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BIOTECHNOLOGY (227 journals)                  1 2 | Last

Showing 1 - 200 of 227 Journals sorted alphabetically
3 Biotech     Open Access   (Followers: 7)
Advances in Bioscience and Biotechnology     Open Access   (Followers: 14)
Advances in Genetic Engineering & Biotechnology     Hybrid Journal   (Followers: 7)
African Journal of Biotechnology     Open Access   (Followers: 6)
Algal Research     Partially Free   (Followers: 9)
American Journal of Biochemistry and Biotechnology     Open Access   (Followers: 69)
American Journal of Bioinformatics Research     Open Access   (Followers: 8)
American Journal of Polymer Science     Open Access   (Followers: 29)
Animal Biotechnology     Hybrid Journal   (Followers: 9)
Annales des Sciences Agronomiques     Full-text available via subscription  
Applied Biochemistry and Biotechnology     Hybrid Journal   (Followers: 42)
Applied Bioenergy     Open Access  
Applied Biosafety     Hybrid Journal  
Applied Microbiology and Biotechnology     Hybrid Journal   (Followers: 62)
Applied Mycology and Biotechnology     Full-text available via subscription   (Followers: 5)
Arthroplasty Today     Open Access   (Followers: 1)
Artificial Cells, Nanomedicine and Biotechnology     Hybrid Journal   (Followers: 2)
Asia Pacific Biotech News     Hybrid Journal   (Followers: 2)
Asian Journal of Biotechnology     Open Access   (Followers: 8)
Asian Pacific Journal of Tropical Biomedicine     Open Access   (Followers: 2)
Australasian Biotechnology     Full-text available via subscription   (Followers: 1)
Banat's Journal of Biotechnology     Open Access  
BBR : Biochemistry and Biotechnology Reports     Open Access   (Followers: 4)
Bio-Algorithms and Med-Systems     Hybrid Journal   (Followers: 1)
Bio-Research     Full-text available via subscription   (Followers: 2)
Bioactive Materials     Open Access   (Followers: 1)
Biocatalysis and Agricultural Biotechnology     Hybrid Journal   (Followers: 4)
Biocybernetics and Biological Engineering     Full-text available via subscription   (Followers: 5)
Bioethics UPdate     Hybrid Journal  
Biofuels     Hybrid Journal   (Followers: 11)
Biofuels Engineering     Open Access   (Followers: 1)
Biological & Pharmaceutical Bulletin     Full-text available via subscription   (Followers: 5)
Biological Cybernetics     Hybrid Journal   (Followers: 10)
Biomarkers and Genomic Medicine     Open Access   (Followers: 5)
Biomarkers in Drug Development     Partially Free   (Followers: 1)
Biomaterials Research     Open Access   (Followers: 4)
BioMed Research International     Open Access   (Followers: 6)
Biomédica     Open Access  
Biomedical Engineering Research     Open Access   (Followers: 7)
Biomedical glasses     Open Access  
Biomedical Reports     Full-text available via subscription  
BioMedicine     Open Access  
Bioprinting     Hybrid Journal  
Bioresource Technology Reports     Hybrid Journal  
Bioscience, Biotechnology, and Biochemistry     Hybrid Journal   (Followers: 22)
Biosimilars     Open Access   (Followers: 1)
Biosurface and Biotribology     Open Access  
Biotechnic and Histochemistry     Hybrid Journal   (Followers: 2)
BioTechniques : The International Journal of Life Science Methods     Full-text available via subscription   (Followers: 28)
Biotechnologia Acta     Open Access   (Followers: 1)
Biotechnologie, Agronomie, Société et Environnement     Open Access   (Followers: 2)
Biotechnology     Open Access   (Followers: 6)
Biotechnology & Biotechnological Equipment     Open Access   (Followers: 5)
Biotechnology Advances     Hybrid Journal   (Followers: 33)
Biotechnology and Applied Biochemistry     Hybrid Journal   (Followers: 44)
Biotechnology and Bioengineering     Hybrid Journal   (Followers: 161)
Biotechnology and Bioprocess Engineering     Hybrid Journal   (Followers: 6)
Biotechnology and Genetic Engineering Reviews     Hybrid Journal   (Followers: 14)
Biotechnology and Health Sciences     Open Access   (Followers: 1)
