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  Subjects -> ENGINEERING (Total: 2312 journals)
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ENGINEERING (1213 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     Hybrid Journal   (Followers: 7)
AASRI Procedia     Open Access   (Followers: 15)
Abstract and Applied Analysis     Open Access   (Followers: 3)
Aceh International Journal of Science and Technology     Open Access   (Followers: 2)
ACS Nano     Full-text available via subscription   (Followers: 254)
Acta Geotechnica     Hybrid Journal   (Followers: 7)
Acta Metallurgica Sinica (English Letters)     Hybrid Journal   (Followers: 5)
Acta Polytechnica : Journal of Advanced Engineering     Open Access   (Followers: 2)
Acta Scientiarum. Technology     Open Access   (Followers: 3)
Acta Universitatis Cibiniensis. Technical Series     Open Access  
Active and Passive Electronic Components     Open Access   (Followers: 7)
Adaptive Behavior     Hybrid Journal   (Followers: 11)
Adıyaman Üniversitesi Mühendislik Bilimleri Dergisi     Open Access  
Adsorption     Hybrid Journal   (Followers: 4)
Advanced Engineering Forum     Full-text available via subscription   (Followers: 6)
Advanced Science     Open Access   (Followers: 5)
Advanced Science Focus     Free   (Followers: 3)
Advanced Science Letters     Full-text available via subscription   (Followers: 9)
Advanced Science, Engineering and Medicine     Partially Free   (Followers: 7)
Advanced Synthesis & Catalysis     Hybrid Journal   (Followers: 18)
Advances in Calculus of Variations     Hybrid Journal   (Followers: 2)
Advances in Catalysis     Full-text available via subscription   (Followers: 6)
Advances in Complex Systems     Hybrid Journal   (Followers: 7)
Advances in Engineering Software     Hybrid Journal   (Followers: 27)
Advances in Fuel Cells     Full-text available via subscription   (Followers: 16)
Advances in Fuzzy Systems     Open Access   (Followers: 5)
Advances in Geosciences (ADGEO)     Open Access   (Followers: 11)
Advances in Heat Transfer     Full-text available via subscription   (Followers: 22)
Advances in Human Factors/Ergonomics     Full-text available via subscription   (Followers: 27)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 10)
Advances in Natural Sciences: Nanoscience and Nanotechnology     Open Access   (Followers: 30)
Advances in Operations Research     Open Access   (Followers: 12)
Advances in OptoElectronics     Open Access   (Followers: 5)
Advances in Physics Theories and Applications     Open Access   (Followers: 12)
Advances in Polymer Science     Hybrid Journal   (Followers: 41)
Advances in Porous Media     Full-text available via subscription   (Followers: 5)
Advances in Remote Sensing     Open Access   (Followers: 40)
Advances in Science and Research (ASR)     Open Access   (Followers: 6)
Aerobiologia     Hybrid Journal   (Followers: 2)
African Journal of Science, Technology, Innovation and Development     Hybrid Journal   (Followers: 6)
AIChE Journal     Hybrid Journal   (Followers: 32)
Ain Shams Engineering Journal     Open Access   (Followers: 5)
Akademik Platform Mühendislik ve Fen Bilimleri Dergisi     Open Access   (Followers: 1)
Alexandria Engineering Journal     Open Access   (Followers: 1)
AMB Express     Open Access   (Followers: 1)
American Journal of Applied Sciences     Open Access   (Followers: 28)
American Journal of Engineering and Applied Sciences     Open Access   (Followers: 11)
American Journal of Engineering Education     Open Access   (Followers: 9)
American Journal of Environmental Engineering     Open Access   (Followers: 17)
American Journal of Industrial and Business Management     Open Access   (Followers: 23)
Analele Universitatii Ovidius Constanta - Seria Chimie     Open Access  
Annals of Combinatorics     Hybrid Journal   (Followers: 3)
Annals of Pure and Applied Logic     Open Access   (Followers: 2)
Annals of Regional Science     Hybrid Journal   (Followers: 8)
Annals of Science     Hybrid Journal   (Followers: 7)
Applicable Algebra in Engineering, Communication and Computing     Hybrid Journal   (Followers: 2)
Applicable Analysis: An International Journal     Hybrid Journal   (Followers: 1)
Applied Catalysis A: General     Hybrid Journal   (Followers: 6)
Applied Catalysis B: Environmental     Hybrid Journal   (Followers: 18)
Applied Clay Science     Hybrid Journal   (Followers: 5)
Applied Computational Intelligence and Soft Computing     Open Access   (Followers: 12)
Applied Magnetic Resonance     Hybrid Journal   (Followers: 4)
Applied Nanoscience     Open Access   (Followers: 8)
Applied Network Science     Open Access   (Followers: 1)
Applied Numerical Mathematics     Hybrid Journal   (Followers: 5)
Applied Physics Research     Open Access   (Followers: 3)
Applied Sciences     Open Access   (Followers: 2)
Applied Spatial Analysis and Policy     Hybrid Journal   (Followers: 5)
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   (Followers: 1)
ASEE Prism     Full-text available via subscription   (Followers: 3)
Asia-Pacific Journal of Science and Technology     Open Access  
Asian Engineering Review     Open Access  
Asian Journal of Applied Science and Engineering     Open Access   (Followers: 1)
Asian Journal of Applied Sciences     Open Access   (Followers: 2)
Asian Journal of Biotechnology     Open Access   (Followers: 8)
Asian Journal of Control     Hybrid Journal  
Asian Journal of Current Engineering & Maths     Open Access  
Asian Journal of Technology Innovation     Hybrid Journal   (Followers: 8)
Assembly Automation     Hybrid Journal   (Followers: 2)
at - Automatisierungstechnik     Hybrid Journal   (Followers: 1)
ATZagenda     Hybrid Journal  
ATZextra worldwide     Hybrid Journal  
Australasian Physical & Engineering Sciences in Medicine     Hybrid Journal   (Followers: 1)
Australian Journal of Multi-Disciplinary Engineering     Full-text available via subscription   (Followers: 2)
Autonomous Mental Development, IEEE Transactions on     Hybrid Journal   (Followers: 9)
Avances en Ciencias e Ingeniería     Open Access  
Balkan Region Conference on Engineering and Business Education     Open Access   (Followers: 1)
Bangladesh Journal of Scientific and Industrial Research     Open Access  
Basin Research     Hybrid Journal   (Followers: 5)
Batteries     Open Access   (Followers: 6)
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: 4)
BER : Manufacturing Survey : Full Survey     Full-text available via subscription   (Followers: 2)
BER : Motor Trade Survey     Full-text available via subscription   (Followers: 1)
BER : Retail Sector Survey     Full-text available via subscription   (Followers: 2)
BER : Retail Survey : Full Survey     Full-text available via subscription   (Followers: 2)
BER : Survey of Business Conditions in Manufacturing : An Executive Summary     Full-text available via subscription   (Followers: 3)
BER : Survey of Business Conditions in Retail : An Executive Summary     Full-text available via subscription   (Followers: 4)
Bharatiya Vaigyanik evam Audyogik Anusandhan Patrika (BVAAP)     Open Access   (Followers: 1)
Biofuels Engineering     Open Access   (Followers: 1)
Biointerphases     Open Access   (Followers: 1)
Biomaterials Science     Full-text available via subscription   (Followers: 10)
Biomedical Engineering     Hybrid Journal   (Followers: 15)
Biomedical Engineering and Computational Biology     Open Access   (Followers: 14)
Biomedical Engineering Letters     Hybrid Journal   (Followers: 5)
Biomedical Engineering, IEEE Reviews in     Full-text available via subscription   (Followers: 18)
Biomedical Engineering, IEEE Transactions on     Hybrid Journal   (Followers: 34)
Biomedical Engineering: Applications, Basis and Communications     Hybrid Journal   (Followers: 5)
Biomedical Microdevices     Hybrid Journal   (Followers: 9)
Biomedical Science and Engineering     Open Access   (Followers: 4)
Biomedizinische Technik - Biomedical Engineering     Hybrid Journal  
Biomicrofluidics     Open Access   (Followers: 4)
BioNanoMaterials     Hybrid Journal   (Followers: 2)
Biotechnology Progress     Hybrid Journal   (Followers: 39)
Boletin Cientifico Tecnico INIMET     Open Access  
Botswana Journal of Technology     Full-text available via subscription   (Followers: 1)
Boundary Value Problems     Open Access   (Followers: 1)
Brazilian Journal of Science and Technology     Open Access   (Followers: 2)
Broadcasting, IEEE Transactions on     Hybrid Journal   (Followers: 10)
Bulletin of Canadian Petroleum Geology     Full-text available via subscription   (Followers: 14)
Bulletin of Engineering Geology and the Environment     Hybrid Journal   (Followers: 14)
Bulletin of the Crimean Astrophysical Observatory     Hybrid Journal  
Cahiers, Droit, Sciences et Technologies     Open Access  
Calphad     Hybrid Journal  
Canadian Geotechnical Journal     Hybrid Journal   (Followers: 30)
Canadian Journal of Remote Sensing     Full-text available via subscription   (Followers: 44)
Case Studies in Engineering Failure Analysis     Open Access   (Followers: 8)
Case Studies in Thermal Engineering     Open Access   (Followers: 4)
Catalysis Communications     Hybrid Journal   (Followers: 6)
Catalysis Letters     Hybrid Journal   (Followers: 2)
Catalysis Reviews: Science and Engineering     Hybrid Journal   (Followers: 8)
Catalysis Science and Technology     Free   (Followers: 8)
Catalysis Surveys from Asia     Hybrid Journal   (Followers: 3)
Catalysis Today     Hybrid Journal   (Followers: 7)
CEAS Space Journal     Hybrid Journal   (Followers: 2)
Cellular and Molecular Neurobiology     Hybrid Journal   (Followers: 3)
Central European Journal of Engineering     Hybrid Journal   (Followers: 1)
CFD Letters     Open Access   (Followers: 6)
Chaos : An Interdisciplinary Journal of Nonlinear Science     Hybrid Journal   (Followers: 2)
Chaos, Solitons & Fractals     Hybrid Journal   (Followers: 3)
Chinese Journal of Catalysis     Full-text available via subscription   (Followers: 2)
Chinese Journal of Engineering     Open Access   (Followers: 2)
Chinese Science Bulletin     Open Access   (Followers: 1)
Ciencia e Ingenieria Neogranadina     Open Access  
Ciencia en su PC     Open Access   (Followers: 1)
Ciencias Holguin     Open Access   (Followers: 1)
CienciaUAT     Open Access  
Cientifica     Open Access  
CIRP Annals - Manufacturing Technology     Full-text available via subscription   (Followers: 11)
CIRP Journal of Manufacturing Science and Technology     Full-text available via subscription   (Followers: 14)
City, Culture and Society     Hybrid Journal   (Followers: 24)
Clay Minerals     Full-text available via subscription   (Followers: 10)
Clean Air Journal     Full-text available via subscription   (Followers: 2)
Coal Science and Technology     Full-text available via subscription   (Followers: 3)
Coastal Engineering     Hybrid Journal   (Followers: 11)
Coastal Engineering Journal     Hybrid Journal   (Followers: 5)
Coatings     Open Access   (Followers: 4)
Cogent Engineering     Open Access   (Followers: 2)
Cognitive Computation     Hybrid Journal   (Followers: 4)
Color Research & Application     Hybrid Journal   (Followers: 2)
COMBINATORICA     Hybrid Journal  
Combustion Theory and Modelling     Hybrid Journal   (Followers: 14)
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: 27)
Composite Interfaces     Hybrid Journal   (Followers: 6)
Composite Structures     Hybrid Journal   (Followers: 271)
Composites Part A : Applied Science and Manufacturing     Hybrid Journal   (Followers: 199)
Composites Part B : Engineering     Hybrid Journal   (Followers: 257)
Composites Science and Technology     Hybrid Journal   (Followers: 194)
Comptes Rendus Mécanique     Full-text available via subscription   (Followers: 2)
Computation     Open Access  
Computational Geosciences     Hybrid Journal   (Followers: 15)
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: 8)
Computer Science and Engineering     Open Access   (Followers: 19)
Computers & Geosciences     Hybrid Journal   (Followers: 30)
Computers & Mathematics with Applications     Full-text available via subscription   (Followers: 7)
Computers and Electronics in Agriculture     Hybrid Journal   (Followers: 5)
Computers and Geotechnics     Hybrid Journal   (Followers: 11)
Computing and Visualization in Science     Hybrid Journal   (Followers: 6)
Computing in Science & Engineering     Full-text available via subscription   (Followers: 33)
Conciencia Tecnologica     Open Access  
Concurrent Engineering     Hybrid Journal   (Followers: 3)
Continuum Mechanics and Thermodynamics     Hybrid Journal   (Followers: 8)
Control and Dynamic Systems     Full-text available via subscription   (Followers: 9)
Control Engineering Practice     Hybrid Journal   (Followers: 43)
Control Theory and Informatics     Open Access   (Followers: 8)
Corrosion Science     Hybrid Journal   (Followers: 25)
Corrosion Series     Full-text available via subscription   (Followers: 6)
CT&F Ciencia, Tecnologia y Futuro     Open Access   (Followers: 1)

        1 2 3 4 5 6 7 | Last

Journal Cover Biotechnology Progress
  [SJR: 0.736]   [H-I: 101]   [39 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 8756-7938 - ISSN (Online) 1520-6033
   Published by John Wiley and Sons Homepage  [1592 journals]
  • Analysis of Recombinant Human Serum Albumin Extraction & Degradation
           in Transgenic Rice Extracts
    • Authors: Kseniya A. Sheshukova; Lisa R. Wilken
      Abstract: Transgenic plant systems have successfully been used to express recombinant proteins, including rice seed-expressed recombinant human serum albumin (rHSA), without the risk of contamination of human pathogens. Developing an efficient extraction process is critical as the step determines recombinant protein concentration and purity, quantity of impurities, and process volume. This paper evaluates the effect of pH and time on the extraction and stability of rHSA. The amount of rHSA in clarified extract after 60 min of solubilization increased with pH from 0.9 mg/g (pH 3.5) to 9.6 mg/g (pH 6.0), but not over time as 10 min was sufficient for solubilization. Total soluble protein (TSP) in extracts also increased with pH from 3.9 mg/g (pH 3.5) to 19.7 mg/g (pH 6.0) in clarified extract. Extraction conditions that maximized rHSA purity were not optimal for rHSA stability and yield. Extraction at pH 3.5 resulted in high purity (78%), however, rHSA degraded over time. Similar purities (78%) were observed in pH 4.0 extracts yet rHSA remained stable. rHSA degradation was not observed in pH 4.5 and 6.0 extracts but higher native protein concentrations decreased purity. Strategies such as pH and temperature adjustment were effective for reducing rHSA degradation in pH 3.5 rice extracts. Low temperature pH 3.5 extraction retained high purity (97%) and rHSA stability. While seed-expressed recombinant proteins are known to be stable for up to 3 years, the degradation of rHSA was notably extensive (56% within 60 min) when extracted at low pH. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-08T03:33:29.21138-05:0
      DOI: 10.1002/btpr.2609
  • Physicochemical and catalytic properties of acylase I from Aspergillus
           melleus immobilized on amino- and carbonyl-grafted Stöber silica
    • Authors: Agnieszka Kołodziejczak-Radzimska; Jakub Zdarta, Teofil Jesionowski
      Abstract: Acylase I from Aspergillus melleus was immobilized on supports consisting of unmodified and modified silica. Modification was performed using 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA). The effectiveness of immobilization was investigated using the standard Bradford method in addition to a number of physicochemical techniques, including spectroscopic methods (FTIR, 29Si and 13C CP MAS NMR), porous structure and elemental analysis, and zeta potential measurement. A determination of catalytic activity was made based on the deacetylation reaction of N-acetyl-l-methionine. Furthermore, the effect of pH and temperature on the catalytic activity of the free and immobilized enzyme, as well as the reusability of the silica-bound aminoacylase, were determined. The immobilized systems demonstrated a high degree of catalytic activity. The best catalytic parameters were those of aminoacylase immobilized on silica modified with APTES (apparent activity 3937 U/g, relative activity 61.6%). This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-08T03:33:19.2538-05:00
