a physiological view on the process and illustrates potential benefits. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:261–270, 2017
      PubDate: 2016-10-31T05:38:06.858889-05:
      DOI: 10.1002/btpr.2377
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