      DOI: 10.1002/btpr.2610
  • Gradual enhancement of ethyl acetate production through promoter
           engineering in Chinese liquor yeast strains
    • Authors: Jian Dong; Kun-Qiang Hong, Ai-Li Hao, Cui-Ying Zhang, Xiao-Meng Fu, Peng-Fei Wang, Dong-Guang Xiao
      Abstract: As content and proportion of ethyl acetate is critical to the flavor and quality of beverages, the concise regulation of the ethyl acetate metabolism is a major issue in beverage fermentations. In the present study, for ethyl acetate yield regulation, we finely modulated the expression of ATF1 through precise and seamless insertion of serially truncated PGK1 promoter from the 3'end by 100bp steps in the Chinese liquor yeast, CLy12a. The three engineered promoters carrying 100-, 200- and 300-bp truncations exhibited reduced promoter strength but unaffected growth. These three promoters were integrated into the CLy12a strain, generating strains CLy12a-P-100, CLy12a-P-200 and CLy12a-P-300, respectively. The transcription levels of CLy12a-P-100, CLy12a-P-200 and CLy12a-P-300 were 20%, 17% and 10% of that of CLy12a-P, respectively. The AATase (Alcohol acetyl transferases, encoded by the ATF1 gene) activity of three engineered strains were 36%, 56% and 62% of that of CLy12a-P. In the liquid fermentation of corn hydrolysate at 30°C, the concentration of ethyl acetate in CLy12a-P-100, CLy12a-P-200 and CLy12a-P-300 were reduced by 28%, 30% and 42%, respectively, compared to CLy12a-P. These results verifying that the ethyl acetate yield could be gradually enhanced by finely modulating the expression of ATF1. The engineered strain CLy12a-P-200 produced the ethyl acetate concentration with the best sensorial quality compared to the other engineered yeast strains. The method proposed in this work supplies a practical proposal for breeding Chinese liquor yeast strains with finely modulated ethyl acetate yield. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:35:54.512383-05:
      DOI: 10.1002/btpr.2605
  • Concomitant reduction of lactate and ammonia accumulation in fed-batch
           cultures: Impact on glycoprotein production and quality
    • Authors: Eric Karengera; Anna Robotham, John Kelly, Yves Durocher, Gregory De Crescenzo, Olivier Henry
      Abstract: Lactate and ammonia accumulation is a major factor limiting the performance of fed-batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by-products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine-free fed-batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2-overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed-batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine-free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 mM and 1 mM, respectively. In flask, this resulted in more than a 2.5-fold increase in IFNα2b titer in comparison to the control glutamine-supplied fed-batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC-MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high quality therapeutic glycoproteins. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:35:52.157494-05:
      DOI: 10.1002/btpr.2607
  • Real-time monitoring of antibody glycosylation site occupancy by in situ
           Raman spectroscopy during bioreactor CHO cell cultures
    • Authors: Meng-Yao Li; Bruno Ebel, Cédric Paris, Fabien Chauchard, Emmanuel Guedon, Annie Marc
      Abstract: The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time- and labor-intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real-time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and non-glycosylated mAbs. By comparing Variable Importance in Projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed-harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and non-glycosylated mAbs could be successfully and simultaneously estimated in real-time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on-line optimization of mAb quality. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:35:43.548707-05:
      DOI: 10.1002/btpr.2604
  • Protein A Chromatography Resin Lifetime - Impact of Feed Composition
    • Authors: Mili Pathak; Katherine Lintern, Daniel G. Bracewell, Anurag S. Rathore
      Abstract: Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This paper focuses on the impact of feed composition on the performance of a typical agarose based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and non-histone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC-MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss or impact on product quality was seen to vary depending upon the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin ageing as a basis for improved process understanding. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:35:36.320702-05:
      DOI: 10.1002/btpr.2608
  • Ectopic expression of EuWRI1, encoding a transcription factor in E.
           ulmoides, changes the seeds oil content in transgenic tobacco
    • Authors: Lu Wang; Xilong Du, YanZhi Feng, Panfeng liu, Jingle Zhu, Lin Zhang, Hongyan Du, Mingguo Ma, Fangdong Li
      Abstract: The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the regulation of fatty acid biosynthesis in developing seeds. In this study, a new WRI1 gene was isolated from seeds of Eucommia ulmoides (E. ulmoides) and named EuWRI1. A close link between gibberellins (GA) signaling and EuWRI1 gene expression was suggested in this study. Functional characterization of EuWRI1 was elucidated through seed-specific expression in tobacco. In transgenic tobacco, the expression of EuWRI1 in eight independent transgenic lines was detected by semi-quantitative RT-PCR. The relative mRNA accumulation of genes encoding enzymes involved in fatty acid biosynthesis (BCCP and KAS1) was also assayed in tobacco seeds. Analysis of the seeds oil content and starch content indicated that the transgenic lines showed a significant increase in seeds oil content, whereas starch content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (16:0), linoleic acid (18:2) and linolenic acid (18:3) increased significantly in seeds of transgenic tobacco lines, but stearic acid (18:0) levels significantly declined. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:31:17.381163-05:
      DOI: 10.1002/btpr.2606
  • Characterizing the Fluid Dynamics of the Inverted Frusto-conical Shaking
    • Authors: Likuan Zhu; Xueting Zhang, Kai Cheng, Zhonghua Lv, Lei Zhang, Qingyong Meng, Shujie Yuan, Boyan Song, Zhenlong Wang
      Abstract: We conducted a three-dimensional computational fluid dynamics (CFD) simulation to calculate the flow field in the Inverted Frusto-conical Shaking Bioreactor with 5 L working volume (IFSB-5L). The CFD models were established for the IFSB-5L at different operating conditions (different shaking speeds and filling volumes) and validated by comparison of the liquid height distribution in the agitated IFSB-5L. The “out of phase” operating conditions were characterized by analyzing the flow field in the IFSB-5L at different filling volumes and shaking speeds. The values of volumetric power consumption (P/VL) and volumetric mass transfer coefficient (kLa) were determined from simulated and experimental results respectively. Finally, the operating condition effect on P/VL and kLa was investigated. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:30:53.551954-05:
      DOI: 10.1002/btpr.2602
  • Short- and Long-Term Effects on mAb-Producing CHO Cell Lines after
    • Authors: Jayashree Subramanian; Rigzen P. S. Aulakh, Parbir S. Grewal, Mark Sanford, Abigail F. J. Pynn, Inn H. Yuk
      Abstract: Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥ 20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:30:30.411462-05:
      DOI: 10.1002/btpr.2599
  • HPV-16 targeted DNA vaccine expression: The role of purification
    • Authors: Ana M. Almeida; Joana Tomás, Patrícia Pereira, João A. Queiroz, Fani Sousa, Ângela Sousa
      Abstract: DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV-16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:30:27.229608-05:
      DOI: 10.1002/btpr.2603
  • Preparation of an injectable doxorubicin surface modified cellulose
           nanofiber gel and evaluation of its anti-tumor and anti-metastasis
           activity in melanoma
    • Authors: Najmeh Alizadeh; Vajihe Akbari, Maryam Nurani, Azade Taheri
      Abstract: Cellulose nanofibers (Cel-NFs) gel can be considered as a useful drug carrier because of its biocompatibility, high specific surface area and high loading capacity of drugs. Injectable Cel-NFs gel could deliver doxorubicin (DOX) for localized chemotherapy of melanoma and suppress melanoma cells migration because of the physical barrier property of Cel-NFs. We prepared DOX surface modified Cel-NFs (DOX-Cel-NFs) gel by the electrostatic attachment of DOX molecules on the surface of Cel-NFs. The increase in the zeta potential of nanofibers and the changes in the FTIR spectra of DOX-Cel-NFs compared to Cel-NFs proved this attachment. DOX-Cel-NFs showed nano-fibrous structure with an average diameter of 22.32 ±10.66 nm after analyzing using FESEM. The suitable injectability of DOX-Cel-NFs gel verified its promising application for the localized chemotherapy. DOX-Cel-NFs gel exhibited a sustained drug release manner. The cytotoxicity results showed that DOX-Cel-NFs were more cytotoxic against melanoma cancer cells than the free DOX during 48 h incubation period. Moreover, DOX-Cel-NFs gel can suppress the melanoma cancer cells migration efficiently. Thus our results emphasize the potential of DOX-Cel-NFs gel as a chemotherapeutic agent for local delivery of DOX in order to treat melanoma and prevent its metastasis. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:30:25.0006-05:00
      DOI: 10.1002/btpr.2598
  • Leveraging Single Pass Tangential Flow Filtration to Enable Decoupling of
           Upstream and Downstream Monoclonal Antibody Processing
    • Authors: Alex Brinkmann; Sanaa Elouafiq, John Pieracci, Matthew Westoby
      Abstract: Decoupling upstream and downstream operations in biopharmaceutical production could enable more flexible manufacturing operations and could allow companies to leverage strategic or financial benefits that would be otherwise unattainable. A decoupling process was developed and scaled-up utilizing single pass tangential flow filtration for volume reduction followed by bulk freezing in single-use bags prior to purification. Single pass tangential flow filtration can be used to continuously concentrate harvested cell culture fluid, reducing the volume by 15-25X with a step yield of greater than 96%. These concentration factors were reproduced with a second product, indicating that the process could be amenable to platform processes. Experimental data indicates that the product tested was stable for at least one year at -40 or -70°C. The concentration of the harvested cell culture fluid, either with or without a subsequent period of frozen storage, had no impact on the product quality attributes that were tested. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:25:30.161425-05:
      DOI: 10.1002/btpr.2601
  • Online measurement of the respiratory activity in shake flasks enables the
           identification of cultivation phases and patterns indicating recombinant
           protein production in various E. coli host strains
    • Authors: Nina Ihling; Natalie Bittner, Sylvia Diederichs, Maximilian Schelden, Anna Korona, Georg Theo Höfler, Alexander Fulton, Karl-Erich Jaeger, Kohsuke Honda, Hisao Ohtake, Jochen Büchs
      Abstract: Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and non-inducing conditions. As model enzymes a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen.Four strains were compared during expression of both enzymes in auto-induction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to non-induced second growth phase, thereby ensuring a fair comparison of different strains.In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-05T03:25:28.050714-05:
      DOI: 10.1002/btpr.2600
  • Preparation and application of sulfated xylan as a flocculant for dye
    • Authors: Zhongming Liu; Dingding Xu, Fangong Kong, Shoujuan Wang, Guihuia Yang, Pedram Fatehi
      Abstract: The main purpose of this paper is to explore the sulfation of xylan to produce an anionic flocculant, sulfated xylan, for removing ethyl violet dye from simulated dye solutions. In this work, xylan was sulfated with chlorosulfonic acid in N, N-dimethylformamide (DMF) solvent and the reaction conditions were optimized using a response surface methodology (RSM). It was observed that the maximum degree of substitution (DS) of 1.1 was obtained for sulfated xylan under the conditions of 3.71 chlorosulfonic acid/xylan molar ratio, 70°C and 7 h reaction time. The resulting sulfated xylan had a charge density of -3.12 mmol/g and molecular weight (Mw) of 22,300 g/mol. Furthermore, elemental and thermogravimetric analyses, Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) confirmed the sulfation of xylan. The application of sulfated xylan as a flocculant for decolorizing the simulated ethyl violet dye wastewater was studied. The results indicated that 97% of dye was removed from 50 mg/L dye solution at the sulfated concentration of 175 mg/L and pH 9, but unmodified xylan was ineffective in flocculating and removing dye segments. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-02T03:25:22.171467-05:
      DOI: 10.1002/btpr.2597
  • Enhanced Expression of a Biosimilar Monoclonal Antibody with a Novel NS0
    • Authors: Darryl Sampey; Pascal Courville, David Acree, Jeffrey Hausfeld, William E. Bentley
      Abstract: The precise product quality and lower cost of goods demands of the growing biosimilars industry are driving biomanufacturing innovation. Biosimilar cell lines that produce complex glycoproteins such as monoclonal antibodies must be both highly productive and express a product with critical quality attributes closely matching those of the innovator reference. In this work, a biomanufacturing platform is described that harnesses the commercially-established NS0 host cell in new ways to create stable, highly productive cell lines with characteristics meeting the current demands. A cholesterol metabolic selection marker and implementation strategy that can be generically applied are shown to yield high expressing cell lines as well as eliminate the need for cholesterol addition, which has been a significant barrier in both stainless steel reactors as well as in single-use plastic systems. Additionally, for the first time, a multiplex selection strategy was implemented that served to increase NS0 cell line specific productivity>10-fold and volumetric yields>6-fold. The best overall performing cell line had a Qp of 28.5 picograms per cell per day was rapidly adapted to a lean production medium. Yields in L-glutamine fed-batch shaker cultures exceeded 500 mg/L. An initial screening of four feeding strategies resulted in a final 13-day yield of over 1.4 g/L in small shaker culture. Overall, this work shows both the strategy to develop biosimilar cell lines and the commercial potential of a novel expression system highly suited for the manufacture of biosimilars of reference biologics currently produced in murine cells. This article is protected by copyright. All rights reserved.
      PubDate: 2018-01-02T03:20:24.234835-05:
      DOI: 10.1002/btpr.2596
  • Probing the importance of clonality: Single cell subcloning of clonally
           derived CHO cell lines yields widely diverse clones differing in growth,
           productivity, and product quality
    • Authors: Peggy Ko; Shahram Misaghi, Zhilan Hu, Dejin Zhan, Joni Tsukuda, Mandy Yim, Mark Sanford, David Shaw, Masaru Shiratori, Brad Snedecor, Michael Laird, Amy Shen
      Abstract: In the past few decades, a large variety of therapeutic antibodies and proteins have been expressed in Chinese hamster ovary (CHO) cells. This mammalian expression system is robust, scalable, relatively inexpensive, and importantly allows for post-translational modifications that are important for some therapeutic proteins. Historically, CHO cell lines were derived from colonies of cells grown in semi-solid or liquid plates using either serum-containing or serum-free media. Current advancements in cell sorting and imaging technologies have allowed for isolating and imaging single cell progenitors at the seeding step, significantly increasing the probability of isolating clonally derived cell lines. However, it is debatable how much population heterogeneity can be eliminated when clonally derived cell lines, originated from a single cell progenitor, are scaled up. To further investigate this phenomenon, we subcloned two different clonally derived (day 0 imaged and visually inspected) cell lines expressing antibody-X. The results showed that when six randomly chosen subclones of each line were evaluated in a production assay, these subclones displayed a range of variation in titer, specific productivity, growth, and product quality attributes. Some subclones displayed variations in transgene copy numbers. Additionally, clonal derivation did not assure stability of the derived cell lines. Our findings show that cell heterogeneity exists in a population even when derived from a single cell progenitor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-12-27T08:42:13.6661-05:00
      DOI: 10.1002/btpr.2594
  • Glutaraldehyde cross-linking increases the stability of Lumbricus
           terrestris erythrocruorin
    • Authors: Athul Rajesh; Devon Zimmerman, Kyle Spivack, Osheiza Abdulmalik, Jacob Elmer
      Abstract: Since donated red blood cells must be constantly refrigerated, they are not available in remote areas and battlefields. We have previously shown that the hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an effective and safe substitute for donated blood that is stable enough to be stored for long periods at the relatively high temperatures that may be encountered in remote areas. The goal of this study was to further increase the thermal stability of LtEc by covalently cross-linking LtEc with glutaraldehyde (gLtEc). Our results show that the melting temperatures of the gLtEc samples steadily increase as the molar ratio of glutaraldehyde to heme increases (from Tm = 57°C for native LtEc up to Tm = 68°C at a ratio of 128:1). In addition, while native LtEc is susceptible to subunit dissociation at alkaline pH (8–10), cross-linking with glutaraldehyde completely prevents dissociation of gLtEc at pH 10. Increasing the molar ratio of glutaraldehyde:heme also significantly increased the oxygen affinity of gLtEc, but this effect was decreased by cross-linking gLtEc in the deoxygenated T state. Finally, while gLtEc samples cross-linked at low G:H ratios (e.g., 2:1) exhibited slight increases in oxidation rate in Tris buffer, no significant difference in oxidation rate was observed between native LtEc and the gLtEc samples in Ringer's Solution, which contains antioxidants. Overall, cross-linking LtEc with glutaraldehyde significantly increases its thermal and structural stability without any loss of function, making gLtEc an attractive blood substitute for deployment in remote areas and battlefields. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-12-20T02:03:24.929738-05:
      DOI: 10.1002/btpr.2593
  • Large-scale culture of a megakaryocytic progenitor cell line with a
           single-use bioreactor system
    • Authors: Retno Wahyu Nurhayati; Yoshihiro Ojima, Takeaki Dohda, Masahiro Kino-oka
      Abstract: The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (kLa) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h−1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2±0.2 × 109 cells L−1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. This article is protected by copyright. All rights reserved.
      PubDate: 2017-12-11T03:00:36.52466-05:0
      DOI: 10.1002/btpr.2595
  • Recent advances on sources and industrial applications of lipases
    • Authors: Nipon Sarmah; D. Revathi, G. Sheelu, K. Yamuna Rani, S. Sridhar, V. Mehtab, C. Sumana
      Abstract: Lipases are the industrially important biocatalysts, which are envisioned to have tremendous applications in the manufacture of a wide range of products. Their unique properties such as better stability, selectivity and substrate specificity position them as the most expansively used industrial enzymes. The research on production and applications of lipases is ever growing and there exists a need to have a latest review on the research findings of lipases. The present review aims at giving the latest and broadest overall picture of research and development on lipases by including the current studies and progressions not only in the diverse industrial application fields of lipases, but also with regard to its structure, classification and sources. Also, a special emphasis has been made on the aspects such as process optimization, modeling, and design that are very critical for further scale-up and industrial implementation. The detailed tabulations provided in each section, which are prepared by the exhaustive review of current literature covering the various aspects of lipase including its production and applications along with example case studies, will serve as the comprehensive source of current advancements in lipase research. This review will be very useful for the researchers from both industry as well as academia in promoting lipolysis as the most promising approaches to intensified, greener and sustainable processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-12-04T09:15:24.914107-05:
      DOI: 10.1002/btpr.2581
  • Sustaining an efficient and effective CHO cell line development platform
           by incorporation of 24-deep well plate screening and multivariate analysis
    • Authors: Alessandro Mora; Sheng (Sam) Zhang, Gerald Carson, Bernard Nabiswa, Patrick Hossler, Seongkyu Yoon
      Abstract: Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24-deep well plate (24-DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24-DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24-DWP achieved the highest productivity improvement upon scaling-up to shake flasks. Product quality of the protein purified from shake flasks and 24-DWP were also compared. The 24-DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N-glycan species, and increase the high-mannose N-glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled-up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24-DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-12-01T01:10:50.874263-05:
      DOI: 10.1002/btpr.2584
  • Strain Engineering to Reduce Acetate Accumulation during Microaerobic
           Growth Conditions in Escherichia coli
    • Authors: Karthik Veeravalli; Tony Schindler, Emily Dong, Masaki Yamada, Ryan Hamilton, Michael W. Laird
      Abstract: Microaerobic (oxygen limited) conditions are advantageous for several industrial applications since a majority of the carbon atoms can be directed for synthesis of desired products. Oxygen limited conditions, however, can result in high levels of undesirable by-products such as acetate which subsequently can have an impact on biomass and product yields. The molecular mechanisms involved in acetate accumulation under oxygen limited conditions are not well understood. Our results indicate that a majority of the genetic modifications known to decrease acetate under aerobic conditions results in similar or even higher acetate under oxygen limitation. Deletion of arcA, whose gene product is a global transcriptional regulator, was the only modification among those evaluated that significantly decreased acetate under both transient and prolonged oxygen limitation. Transcriptome results indicate that the arcA deletion results in an increased expression of the operon involving acs and actP (whose gene products are involved in acetate assimilation and uptake respectively) and some genes in the TCA cycle, thereby promoting increased acetate assimilation. These results provide useful cues for strain design for improved manufacturing of biopharmaceuticals under oxygen limited conditions. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-30T03:30:40.165783-05:
      DOI: 10.1002/btpr.2592
  • Optimizing performance of semi-continuous cell culture in an ambr15™
           microbioreactor using dynamic flux balance modeling
    • Authors: William Kelly; Sorelle Veigne, Xianhua Li, Shyam Sundar Subramanian, Zuyi Huang, Eugene Schaefer
      Abstract: The ambr bioreactors are single-use microbioreactors for cell line development and process optimization. With operating conditions for large-scale biopharmaceutical production properly scaled down, microbioreactors such as the ambr15™ can potentially be used to predict the effect of process changes such as modified media or different cell lines. While there have been some recent studies evaluating the ambr15™ technology as a scale-down model for fed-batch operations, little has been reported for semi-continuous or continuous operation. Gassing rates and dilution rates in the ambr15™ were varied in this study to attempt to replicate performance of a perfusion process at the 5 L scale. At both scales, changes to metabolite production and consumption, and cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15™ bioreactor that produced metabolic shifts and specific metabolic and protein production rates that are characteristic of the corresponding 5 L perfusion process. A dynamic flux balance (DFB) model was employed to understand and predict the metabolic changes observed. The DFB model predicted trends observed experimentally, including lower specific glucose consumption and a switch from lactate production to consumption when dissolved CO2 was maintained at higher levels in the broth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-30T01:20:41.010516-05:
      DOI: 10.1002/btpr.2585
  • Sulfanilic acid-modified chitosan mini-spheres and their application for
           lysozyme purification from egg white
    • Authors: Daniela B. Hirsch; María F. Baieli, Nicolás Urtasun, Juan M. Lázaro- Martínez, Romina J. Glisoni, María V. Miranda, Osvaldo Cascone, Federico J. Wolman
      Abstract: A cation exchange matrix with zwitterionic and multi-modal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico-chemically characterized by ss-NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g−1 while the dissociation constant was 0.074 ± 0.012 mg mL−1. The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP-HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T03:37:24.032214-05:
      DOI: 10.1002/btpr.2588
  • Sodium bicarbonate-gelled chitosan beads as mechanically stable carriers
           for the covalent immobilization of enzymes
    • Authors: Marwa I. Wahba
      Abstract: The poor mechanical stability of chitosan has long impeded its industrial utilization as an immobilization carrier. In this study, the mechanical properties of chitosan beads were greatly improved through utilizing the slow rate of the sodium bicarbonate-induced chitosan gelation and combining it with the chemical cross-linking action of glutaraldehyde (GA). The GA-treated sodium bicarbonate-gelled chitosan beads exhibited much better mechanical properties and up to 2.45-fold higher observed activity of the immobilized enzyme (β-D-galactosidase (β-gal)) when compared to the GA-treated sodium tripolyphosphate (TPP)-gelled chitosan beads. The differences between the sodium bicarbonate-gelled and the TPP-gelled chitosan beads were proven visually and also via scanning electron microscopy, elemental analysis, and differential scanning calorimetry. Moreover, the optimum pH, the optimum temperature, the apparent Km, and the apparent Vmax of the β-gals immobilized onto the two aforementioned types of chitosan beads were determined and compared. A reusability study was also performed. This study proved the superiority of the sodium bicarbonate-gelled chitosan beads as they retained 72.22±4.57% of their initial observed activity during the 13th reusability cycle whereas the TPP-gelled beads lost their activity during the first four reusability cycles, owing to their fragmentation. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T03:26:57.889278-05:
      DOI: 10.1002/btpr.2587
  • Deconvolution of images from 3D printed cells in layers on a chip
    • Authors: Sean Yu; Pranav Joshi, Yi Ju Park, Kyeong-Nam Yu, Moo-Yeal Lee
      Abstract: Layer-by-layer cell printing is useful in mimicking layered tissue structures inside the human body and has great potential for being a promising tool in the field of tissue engineering, regenerative medicine, and drug discovery. However, imaging human cells cultured in multiple hydrogel layers in 3D-printed tissue constructs is challenging as the cells are not in a single focal plane. Although confocal microscopy could be a potential solution for this issue, it compromises the throughput which is a key factor in rapidly screening drug efficacy and toxicity in pharmaceutical industries. With epifluorescence microscopy, the throughput can be maintained at a cost of blurred cell images from printed tissue constructs. To rapidly acquire in-focus cell images from bioprinted tissues using an epifluorescence microscope, we created two layers of Hep3B human hepatoma cells by printing green and red fluorescently labeled Hep3B cells encapsulated in two alginate layers in a microwell chip. In-focus fluorescent cell images were obtained in high throughput using an automated epifluorescence microscopy coupled with image analysis algorithms, including three deconvolution methods in combination with three kernel estimation methods, generating a total of nine deconvolution paths. As a result, a combination of Inter-Level Intra-Level Deconvolution (ILILD) algorithm and Richardson-Lucy (RL) kernel estimation proved to be highly useful in bringing out-of-focus cell images into focus, thus rapidly yielding more sensitive and accurate fluorescence reading from the cells in different layers. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T03:26:51.36976-05:0
      DOI: 10.1002/btpr.2591
  • Interactions between protein molecules and the virus removal membrane
           surface: Effects of immunoglobulin G adsorption and conformational changes
           on filter performance
    • Authors: Ryo Hamamoto; Hidemi Ito, Makoto Hirohara, Ryongsok Chang, Tomoko Hongo-Hirasaki, Tomohiro Hayashi
      Abstract: Membrane fouling commonly occurs in all filter types during virus filtration in protein-based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose-based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post-adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose-based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T03:26:27.529473-05:
      DOI: 10.1002/btpr.2586
  • Bispecific Antibody Process Development: Assembly and Purification of Knob
           and Hole Bispecific Antibodies
    • Authors: Glen Giese; Ambrose Williams, Maricel Rodriguez, Josefine Persson
      Abstract: Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half-antibodies targeting two different antigens were expressed separately in E. coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and re-oxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, non-covalently linked homodimers, covalently linked homodimers, and non-covalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high-throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product-related and process-related impurities. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T03:25:23.904763-05:
      DOI: 10.1002/btpr.2590
  • A factorial design to identify process parameters affecting whole
           mechanically-disrupted rat pancreata in a perfusion bioreactor
    • Authors: Jamie Sharp; Tim WGM Spitters, Patrick Vermette
      Abstract: Few studies report whole pancreatic tissue culture, as it is a difficult task using traditional culture methods. Here, a factorial design was used to investigate the singular and combinational effects of flow, dissolved oxygen concentration (D.O.) and pulsation on whole mechanically-disrupted rat pancreata in a perfusion bioreactor. Whole rat pancreata were cultured for 72h under defined bioreactor process conditions. Secreted insulin was measured and histological (haematoxylin and eosin (H&E)) as well as immunofluorescent insulin staining were performed and quantified. The combination of flow and D.O. had the most significant effect on secreted insulin at 5h and 24h. The D.O. had the biggest effect on tissue histological quality, and pulsation had the biggest effect on the number of insulin-positive structures. Based on the factorial design analysis, bioreactor conditions using high flow, low D.O. and pulsation were selected to further study glucose-stimulated insulin secretion. Here, mechanically-disrupted rat pancreata were cultured for 24h under these bioreactor conditions and were then challenged with high glucose concentration for 6h and high glucose + IBMX (an insulin secretagogue) for a further 6h. These cultures secreted insulin in response to high glucose concentration in the first 6h, however stimulated-insulin secretion was markedly weaker in response to high glucose concentration + IBMX thereafter. After this bioreactor culture period, higher tissue metabolic activity was found compared to that of non-bioreacted static controls. More insulin- and glucagon-positive structures, and extensive intact endothelial structures were observed compared to non-bioreacted static cultures. H&E staining revealed more intact tissue compared to static cultures. This article is protected by copyright. All rights reserved.
      PubDate: 2017-11-29T03:15:24.635253-05:
      DOI: 10.1002/btpr.2589
  • Introduction to special section on using pools to generate Good Laboratory
           Practice tox or other non-clinical material to accelerate development
    • Authors: Christine DeMaria; Trent Munro
      PubDate: 2017-11-28T00:25:30.196975-05:
      DOI: 10.1002/btpr.2583
  • Biotransformation of β-hydroxypyruvate and glycolaldehyde to
           l-erythrulose by Pichia pastoris strain GS115 overexpressing native
    • Authors: Yu-Chia Wei; Stephanie Braun-Galleani, Maria José Henríquez, Sahan Bandara, Darren Nesbeth
      Abstract: Transketolase is a proven biocatalytic tool for asymmetric carbon-carbon bond formation, both as a purified enzyme and within bacterial whole-cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole-cell biocatalysis was investigated using a transketolase-overexpressing strain to catalyze formation of l-erythrulose from β-hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1-4_0150 was subcloned downstream of the methanol-inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed-batch fermentation achieved a maximum l-erythrulose space time yield (STY) of 46.58 g L−1 h−1, specific activity of 155 U gCDW−1, product yield on substrate (Yp/s) of 0.52 mol mol−1 and product yield on catalyst (Yp/x) of 2.23g gCDW−1. We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l-erythrulose. © 2017 The
      Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-20T09:40:50.560093-05:
      DOI: 10.1002/btpr.2577
  • Impact of media and antifoam selection on monoclonal antibody production
           and quality using a high throughput micro-bioreactor system
    • Authors: Sai Rashmika Velugula-Yellela; Abasha Williams, Nicholas Trunfio, Chih-Jung Hsu, Brittany Chavez, Seongkyu Yoon, Cyrus Agarabi
      Abstract: Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology-derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer-based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro-scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro-scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX-Cell Advanced, and OptiCHO media, and 204, C, EX-Cell, SE-15, and Y-30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX-Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25-35 × 106 cells-d/mL, while maintaining specific antibody production (Qp > 2 pg/cell-d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX-Cell, and SE-15 were capable of providing adequate control of foaming while antifoam 204 and Y-30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The
      Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-16T08:25:32.885899-05:
      DOI: 10.1002/btpr.2575
  • High yield purification and first structural characterization of the
           full-length bacterial toxin CNF1
    • Authors: Andrea Colarusso; Marco Caterino, Alessia Fabbri, Carla Fiorentini, Alessandro Vergara, Filomena Sica, Ermenegilda Parrilli, Maria Luisa Tutino
      Abstract: The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-14T07:45:49.448998-05:
      DOI: 10.1002/btpr.2574
  • Effect of propeptide amino acid substitution in γ-carboxylation, activity
           and expression of recombinant human coagulation factor IX
    • Authors: Jafar Vatandoost; Omid Zarei Sani
      Abstract: The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre–propeptide sequences. The propeptide is connected to γ-carboxylase enzyme through the γ-carboxylase recognition site for the direct γ-carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ-carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ-carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT-hFIX-M14 expression cassette, containing cDNA of hFIX with substituted −14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4-fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD-14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated 2-fold enhanced recovery in the S2-expressing hFIXD-14A relative to that expressed native hFIX. These results show that changing −14 residues leads to a decrease in the binding affinity to substrate, increase in γ-carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-11T08:35:30.826114-05:
      DOI: 10.1002/btpr.2582
  • Microfluidic device-assisted etching of p-HEMA for cell or protein
    • Authors: Frank H. Kung; David Sillitti, David I. Shreiber, Jeffrey D. Zahn, Bonnie L. Firestein
      Abstract: The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2-hydroxyethyl methacrylate) (p-HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p-HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p-HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p-HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p-HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-11T08:35:28.494816-05:
      DOI: 10.1002/btpr.2576
  • Modeling of microalgal shear-induced flocculation and sedimentation using
           a coupled CFD-population balance approach
    • Authors: Mohammad Golzarijalal; Farzin Zokaee Ashtiani, Bahram Dabir
      Abstract: In this study, shear-induced flocculation modeling of Chlorella sp. microalgae was conducted by combination of population balance modeling and CFD. The inhomogeneous Multiple Size Group (MUSIG) and the Euler–Euler two fluid models were coupled via Ansys-CFX-15 software package to achieve both fluid and particle dynamics during the flocculation. For the first time, a detailed model was proposed to calculate the collision frequency and breakage rate during the microalgae flocculation by means of the response surface methodology as a tool for optimization. The particle size distribution resulted from the model was in good agreement with that of the jar test experiment. Furthermore, the subsequent sedimentation step was also examined by removing the shear rate in both simulations and experiments. Consequently, variation in the shear rate and its effects on the flocculation behavior, sedimentation rate and recovery efficiency were evaluated. Results indicate that flocculation of Chlorella sp. microalgae under shear rates of 37, 182, and 387 s−1 is a promising method of pre-concentration which guarantees the cost efficiency of the subsequent harvesting process by recovering more than 90% of the biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-11T08:31:15.560897-05:
      DOI: 10.1002/btpr.2580
  • Impact of fly ash pretreatment on aerobic treatment of thermomechanical
           pulping spent liquor
    • Authors: Xiaoqian Chen; Chuanling Si, Pedram Fatehi
      Abstract: This study investigated the impact of biomass-based fly ash (FA) pretreatment on the biodegradability of a thermomechanical pulping spent liquor (TMPL) in an aerobic system. In this study, FA was mixed with TMPL under the conditions of 6 wt.% based on TMPL, 25°C and 10 h, which removed a part of recalcitrant organic materials and resulted in 68.0, 40.0, 60.1, 81.2 and 48.3% reductions in chemical oxygen demand (COD), biochemical oxygen demand (BOD), total organic carbon (TOC), lignin and sugar, respectively. FA-pretreated TMPL pressate (FA-TMPL) was biologically treated in an aerobic system of sequencing batch reactor (SBR). The performance of the biological treatment with and without FA pretreatment was studied in two parallel SBRs over three months. The combination of FA and biological treatments removed 97.3% of COD, 98.3% of BOD, 96.3% of lignin, 99.5% of sugar, and 98.1% of TOC. Without FA pretreatment, the biological system removed 87.3% of COD, 89% of BOD, 81.6% of lignin, 98.6% of sugars, and 90.5% of TOC. The results also confirmed that the settling ability of sludge, which was indicated as a sludge volume index, was reduced from 109.3 mL/g to 53.5 mL/g. In addition, the advantages of using FA pretreatment in aerobic systems were discussed in detail. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-10T10:30:49.217505-05:
      DOI: 10.1002/btpr.2578
  • Accelerating patient access to novel biologics using stable pool-derived
           product for non-clinical studies and single clone-derived product for
           clinical studies
    • Authors: Trent P. Munro; Kim Le, Huong Le, Li Zhang, Jennitte Stevens, Neil Soice, Sabrina A. Benchaar, Robert W. Hong, Chetan T. Goudar
      Abstract: Cell cloning and subsequent process development activities are on the critical path directly impacting the timeline for advancement of next generation therapies to patients with unmet medical needs. The use of stable cell pools for early stage material generation and process development activities is an enabling technology to reduce timelines. To successfully use stable pools during development, it is important that bioprocess performance and requisite product quality attributes be comparable to those observed from clonally derived cell lines. To better understand the relationship between pool and clone derived cell lines, we compared data across recent first in human (FIH) programs at Amgen including both mAb and Fc-fusion modalities. We compared expression and phenotypic stability, bioprocess performance, and product quality attributes between material derived from stable pools and clonally derived cells. Overall, our results indicated the feasibility of matching bioprocess performance and product quality attributes between stable pools and subsequently derived clones. These findings support the use of stable pools to accelerate the advancement of novel biologics to the clinic. © 2017 The
      Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-10T10:25:28.071329-05:
      DOI: 10.1002/btpr.2572
  • Ethanol addition on inactivation of Saccharomyces pastorianus by a
           two-stage system with low-pressure carbon dioxide microbubbles can
           accelerate the cell membrane injury
    • Authors: Fumiyuki Kobayashi; Sachiko Odake
      Abstract: The effect of ethanol on the inactivation of Saccharomyces pastorianus by a two-stage system with low-pressure carbon dioxide microbubbles (two-stage MBCO2) was investigated. Zero and>5 log reductions of S. pastorianus populations suspended in physiological saline (PS) containing 0% and 10% ethanol, respectively, occurred by the two-stage MBCO2 at a mixing vessel pressure of 1 MPa and a heating coil temperature of 40°C. Conversely, the detected number of surviving S. pastorianus cells in PS containing 5% ethanol was higher in yeast and mold agar (YMA, an optimum agar) than YMA with 2.5% sodium chloride, followed by yeast nitrogen base agar (YNBA, a minimum agar). The fluorescence polarization of S. pastorianus in PS containing 5% and 10% ethanol increased similarly with exposure time in the heating coil of two-stage MBCO2 and was correlated with the surviving cell number measured in YNBA. The intracellular pH (pHin) of S. pastorianus in PS containing 5% ethanol decreased linearly with exposure time in the heating coil of two-stage MBCO2. Also, the pHin-lowering of S. pastorianus in PS containing 10% ethanol was drastically caused by two-stage MBCO2 at 1 min exposure time in the heating coil but then stayed constant until 5 min, agreeing with the inactivation efficiency. Therefore, ethanol in S. pastorianus suspension was suggested to accelerate the cell membrane injury caused by two-stage MBCO2. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-10T10:25:25.559278-05:
      DOI: 10.1002/btpr.2579
  • The possible causal relationship between fragmentation of genomic DNA and
           formation of viable, but non-culturable probiotic bacteria upon storage in
           dry state
    • Authors: Marie-Louise R. W. Hansen; Morten Arendt Ramsussen, Thomas Skov, Anders Clausen, Jens Risbo
      Abstract: In this study, the aim was to establish if loss of DNA integrity is a cause of loss of culturability for probiotic bacteria during storage in dry state. The number of colony forming units (CFU), number of metabolically active cells, and DNA integrity during dry storage of probiotic strains, B. animalis subsp. lactis BB-12 and L. acidophilus LA-5, were investigated. The probiotic strains were freeze-dried and stored at 20°C, with and without oxygen present, and at water activity levels 0.22 or 0.32. Dry storage resulted in a decrease in CFU during the entire storage period. The number of metabolically active cells was unchanged during storage of B. animalis subsp. lactis BB-12, but did decrease during the first week of storage of L. acidophilus LA-5. Loss of DNA integrity was evident for both strains during storage and correlated well with the loss of CFU. Both loss of CFU and loss of DNA integrity were significantly greater for both strains when oxygen was present and when aw was increased. Statistical analysis indicates a possible causal relationship between DNA degradation and loss of culturability and this idea is consistent with the function of DNA at cell division. The study contributes with new knowledge of the cause for loss of CFU during dry storage of probiotic bacteria, which possibly can aid in the improvement of preservation techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-06T01:40:28.407861-05:
      DOI: 10.1002/btpr.2573
  • Cold shocks of Anammox biofilm stimulate nitrogen removal at low
    • Authors: V. Kouba; R. Darmal, D. Vejmelkova, P. Jenicek, J. Bartacek
      Abstract: The adaptation of Anammox (ANaerobic AMMonium OXidation) to low temperatures (10–15°C) is crucial for sustaining energy-efficient nitrogen removal from the mainstream of municipal wastewater. But, current adaptation methods take months or even years. To speed up the adaption of Anammox to low temperatures, this study describes a new approach: exposing Anammox microorganisms to an abrupt temporary reduction of temperature, i.e., cold shock. Anammox biomass in a moving bed biofilm reactor was subjected to three consecutive cold shocks (reduction from 24 ± 2 to 5.0 ± 0.2°C), each taking eight hours. Before the cold shocks, Anammox activity determined in ex situ tests using the temperature range of 12.5–19.5°C was 0.005–0.015 kg-N kg-VSS−1 day−1. Cold shocks increased the activity of Anammox at 10°C to 0.054 kg-N kg-VSS−1 day−1 after the third shock, which is similar to the highest activities obtained for cold-enriched or adapted Anammox reported in the literature (0.080 kg-N kg-VSS−1 day−1). Fluorescence in situ hybridization analysis showed that Ca. Brocadia fulgida was the dominant species. Thus, cold shocks are an intriguing new strategy for the adaptation of Anammox to low temperature. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-03T01:00:34.349454-05:
      DOI: 10.1002/btpr.2570
  • Differential transcriptomic analysis reveals hidden light response in
           Streptomyces lividans
    • Authors: Joachim Koepff; Holger Morschett, Tobias Busche, Anika Winkler, Jörn Kalinowski, Wolfgang Wiechert, Marco Oldiges
      Abstract: Recently, a comprehensive screening workflow for the filamentous bacterium Streptomyces lividans, a highly performant source for pharmaceutically active agents was introduced. This framework used parallelized cultivation in microtiter plates to efficiently accelerate early upstream process development. Focusing on growth performance, cultivation was successfully scaled-up to 1 L stirred tank reactors. However, metabolic adaptation was observed on the transcriptomic level as among others, several genes incorporated in light response were upregulated during bioreactor cultivation. Despite it was assumed that this was attributed to the fact that reactor cultivations were performed in glass vessels exposed to daylight and artificial room light, this setup did not allow distinguishing exclusively between light and other effects. Upon that, the present study directly investigates the influence of light by defined illumination of microtiter plate cultures. Almost identical growth performance was observed for cultures grown in the dark or with illumination. Transcriptomics revealed the upregulation of seven genes of which 6 have previously been described to be relevant for carotenoid synthesis and its regulation. These pigments are effective quenchers of reactive oxygen species. The seventh transcript coded for a photo-lyase incorporated in UV-damage repair of DNA further confirming induced light response. However, this was fully compensated by metabolic adaptation on the transcriptomic level and overall process performance was maintained. Consequently, environmental conditions need extremely careful control and evaluation during in-depth omics analysis of bioprocesses. Otherwise metabolic adaptation induced by such issues can easily be misinterpreted, especially during studies addressing cultivation system comparisons. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-11-03T01:00:24.408001-05:
      DOI: 10.1002/btpr.2566
  • Evaluating manufacturing process profile comparability with multivariate
           equivalence testing: Case study of cell-culture small scale model transfer
    • Authors: Yang Cao; Daniel Obeng, Guodong Hui, Luting Xue, Yukun Ren, Xianjie Yu, Fei Wang, Chad Atwell
      Abstract: This article studies the Generalized Mahalanobis Distance (GMD) approach proposed by Hoffelder which measures the dissimilarity of two multivariate Gaussian distributions with arbitrary covariance matrices and unequal sample sizes. This investigation demonstrated that, with appropriate adjustment, the GMD approach can achieve the targeted nominal Type I error and provide sufficient power for testing equivalence between two profile populations. The adjusted GMD approach was applied to examine the equivalence of harvest profiles between a 12L small scale model and 2000L manufacturing scale in a transfer study performed at Sanofi Specialty Care Framingham Biologics. The harvest profiles were evaluated based on three critical parameters (Productivity, Lactate Production, and Total Cell Density) and deemed practically equivalent using a pre-defined equivalence margin per the adjusted GMD approach. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-30T08:07:09.728517-05:
      DOI: 10.1002/btpr.2571
  • Lentivirus-mediated disintegrin and metalloproteinase 17 RNA interference
           reversed the acquired resistance to gefitinib in lung adenocarcinoma cells
           in vitro
    • Authors: Ya-Qing Li; Yuan-Shun Liu, Xi-Wang Ying, Hong-Bin Zhou, Zhehua Wang, Sheng-Chang Wu, Jian-Ping Yan, Yu-Ting Jing, Yang Yang
      Abstract: Objective: The aim of the study is to evaluate the effects of silencing a disintegrin and metalloproteinase 17 (ADAM17) gene expression by lentivirus-mediated RNA interference (RNAi) in the gefitinib-resistant lung adenocarcinoma cells, and then to explore whether the recombinant lentivirus mediated ADAM17 RNAi reversed the acquired resistance of lung adenocarcinoma to gefitinib in vitro.Methods: The gefitinib-resistant RPC-9 cells were established and the mutations of EGFR were detected by gene sequencing. The ADAM17 shRNA expression vectors were constructed and packaged to recombinant lentivirus. The cell proliferation viability was detected by MTT, and cellular apotosis was analyzed by flow cytometry assay. The expression levels of ADAM17, EGFR and the phosphorylated EGFR were respectively detected by reverse transcription polymerase chain reaction and western blot. TGF-α production in the supernatant was detected by enzyme-linked immunosorbent assay.Results: The gefitinib-resistant RPC-9 cells in which mutated EGFR (exon 20) carried 790T > T/M mutation were established. When the concentrations of gefitinib were less than 10μmol/L, there were no significant changes in the apoptosis and cellular proliferation of RPC-9 with the dose-escalation of gefitinib. The cell proliferation viability of RPC-9 was significantly decreased by lentivirus mediated ADAM17 RNAi (P  0.05). Gefitinib had no significant effects on TGF alpha production in the supernatants (P > 0.05). Gefitinib did not inhibit EGFR expression in gefitinib-sensitive PC-9 and gefitinib-resistant RPC-9 cells (P > 0.05). The phosphorylation of EGFR in gefitinib-sensitive PC-9 cells was significantly inhibited by gefitinib (P  0.05). Lentivirus mediated ADAM17 RNAi significantly inhibited the mRNA and protein expression of ADAM17 in gefitinib-resistant RPC-9 cells (P 
      PubDate: 2017-10-27T01:10:45.959625-05:
      DOI: 10.1002/btpr.2564
  • Development of a high-throughput microscale cell disruption platform for
           Pichia pastoris in rapid bioprocess design
    • Authors: Benjamin A. F. Bláha; Stephen A. Morris, Olotu W. Ogonah, Sophie Maucourant, Vincenzo Crescente, William Rosenberg, Tarit K. Mukhopadhyay
      Abstract: The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-16T09:52:17.221528-05:
      DOI: 10.1002/btpr.2555
  • Polyhydroxyalkanoates: Properties and chemical modification approaches for
           their functionalization
    • Authors: Zulfiqar Ali Raza; Shahina Riaz, Ibrahim M. Banat
      Abstract: Polyhydroxyalkanoates (PHAs) have become an attractive biomaterial in research in the past few years due to their extensive potential industrial applications. Being long chain hydroxyl fatty acid molecules, the PHAs are hydrophobic in nature, and have less functional groups. These features limit their applications in various areas. To enhance their usage, these polymers may need to be modified including surface and chemical modifications. Such modifications may alter their mechanical properties, surface structure, amphiphilic character and rate of degradation to fulfil the requirements for their future applications. Chemical modifications allow incorporation of functional groups to PHAs that could not be introduced through biotechnological methods. These chemically reformed PHAs, with enhanced properties, could be used for broad range of applications. This review aims to introduce different chemical modification approaches including some recent methods that had not been explored or discussed so far for PHAs as possible technologies for widening the range of product and application potentials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-16T09:51:51.453192-05:
      DOI: 10.1002/btpr.2565
  • A uniform-shear rate microfluidic bioreactor for real-time study of
           proplatelet formation and rapidly-released platelets
    • Authors: Andres F. Martinez; Richard D. McMahon, Marc Horner, William M. Miller
      Abstract: Platelet transfusions, with profound clinical importance in blood clotting and wound healing, are entirely derived from human volunteer donors. Hospitals rely on a steady supply of donations, but these methods are limited by a 5-day shelf life, the potential risk of contamination, and differences in donor/recipient histocompatibility. These challenges invite the opportunity to generate platelets ex vivo. Although much progress has been made in generating large numbers of culture-derived megakaryocytes (Mks, the precursor cells to platelets), stimulating a high percentage of Mks to undergo platelet release remains a major challenge. Recent studies have demonstrated the utility of shear forces to enhance platelet release from cultured Mks. In this study, we performed a computational fluid dynamics (CFD) analysis of several published platelet microbioreactor systems, and used the results to develop a new 7-µm slit bioreactor—with well-defined flow patterns and uniform shear profiles. This uniform-shear-rate bioreactor (USRB-7µm) permits real-time visualization of the proplatelet (proPLT) formation process and the rapid-release of individual platelet-like-particles (PLPs), which has been observed in vivo, but not previously reported for platelet bioreactors. We showed that modulating shear forces and flow patterns had an immediate and significant impact on PLP generation. Surprisingly, using a single flow instead of dual flows led to an unexpected six-fold increase in PLP production. By identifying particularly effective operating conditions within a physiologically relevant environment, this USRB-7µm will be a useful tool for the study and analysis of proPLT/PLP formation that will further understanding of how to increase ex vivo platelet release. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-13T09:56:58.353151-05:
      DOI: 10.1002/btpr.2563
  • Heterologous erythromycin production across strain and plasmid
    • Authors: Lei Fang; Marc Guell, George M. Church, Blaine A. Pfeifer
      Abstract: The establishment of erythromycin production within the heterologous host E. coli marked an accomplishment in genetic transfer capacity. Namely, over 20 genes and 50 kb of DNA was introduced to E. coli for successful heterologous biosynthetic reconstitution. However, the prospect for production levels that approach those of the native host requires the application of engineering tools associated with E. coli. In this report, metabolic and genomic engineering were implemented to improve the E. coli cellular background and the plasmid platform supporting heterologous erythromycin formation. Results include improved plasmid stability and metabolic support for biosynthetic product formation. Specifically, the new plasmid design for erythromycin formation allowed for ≥89% stability relative to current standards (20% stability). In addition, the new strain (termed LF01) designed to improve carbon flow to the erythromycin biosynthetic pathway provided a 400% improvement in titer level. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-13T09:55:39.609575-05:
      DOI: 10.1002/btpr.2567
  • Production of free fatty acids from switchgrass using recombinant
           Escherichia coli
    • Authors: Jung-Eun Lee; Praveen V. Vadlani, Yadhu N. Guragain, Ka-Yiu San, Doo-Hong Min
      Abstract: Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake-flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass-based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-13T09:55:35.349691-05:
      DOI: 10.1002/btpr.2569
  • Biogenic synthesis of copper oxide nanoparticles using olea europaea leaf
           extract and evaluation of their toxicity activities: An in vivo and in
           vitro study
    • Authors: Ghassan M. Sulaiman; Amer T. Tawfeeq, Marwa D. Jaaffer
      Abstract: Copper oxide nanoparticles (CUNPs) were synthesized using Olea europaea leaf extract as reducing and protecting agent. The formation of nanoparticles was observed through a color change from yellowish to brownish black. The CUNPs were confirmed with UV–Vis spectrophotometer, which revealed a peak absorbance at 289 nm. The synthesized CUNPs were characterized by XRD, FTIR, SEM, and TEM. The XRD pattern revealed that CUNPs were crystalline in nature with a diameter around 20 nm. FTIR spectral analysis showed that CUNPs were capped with plant constituents. From SEM and TEM analyses, the CUNPs were generally found to be spherical in shape, and the size range was 20–50 nm. Free radical scavenging potential of CUNPs against DPPH was confirmed by its stable antioxidant effects. In addition, the toxicity of CUNPs in mice was also assessed by body weight and weights of liver, kidneys, spleen, and thymus. The immune response in mice was signaled through an obvious change in spleen and thymus index, with a decrease of ADA enzyme activity in serum, spleen, and thymus after CUNPs treatment. The CUNPs were found to exert cell growth arrest against AMJ-13 and SKOV-3 cancer cells in a dose-dependent manner and induce cell death by apoptosis. Less significant cytotoxic effect was observed in normal dermal fibroblast cells. These findings suggest that CUNPs may have the potential to be anticancer agents. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-12T10:15:38.383448-05:
      DOI: 10.1002/btpr.2568
  • Polymerization of coniferyl alcohol by Mn3+-mediated (enzymatic)
           oxidation: Effects of H2O2 concentration, aqueous organic solvents, and pH
    • Authors: Roberto Taboada-Puig; Thelmo A. Lú-Chau, María T. Moreira, Gumersindo Feijoo, Juan M. Lema, Kurt Fagerstedt, Taina Ohra-Aho, Tiina Liitiä, Harri Heikkinen, Jarmo Ropponen, Tarja Tamminen
      Abstract: The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)-malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn2+ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5 ≤ pH ≤ 6.0 and with 0.75 ≤ H2O2:CA ratio ≤ 1. The results obtained from the Mn3+-malonate-mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis-GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H2O2/CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of β–β linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-10T09:46:53.435857-05:
      DOI: 10.1002/btpr.2562
  • Development and characterization of an automated imaging workflow to
           generate clonally-derived cell lines for therapeutic proteins
    • Authors: David Shaw; Mandy Yim, Joni Tsukuda, John C. Joly, Andy Lin, Brad Snedecor, Michael W. Laird, Steven E. Lang
      Abstract: In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high-throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally-derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single-cell to>99.9%. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-10T09:46:28.231888-05:
      DOI: 10.1002/btpr.2561
  • Beating the odds: The poisson distribution of all input cells during
           limiting dilution grossly underestimates whether a cell line is
           clonally-derived or not
    • Authors: Yizhou Zhou; David Shaw, Cynthia Lam, Joni Tsukuda, Mandy Yim, Danming Tang, Salina Louie, Michael W. Laird, Brad Snedecor, Shahram Misaghi
      Abstract: Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-09T00:50:33.174468-05:
      DOI: 10.1002/btpr.2560
  • Efficient secretion of three fungal laccases from Saccharomyces cerevisiae
           and their potential for decolorization of textile industry effluent—A
           comparative study
    • Authors: Zuzana Antošová; Klára Herkommerová, Iva Pichová, Hana Sychrová
      Abstract: Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-05T09:20:30.317205-05:
      DOI: 10.1002/btpr.2559
  • Cryoconcentration of flavonoid extract for enhanced biophotovoltaics and
           pH sensitive thin films
    • Authors: A. Demirbas; K. Groszman, M. Pazmiño-Hernandez, D. C. Vanegas, B. Welt, J. A. Hondred, N. T. Garland, J. C. Claussen, E. S. McLamore
      Abstract: Flavonoids are important value added products for dye sensitized solar cells biosensors, functional foods, medicinal supplements, nanomaterial synthesis, and other applications. Brassica oleracea contains high levels of anthocyanins in leaf sap vacuoles, and there are many viable extraction techniques that vary in terms of simplicity, environmental impact, cost, and extract photochemical/electrochemical properties. The efficiency of value added biotechnologies from flavonoid is a function of anthocyanin activity/concentration and molecule stability (i.e., ability to retain molecular resonance under a wide range of conditions). In this paper, we show that block cryoconcentration and partial thawing of anthocyanin from B. oleracea is a green, facile, and highly efficient technique that does not require any special equipment or protocols for producing enhanced value added products. Cryoconcentration increased anthocyanin activity and total phenol content approximately 10 times compared with common extraction techniques. Cryoconcentrated extract had enhanced electrochemical properties (higher oxidation potential), improved chroma, and higher UV absorbance than extract produced with other methods for a pH range of 2–12, with minimal effect on the diffusion coefficient of the extract. As a proof of concept for energy harvesting and sensor applications, dye sensitized solar cells and pH-sensitive thin films were prepared and tested. These devices were comparable with other recently published biotechnologies in terms of efficacy, but did not require expensive/environmentally detrimental extraction or concentration methods. This low cost, biorenewable, and simple method can be used for development of a variety of value added products. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-10-04T01:25:40.195569-05:
      DOI: 10.1002/btpr.2557
  • Effects of pH, conductivity, host cell protein, and DNA size distribution
           on DNA clearance in anion exchange chromatography media
    • Authors: Melani C. Stone; Jon Borman, Gisela Ferreira, P. David Robbins
      Abstract: Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process-related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ-650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6–8), conductivity (2–15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a “size exclusion qPCR assay.” Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The
      Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-30T01:55:22.84332-05:0
      DOI: 10.1002/btpr.2556
  • Isotope labeling to determine the dynamics of metabolic response in CHO
           cell perfusion bioreactors using MALDI-TOF-MS
    • Authors: Daniel J. Karst; Robert F. Steinhoff, Marie R. G. Kopp, Miroslav Soos, Renato Zenobi, Massimo Morbidelli
      Abstract: The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13C6 glucose in the bioreactor and thus the intracellular 13C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-07T23:26:21.521975-05:
      DOI: 10.1002/btpr.2539
  • Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the
    • Authors: Chapman Wright; Christina Alves, Rashmi Kshirsagar, John Pieracci, Scott Estes
      Abstract: The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high-quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time-to-market led the cell engineering group to develop an expression system that can enable this strategy. We have developed a clonal Chinese Hamster Ovary (CHO) host cell line that can routinely produce consistent antibody material at high titers throughout the cell line generation process. This host line enables faster delivery of early phase material through use of the highly productive stable pool or a mixture of high performance clones. Due to unique characteristics of this cell line, the product quality of material from early cell populations is very comparable to material from the final clones. This lends itself to a “fast-to-tox” strategy whereby toxicology studies can be performed with representative material from an earlier cell population, thus accelerating the clinical timelines. Our new clonal host offers robust and consistent performance that enables a highly productive, flexible process and faster preclinical timelines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-07T23:20:50.5139-05:00
      DOI: 10.1002/btpr.2548
  • Response to “Statistical analysis of data from limiting dilution cloning
           to assess monoclonality in generating manufacturing cell lines”
    • Authors: James Savery; Krista Evans
      PubDate: 2017-09-06T09:17:56.971447-05:
      DOI: 10.1002/btpr.2547
  • Effect of zinc chloride and PEG concentrations on the critical flux during
           tangential flow microfiltration of BSA precipitates
    • Authors: Zhao Li; Andrew L. Zydney
      Abstract: There is renewed interest in the possibility of using precipitation for initial capture of high value therapeutic proteins as part of an integrated continuous downstream process. These precipitates can be continuously washed using tangential flow filtration, with long term operation achieved by operating the membrane modules below the critical filtrate flux for fouling. Our hypothesis was that the critical flux for the precipitated protein would be a function of the properties of the precipitate as determined by the precipitation conditions. We evaluated the critical flux using a flux-stepping procedure for model protein precipitates (bovine serum albumin) generated using a combination of a crosslinking agent (zinc chloride) and an excluded volume precipitant (polyethylene glycol [PEG]). The critical flux varied with shear rate to approximately the 1/3 power, consistent with predictions of the classical polarization model. The critical flux increased significantly with increasing zinc chloride concentration, going from 60 L/m2/h for a 2 mM ZnCl2 solution to 200 L/m2/h for an 8 mM ZnCl2 solution. In contrast, the critical flux achieved a maximum value at an intermediate PEG concentration. Independent measurements of the effective size and viscosity of the protein precipitates were used to obtain additional understanding of the effects of ZnCl2 and PEG on the precipitation and the critical flux. These results provide important insights into the development of effective tangential flow filtration systems for processing large quantities of precipitated protein as would be required for large scale continuous protein purification by precipitation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-04T23:51:18.987653-05:
      DOI: 10.1002/btpr.2545
  • Experimental design and metabolic flux analysis tools to optimize
           industrially relevant Haemophilus influenzae type b growth medium
    • Authors: Mateus Ribeiro da Silva; Carla Andreia Freixo Portela, Silvia Maria Ferreira Albani, Paola Rizzo de Paiva, Martha Massako Tanizaki, Teresa Cristina Zangirolami
      Abstract: Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-04T23:51:15.967435-05:
      DOI: 10.1002/btpr.2546
  • Yersiniabactin metal binding characterization and removal of nickel from
           industrial wastewater
    • Authors: Nicholas J. Moscatello; Blaine A. Pfeifer
      Abstract: Yersiniabactin (Ybt) is a metal-binding natural product that has been re-purposed for water treatment. The early focus of this study was the characterization of metal binding breadth attributed to Ybt. Using LC-MS analysis of water samples exposed to aqueous and surface-localized Ybt, quantitative assessment of binding was completed with metals that included Pd2+, Mg2+, and Zn2+. In total, Ybt showed affinity for 10 metals. Next, Ybt-modified XAD-16N resin (Ybt-XAD) was utilized to quantify the affinity for metal removal, showing a rank order of Fe3+ > Ga3+ > Ni2+ > Cu2+ > Cr2+≈Zn2+ > Co2+ > Pd2+ > Mg2+ > Al3+, and in the applied treatment of wastewater from a local precious metal plating company, showing selective removal of nickel from the aqueous effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-04T23:46:21.104292-05:
      DOI: 10.1002/btpr.2542
  • pH variations during diafiltration due to buffer nonidealities
    • Authors: Youngbin Baek; Deyu Yang, Nripen Singh, Abhiram Arunkumar, Sanchayita Ghose, Zheng Jian Li, Andrew L. Zydney
      Abstract: Diafiltration is used for final formulation of essentially all biotherapeutics. Several studies have demonstrated that buffer/excipient concentrations in the final diafiltered product can be different than that in the diafiltration buffer due to interactions between buffer species and the protein product. However, recent work in our lab has shown variations in solution pH that are largely independent of the protein concentration during the first few diavolumes. Our hypothesis is that these pH variations are due to nonidealities in the acid-base equilibrium coefficient. A model was developed for the diafiltration process accounting for the ionic strength dependence of the pKa. Experimental results obtained using phosphate and histidine buffers were in excellent agreement with model predictions. A decrease in ionic strength leads to an increase in the pKa for the phosphate buffer, causing a shift in the solution pH, even under conditions where the initial feed and the diafiltration buffer are at the same pH. This effect could be eliminated by matching the ionic strength of the feed and diafiltration buffer. The experimental data and model provide new insights into the factors controlling the pH profile during diafiltration processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-09-04T23:45:47.902468-05:
      DOI: 10.1002/btpr.2544
  • A system identification approach for developing model predictive
           controllers of antibody quality attributes in cell culture processes
    • Authors: Brandon Downey; John Schmitt, Justin Beller, Brian Russell, Anthony Quach, Elizabeth Hermann, David Lyon, Jeffrey Breit
      Abstract: As the biopharmaceutical industry evolves to include more diverse protein formats and processes, more robust control of Critical Quality Attributes (CQAs) is needed to maintain processing flexibility without compromising quality. Active control of CQAs has been demonstrated using model predictive control techniques, which allow development of processes which are robust against disturbances associated with raw material variability and other potentially flexible operating conditions. Wide adoption of model predictive control in biopharmaceutical cell culture processes has been hampered, however, in part due to the large amount of data and expertise required to make a predictive model of controlled CQAs, a requirement for model predictive control. Here we developed a highly automated, perfusion apparatus to systematically and efficiently generate predictive models using application of system identification approaches. We successfully created a predictive model of %galactosylation using data obtained by manipulating galactose concentration in the perfusion apparatus in serialized step change experiments. We then demonstrated the use of the model in a model predictive controller in a simulated control scenario to successfully achieve a %galactosylation set point in a simulated fed-batch culture. The automated model identification approach demonstrated here can potentially be generalized to many CQAs, and could be a more efficient, faster, and highly automated alternative to batch experiments for developing predictive models in cell culture processes, and allow the wider adoption of model predictive control in biopharmaceutical processes. © 2017 The
      Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-08-24T00:45:55.324724-05:
      DOI: 10.1002/btpr.2537
  • Quantitative Evaluation of Recombinant Protein Packaged into Outer
           Membrane Vesicles of Escherichia coli Cells
    • Authors: Yoshihiro Ojima; Kyota Yamaguchi, Masahito Taya
      Abstract: Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-08-21T10:25:31.992603-05:
      DOI: 10.1002/btpr.2536
  • Optimal control for micro-algae on a raceway model
    • Authors: Todd Hurst; Volker Rehbock
      Abstract: We apply numerical optimal control methods to an existing algae growth model with the aim to determine the best performance of the model under known conditions using a variety of decision variables. We transform the system of differential algebraic equations in the existing model to a system of ordinary differential equations which introduces dynamics for average light intensity and chlorophyll. In addition, we allow for variable nitrogen concentration of the inflow as well as variable initial nitrogen concentration of the raceway. Our main focus is on optimizing of the production of lipids. We calculate both open and closed loop optimal controllers and test their robustness. Finally, we also consider raceway depth as a decision variable. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-08-11T20:35:49.794445-05:
      DOI: 10.1002/btpr.2532
  • High throughput automated microbial bioreactor system used for clone
           selection and rapid scale-down process optimization
    • Authors: M. Lourdes Velez-Suberbie; John P. J. Betts, Kelly L. Walker, Colin Robinson, Barney Zoro, Eli Keshavarz-Moore
      Abstract: High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The
      Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-08-10T00:21:09.285015-05:
      DOI: 10.1002/btpr.2534
  • Characterization of a cathepsin D protease from CHO cell-free medium and
           mitigation of its impact on the stability of a recombinant therapeutic
    • Authors: Amareth Lim; Brandon L. Doyle, Gerard M. Kelly, Angelia M. Reed-Bogan, Lawrence H. Breen, Parviz A. Shamlou, Peter K. Lambooy
      Abstract: During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH. The presence of residual levels of protease activity in purified protein batches affected the inherent activity of the product when stored as a solution. To develop a robust purification strategy to minimize this undesirable impact, identification and characterization of this protease was essential to ultimately ensure that a solution formulation was stable for many years. A protease was isolated from CHO cell-free medium (CFM) using a combination of immobilized pepstatin-A agarose chromatography and size exclusion chromatography (SEC). The isolated protease has significant proteolytic activity at pH ∼ 3 to neutral pH and was identified as cathepsin D by mass spectrometry. Analytical SEC, chip-based capillary gel electrophoresis, imaged capillary isoelectric focusing (cIEF), and circular dichroism (CD) spectropolarimetry analyses were performed for additional characterization of the protease. The identification and characterization of this protease enabled the development of a robust purification process by implementation of a controlled temperature inactivation unit operation (heat inactivation) that enabled essentially complete inactivation of the protease, resulting in the production of a stable drug product that had not been possible using column chromatography alone. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-08-09T00:50:24.213097-05:
      DOI: 10.1002/btpr.2530
  • Novel fluid shear-based dissociation device for improved single cell
           dissociation of spheroids and cell aggregates
    • Authors: Ursula L. Triantafillu; Jaron N. Nix, Yonghyun Kim
      Abstract: Biological industries commonly rely on bioreactor systems for the large-scale production of cells. Cell aggregation, clumping, and spheroid morphology of certain suspension cells make their large-scale culture challenging. Growing stem cells as spheroids is indispensable to retain their stemness, but large spheroids (>500 µm diameter) suffer from poor oxygen and nutrient diffusion, ultimately resulting in premature cell death in the centers of the spheroids. Despite this, most large-scale bioprocesses do not have an efficient method for dissociating cells into single cells, but rely on costly enzymatic dissociation techniques. Therefore, we tested a proof-of-concept fluid shear-based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates. Our prototype was able to dissociate cells while retaining high viability and low levels of apoptosis. The dissociator also did not impact long-term cell growth or spheroid formation. Thus, the dissociator introduced here has the potential to replace traditional dissociation methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-31T23:46:19.908042-05:
      DOI: 10.1002/btpr.2528
  • High-performance of Agaricus blazei fungus for the biological pretreatment
           of elephant grass
    • Authors: Thais Dal Picolli; Kaliane Regalin Aver, Roselei Claudete Fontana, Marli Camassola
      Abstract: Biological pre-treatment seems to be promising being an eco-friendly process, with no inhibitor generated during the process. The potential for elephant grass pre-treatment with white degradation fungi Pleurotus ostreatus, Agaricus blazei, Lentinula edodes, Pleurotus citrinopileatus, and Pleurotus djamor, in isolated or mixed cultures of these strains, was evaluated. The highest activities of enzymes involved in the degradation of lignocellulosic biomass (laccases, endoglucanases, xylanases, and β-glucosidases) were observed for A. blazei, L. edodes and the combination of P. ostreatus and A. blazei. In the enzymatic hydrolysis, there was greater release of reducing sugars in the pre-treated elephant grass samples by A. blazei during 10 days (338.91 ± 7.39 mg g−1 of biomass). For this sample, higher lignin reductions, 24.81 and 57.45%, after 15 and 35 days of incubation, respectively, were also verified. These data indicate the potential of macromycetes such as A. blazei to perform biological pre-treatments. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-31T23:46:15.908154-05:
      DOI: 10.1002/btpr.2529
  • Biophysical characterization of layer-by-layer synthesis of aptamer-drug
           microparticles for enhanced cell targeting
    • Authors: Kei X. Tan; Michael K. Danquah, Amandeep Sidhu, Sie Yon Lau, Clarence M. Ongkudon
      Abstract: Targeted delivery of drug molecules to specific cells in mammalian systems demonstrates a great potential to enhance the efficacy of current pharmaceutical therapies. Conventional strategies for pharmaceutical delivery are often associated with poor therapeutic indices and high systemic cytotoxicity, and this result in poor disease suppression, low surviving rates, and potential contraindication of drug formulation. The emergence of aptamers has elicited new research interests into enhanced targeted drug delivery due to their unique characteristics as targeting elements. Aptamers can be engineered to bind to their cognate cellular targets with high affinity and specificity, and this is important to navigate active drug molecules and deliver sufficient dosage to targeted malignant cells. However, the targeting performance of aptamers can be impacted by several factors including endonuclease-mediated degradation, rapid renal filtration, biochemical complexation, and cell membrane electrostatic repulsion. This has subsequently led to the development of smart aptamer-immobilized biopolymer systems as delivery vehicles for controlled and sustained drug release to specific cells at effective therapeutic dosage and minimal systemic cytotoxicity. This article reports the synthesis and in vitro characterization of a novel multi-layer co-polymeric targeted drug delivery system based on drug-loaded PLGA-Aptamer-PEI (DPAP) formulation with a stage-wise delivery mechanism. A thrombin-specific DNA aptamer was used to develop the DPAP system while Bovine Serum Albumin (BSA) was used as a biopharmaceutical drug in the synthesis process by ultrasonication. Biophysical characterization of the DPAP system showed a spherical shaped particulate formulation with a unimodal particle size distribution of average size ∼0.685 µm and a zeta potential of +0.82 mV. The DPAP formulation showed a high encapsulation efficiency of 89.4 ± 3.6%, a loading capacity of 17.89 ± 0.72 mg BSA protein/100 mg PLGA polymeric particles, low cytotoxicity and a controlled drug release characteristics in 43 days. The results demonstrate a great promise in the development of DPAP formulation for enhanced in vivo cell targeting. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-29T00:20:39.831998-05:
      DOI: 10.1002/btpr.2524
  • A three plus three parameters mechanistic model for viral filtration
    • Authors: Prakhar Misra; Abhishek Sinha, Anurag S. Rathore, Anupam Shukla, Fasil Q. Mir
      Abstract: Viral filtration is an expensive regulatory requirement in downstream processing of monoclonal antibodies (mAbs). This process step is typically operated with an overdesigned filter in order to account for any batch to batch variability in the filter, as well as the feed characteristics. Here, we propose a simple, six-parameter mechanistic model for viral filtration where three parameters are membrane-specific while the other three depend on feed characteristics and membrane-feed interactions. Viruses are considered as passive particles which are retained by the membrane on the basis of size exclusion. The model envisages that the viral filter contains two kind of pores: virus-retentive, small-sized pores and non-retentive, large-sized pores. The small-sized pores get blocked during filtration resulting in decrease in active membrane area, while the large-sized pores get constricted during filtration. The length of constricted part increases during filtration and contributes to increase in hydraulic resistance of the filter. Rate of these processes (blocking and constriction) are assumed to be proportional to the instantaneous rate of retention of the viral particles. The general nature of the model is validated with the experimental data on viral filtration for four different commercial membranes used in biotech industries as well as different model viruses. The proposed model has been demonstrated to describe the behavior of filters with very good accuracy. The best-fit model parameter values indicate about the various phenomena that are responsible for differences in the behavior of the membranes as well as change in retention and flux with feed concentration. The proposed model can be used for improving design of virus filters as well as in appropriate sizing of the filters during processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-21T00:20:35.772095-05:
      DOI: 10.1002/btpr.2523
  • Cyclodextrins increase phytosterol and tocopherol levels in suspension
           cultured cells obtained from mung beans and safflower
    • Authors: Lorena Almagro; Libertad Raquel Tudela, Ana Belén Sabater-Jara, Begoña Miras-Moreno, Maria A. Pedreño
      Abstract: In this work, suspension-cultured cells of mung beans and safflower were used in order to analyze the effect of methyl jasmonate and/or cyclodextrins, on bioactive compound production such as phytosterols and tocopherols. The results indicated that mung bean suspension-cultured cells produced higher amount of total phytosterols and tocopherols. In particular, mung bean suspension-cultured cells produced almost 220-fold higher levels of tocopherols than safflower suspension-cultured cells in the best conditions. However, while cyclodextrins were able to enhance extracellular production of phytosterols, in the case of tocopherols, they only increased their intracellular accumulation. Our results showed that mung bean cells could be used as a highly efficient system for the production of phytosterols and tocopherols which have a wide range of biological activities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-21T00:20:25.567057-05:
      DOI: 10.1002/btpr.2525
  • Amylosucrase-mediated β-carotene encapsulation in amylose
    • Authors: Carlos Andres Morales Letona; Cheon-Seok Park, Young-Rok Kim
      Abstract: The β-carotene embedded amylose microparticles (BC-AmMPs) were prepared in one-step by utilizing the unique catalytic activity of amylosucrase from Deinococcus geothermalis (DgAS), which synthesizes linear amylose chains using sucrose as the sole substrate. Synthesized amylose chains self-assembled with β-carotene to form well-defined spherical microparticles with an encapsulation yield of 65%. The BC-AmMPs produced (average diameter ∼8 µm) were bright orange due to the embedded β-carotene, and this was confirmed by Raman analysis. XRD showed BC-AmMPs had a B-type amylose crystal structure with a degree of crystallinity lower than that of AmMPs. This lower crystallinity of AmMP after BC encapsulation was confirmed by DSC analysis. Decreased enthalpy of gelatinization (ΔHgel) of BC-AmMP implied that molecular order within the amylose microstructure was influenced by the presence of BC. The stability of BC against environmental stresses, such as UV light and oxidative stress, was significantly enhanced by its encapsulation. The authors propose a new approach to the preparation of an amylose based carrier system for active compounds or expensive food ingredients with poor stabilities during storage or processing. Given that amylose is a safe food material, the devised encapsulation system will find wide range of practical applications in the food industry. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-14T04:15:40.588448-05:
      DOI: 10.1002/btpr.2521
  • Different RNA and protein expression of surface markers in rabbit amniotic
           fluid-derived mesenchymal stem cells
    • Authors: Michal Kovac; Jaromir Vasicek, Barbora Kulikova, Miroslav Bauer, Jozef Curlej, Andrej Balazi, Peter Chrenek
      Abstract: Over the years, there has been much confusion in defining molecular markers of mesenchymal stem cells (MSCs) for other than human species due to a lack of species-specific antibodies. Therefore, the aim of our study was to define rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs) and to reflect upon the current identification of AF-MSCs by providing a summary of detected surface markers in different species. The expression of rAF-MSC surface markers was analyzed at the protein and mRNA level. Flow cytometry analyses showed that rAF-MSCs were positive for CD29 and CD44, low positive for CD90, but negative for CD73, CD105, and CD166. Interestingly, RT-PCR (reverse transcription-polymerase chain reaction) exposed a discprepancy between transcribed mRNA and protein expression, as the cells expressed mRNA of all MSC markers: CD29, CD44, CD73, CD90, CD105, and CD166. Our results also confirmed the mesenchymal nature of isolated cells by morphology, ultrastructure, and intracellular marker expression profile. In addition, the expression of few pluripotency markers was also detected. We also found that passaging did not affect apoptosis and viability. Similarly, changes in karyotype were not observed during passaging. In conclusion, the provided characteristics may be used as a comprehensive set of criteria to define and characterize rAF-MSCs, required for the identification of these cells in preclinical investigations. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-08T02:31:38.981266-05:
      DOI: 10.1002/btpr.2519
  • High throughput screening identifies novel, cell cycle-arresting small
           molecule enhancers of transient protein expression
    • Authors: Hermann-Josef Meyer; Rebecca Turincio, Shirley Ng, Juan Li, Blair Wilson, Pamela Chan, Mark Zak, Dorothea Reilly, Maureen H. Beresini, Athena W. Wong
      Abstract: Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 μL scaled-down process, we validated 164 SMs to improve yields by up to twofold. The titer increase mediated by the SMs varied for different antibodies. SM dose optimizations resulted in almost threefold higher titers. The top 2, structurally distinct SM hits, increased antibody titers more than twofold in a 1 mL production process. Averaged across three antibodies of different expression levels, the compounds enhanced transient productivity by ∼80%. Intriguingly, both compounds arrested cells in the G2/M cell cycle phase leading to a decrease in growth and nutrient consumption, while elevating titer, nuclear plasmid DNA (pDNA) copy numbers, and mRNA levels. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-08T02:31:19.53492-05:0
      DOI: 10.1002/btpr.2517
  • Preliminary evaluation of Pleurotus ostreatus for the removal of selected
           pharmaceuticals from hospital wastewater
    • Authors: Laura Palli; Francesc Castellet-Rovira, Miriam Pérez-Trujillo, Donatella Caniani, Montserrat Sarrà-Adroguer, Riccardo Gori
      Abstract: The fungus Pleurotus ostreatus was investigated to assess its ability to remove diclofenac, ketoprofen, and atenolol spiked at 10 mg/L each one in hospital wastewater. The degradation test was carried out in a fluidized bed bioreactor testing both the batch and the continuous mode (hydraulic retention time in the range 1.63–3 days). In batch mode, diclofenac disappeared in less than 24 h, ketoprofen was degraded up to almost 50% in 5 days while atenolol was not removed. In continuous mode, diclofenac and ketoprofen removals were about 100% and 70% respectively; atenolol degradation was negligible during the first 20 days but it increased up to 60% after a peak of laccase production and notable biomass growth. In order to identify the enzymatic system involved, further experiments were carried out in flasks. Purified laccase completely transformed atenolol and diclofenac in less than 5 h, but not ketoprofen. In vivo experiments suggested that cytochrome P450 could be involved in diclofenac and ketoprofen degradation, while partial correlation studies confirmed the role of laccase in atenolol and diclofenac degradation. Two intermediates of diclofenac and ketoprofen were detected by nuclear magnetic resonance. Moreover P. ostreatus was able to reduce chemical oxygen demand of the hospital wastewater which is an important advantage comparing to other fungi in order to develop a wastewater treatment process. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-08T02:30:52.041143-05:
      DOI: 10.1002/btpr.2520
  • Improving the baculovirus expression vector system with vankyrin-enhanced
    • Authors: Kendra H. Steele; Barbara J. Stone, Kathleen M. Franklin, Angelika Fath-Goodin, Xiufeng Zhang, Haobo Jiang, Bruce A. Webb, Christoph Geisler
      Abstract: The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High FiveTM cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-06T23:27:30.828516-05:
      DOI: 10.1002/btpr.2516
  • Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and
           composition under various production conditions
    • Authors: Somruethai Bouyam; Wanna Choorit, Sarote Sirisansaneeyakul, Yusuf Chisti
      Abstract: The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon-to-nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L−1 glucose, 2.5 g L−1 KNO3) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500-mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L−1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g−1 and 305 mg L−1 day−1, respectively. A repeated fed-batch culture with four cycles of feeding with the fivefold concentrated medium in a 3-L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L−1 was obtained with a TFA productivity of 223 mg L−1 day−1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-07-06T23:25:50.324612-05:
      DOI: 10.1002/btpr.2518
  • Evaluation of piggyBac-mediated CHO pools to enable material generation to
           support GLP toxicology studies
    • Authors: Yashas Rajendra; Sowmya Balasubramanian, Neil A. McCracken, Dawn L. Norris, Zhirui Lian, Matthew G. Schmitt, Christopher C. Frye, Gavin C. Barnard
      Abstract: Generating purified protein for GLP toxicology studies (GLP-Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100 g), typically produced in a single 500–2,500 L bioreactor, using the final CHO clonally derived cell line (CDCL). One approach currently used to save time is to manufacture GLP-Tox material using pools of high-producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers>7 g/L using piggyBac-mediated gene integration (PB CHO pools). In this study, we wanted to leverage high titer PB CHO pools to produce GLP-Tox material. A detailed product quality attribute (PQA) assessment was conducted comparing PB CHO pools to pooled Top4 CDCLs. Four mAbs were evaluated. First, we found that PB CHO pools expressed all four mAbs at high titers (2.8–4.4 g/L in shake flasks). Second, all four PB CHO pools were aged to 55 generations (Gen). All four PB CHO Pools were found to be suitable over 55 Gen. Finally, we performed bioreactor scale-up. PB CHO pool titers (3.7–4.8 g/L) were similar or higher than the pooled Top 4 CDCLs in 5 L bioreactors (2.4–4.1 g/L). The PQAs of protein derived from PB CHO pools were very similar to pooled Top 4 CHO CDCLs according to multiple orthogonal techniques including peptide mapping analysis. Taken together, these results demonstrate the technical feasibility of using PB CHO pools to manufacture protein for GLP-Tox. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-06-29T01:50:33.629523-05:
      DOI: 10.1002/btpr.2495
  • Linkage of microbial kinetics and bacterial community structure of MBR and
           hybrid MBBR–MBR systems to treat salinity-amended urban wastewater
    • Authors: Alejandro Rodriguez-Sanchez; Juan Carlos Leyva-Diaz, Alejandro Gonzalez-Martinez, Jose Manuel Poyatos
      Abstract: Three pilot-scale bioreactors were started up and operated under salinity-amended urban wastewater feeding. The bioreactors were configured as membrane bioreactor and two different hybrid, moving bed biofilm reactor-membrane bioreactor and operated with a hydraulic retention time of 9.5 h, a solid residence time of 11.75 days and a total solids concentration of 2500 mg L−1. The three systems showed excellent performance in suspended solids, BOD5, and COD removal (values of 96–100%, 97–99%, and 88–90%, respectively), but poor nitrogen removal (values of 20–30%). The bacterial community structure during the start-up phase and the stabilization phase were different, as showed by β-diversity analyses. The differences between aerobic and anoxic biomass—and between suspended and attached biomass—were higher at the start-up phase than at the stabilization phase. The start-up phase showed high abundances of Chiayiivirga (mean values around 3–12% relative abundance) and Luteimonas (5–8%), but in the stabilization phase, the domination belonged to Thermomonas (3–14%), Nitrobacter (3–7%), Ottowia (3–11.5%), and Comamonas (2–6%), among others. Multivariate redundancy analyses showed that Thermomonas and Nitrosomonas were positively correlated with fast autotrophic kinetics, while Caulobacter and Ottowia were positively correlated with fast heterotrophic kinetics. Nitrobacter, Rhodanobacter, and Comamonas were positively correlated with fast autotrophic and heterotrophic kinetics. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-06-21T01:41:45.702528-05:
      DOI: 10.1002/btpr.2513
  • Improvement of covalent immobilization procedure of β-galactosidase from
           Kluyveromyces lactis for galactooligosaccharides production: Modeling and
           kinetic study
    • Authors: Flor González-Cataño; Luz Tovar-Castro, Eduardo Castaño-Tostado, Carlos Regalado-Gonzalez, Blanca García-Almendarez, Anaberta Cardador-Martínez, Silvia Amaya-Llano
      Abstract: Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β-galactosidase (EC from Kluyveromyces lactis covalently immobilized to a polysiloxane-polyvinyl alcohol (POS-PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL−1 initial lactose concentration and 6 U mL−1 enzyme concentration, obtaining 25.46 ± 0.01 gL−1 yield of trisaccharides. Although below the HPLC-IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL−1 of trisaccharides and 13.79 ± 0.21 gL−1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady-state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-06-18T23:50:37.081404-05:
      DOI: 10.1002/btpr.2509
  • Measuring the aggregation of CHO cells prior to single cell cloning allows
           a more accurate determination of the probability of clonality
    • Authors: Kerensa J. Klottrup; Guillermo Miro-Quesada, Layla Flack, Ivan Pereda, Pamela Hawley-Nelson
      Abstract: The manufacturing process for biotherapeutics is closely regulated by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory agencies worldwide. To ensure consistency of the product of a manufacturing cell line, International Committee on Harmonization guidelines (Q5D, 1997) state that the cell substrate should be derived from a single cell progenitor, i.e., clonal.Cell lines in suspension culture may naturally revert to cell adhesion in the form of doublets, triplets and higher order structures of clustered cells. We can show evidence of a single colony from limiting dilution cloning or in semi-solid media, but we cannot determine the number of cells from which the colony originated. To address this, we have used the ViCELL® XR (Beckman Coulter, High Wycombe, UK) cell viability analyzer to determine the proportion of clusters of two or more cells in a sample of the cell suspension immediately prior to cloning. Here, we show data to define the accuracy of the ViCELL for characterizing a cell suspension and summarize the statistical model combining two or more rounds of cloning to derive the probability of clonality. The resulting statistical model is applied to cloning in semi-solid medium, but could equally be applied to a limiting dilution cloning process. We also describe approaches to reduce cell clusters to generate a cell line with a high probability of clonality from a CHO host lineage. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-06-17T18:18:10.477946-05:
      DOI: 10.1002/btpr.2500
  • Enhanced expression of cysteine-rich antimicrobial peptide snakin-1 in
           Escherichia coli using an aggregation-prone protein coexpression system
    • Authors: Md. Ruhul Kuddus; Megumi Yamano, Farhana Rumi, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
      Abstract: Snakin-1 (SN-1) is a cysteine-rich plant antimicrobial peptide and the first purified member of the snakin family. SN-1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN-1 in Escherichia coli by a previously developed coexpression method using an aggregation-prone partner protein. Our goal was to increase the productivity of SN-1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN-1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN-1, the identity of which was verified by MALDI-TOF MS and NMR studies. The purified recombinant SN-1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation-prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN-1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-06-12T06:55:26.603344-05:
      DOI: 10.1002/btpr.2508
  • Strategic deployment of CHO expression platforms to deliver Pfizer's
           Monoclonal Antibody Portfolio
    • Authors: John J. Scarcelli; Tanya Q. Shang, Tim Iskra, Martin J. Allen, Lin Zhang
      Abstract: Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017
      PubDate: 2017-06-02T00:10:33.496632-05:
      DOI: 10.1002/btpr.2493
  • Comparative Study of Therapeutic Antibody Candidates Derived from
           Mini-Pool and Clonal Cell Lines
    • Authors: Lianchun Fan; Giovanni Rizzi, Kathleen Bierilo, Jun Tian, Joon Chong Yee, Reb Russel, Tapan Das
      Abstract: The long journey of developing a drug from initial discovery target identification to regulatory approval often leaves many patients with missed window of opportunities. Both regulatory agencies and biopharmaceutical industry continue to develop creative approaches to shorten the time of new drug development in order to deliver life-saving medicine to patients. Historically, drug substance materials to support the toxicology and early phase clinical study can only be manufactured after creating the final Master Cell Bank (MCB) of the clonally-derived cell line, which normally takes 1-2 years. With recent advances in cell line development, cell culture process and analytical technologies, generating more homogeneous bulk/mini-pool population with higher productivity has become a norm, thereby making it possible to shorten the timeline to initiate First in Human (FIH) trial by using bulk/mini-pool generated materials to support toxicology and FIH studies. In this study, two monoclonal antibodies of different subclasses (IgG1 and IgG4) were expressed from the mini-pool cells as well as clonally-derived cell lines generated from the same mini-pool. Cell growth, productivity, and product quality were compared between the materials generated from the mini-pool and clonally-derived cell line. The results demonstrate the similarity of the antibody products generated from mini-pool cells and clonally-derived cell lines from the same mini-pool, and strongly support the concept and feasibility of using antibody materials produced from mini-pool cultures for toxicology and FIH studies. The strategy to potentially shorten the FIH timeline is discussed. This article is protected by copyright. All rights reserved.
      PubDate: 2017-04-10T03:35:43.248139-05:
      DOI: 10.1002/btpr.2477
  • Issue Information
    • Pages: 1431 - 1434
      PubDate: 2017-12-26T23:34:06.589544-05:
      DOI: 10.1002/btpr.2357
  • A strategy to accelerate protein production from a pool of clones in
           Chinese hamster ovary cells for toxicology studies
    • Authors: Zhilan Hu; Wendy Hsu, Abby Pynn, Domingos Ng, Donna Quicho, Yilma Adem, Zephie Kwong, Brad Mauger, John Joly, Bradley Snedecor, Michael W. Laird, Dana C. Andersen, Amy Shen
      Pages: 1449 - 1455
      Abstract: In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies—mAb1, mAb2, and mAb3—at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449–1455, 2017
      PubDate: 2017-04-26T04:05:43.212884-05:
      DOI: 10.1002/btpr.2467
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