Biotechnology and Molecular Biology Reviews     Open Access   (Followers: 1)
Biotechnology Annual Review     Full-text available via subscription   (Followers: 7)
Biotechnology for Biofuels     Open Access   (Followers: 10)
Biotechnology Frontier     Open Access   (Followers: 2)
Biotechnology Journal     Hybrid Journal   (Followers: 15)
Biotechnology Law Report     Hybrid Journal   (Followers: 4)
Biotechnology Letters     Hybrid Journal   (Followers: 33)
Biotechnology Progress     Hybrid Journal   (Followers: 39)
Biotechnology Reports     Open Access  
Biotechnology Research International     Open Access   (Followers: 2)
Biotechnology Techniques     Hybrid Journal   (Followers: 10)
Biotecnología Aplicada     Open Access  
Biotribology     Hybrid Journal  
BMC Biotechnology     Open Access   (Followers: 15)
Chinese Journal of Agricultural Biotechnology     Full-text available via subscription   (Followers: 3)
Communications in Mathematical Biology and Neuroscience     Open Access  
Computational and Structural Biotechnology Journal     Open Access   (Followers: 2)
Computer Methods and Programs in Biomedicine     Hybrid Journal   (Followers: 8)
Contributions to Tobacco Research     Open Access   (Followers: 3)
Copernican Letters     Open Access   (Followers: 1)
Critical Reviews in Biotechnology     Hybrid Journal   (Followers: 20)
Crop Breeding and Applied Biotechnology     Open Access   (Followers: 4)
Current Bionanotechnology     Hybrid Journal  
Current Biotechnology     Hybrid Journal   (Followers: 3)
Current Opinion in Biomedical Engineering     Hybrid Journal   (Followers: 1)
Current Opinion in Biotechnology     Hybrid Journal   (Followers: 55)
Current Pharmaceutical Biotechnology     Hybrid Journal   (Followers: 9)
Current Research in Bioinformatics     Open Access   (Followers: 14)
Current trends in Biotechnology and Pharmacy     Open Access   (Followers: 9)
EBioMedicine     Open Access  
Electronic Journal of Biotechnology     Open Access   (Followers: 1)
Entomologia Generalis     Full-text available via subscription  
Environmental Science : Processes & Impacts     Full-text available via subscription   (Followers: 4)
Experimental Biology and Medicine     Hybrid Journal   (Followers: 3)
Folia Medica Indonesiana     Open Access  
Food Bioscience     Hybrid Journal  
Food Biotechnology     Hybrid Journal   (Followers: 12)
Food Science and Biotechnology     Hybrid Journal   (Followers: 9)
Frontiers in Bioengineering and Biotechnology     Open Access   (Followers: 6)
Frontiers in Systems Biology     Open Access   (Followers: 2)
Fungal Biology and Biotechnology     Open Access   (Followers: 1)
GM Crops and Food: Biotechnology in Agriculture and the Food Chain     Full-text available via subscription   (Followers: 1)
GSTF Journal of BioSciences     Open Access  
HAYATI Journal of Biosciences     Open Access  
Horticulture, Environment, and Biotechnology     Hybrid Journal   (Followers: 11)
IEEE Transactions on Molecular, Biological and Multi-Scale Communications     Hybrid Journal   (Followers: 1)
IET Nanobiotechnology     Hybrid Journal   (Followers: 2)
IIOAB Letters     Open Access  
IN VIVO     Full-text available via subscription   (Followers: 4)
Indian Journal of Biotechnology (IJBT)     Open Access   (Followers: 2)
Indonesia Journal of Biomedical Science     Open Access   (Followers: 1)
Indonesian Journal of Biotechnology     Open Access   (Followers: 1)
Industrial Biotechnology     Hybrid Journal   (Followers: 18)
International Biomechanics     Open Access  
International Journal of Bioinformatics Research and Applications     Hybrid Journal   (Followers: 15)
International Journal of Biomechatronics and Biomedical Robotics     Hybrid Journal   (Followers: 4)
International Journal of Biomedical Research     Open Access   (Followers: 2)
International Journal of Biotechnology     Hybrid Journal   (Followers: 5)
International Journal of Biotechnology and Molecular Biology Research     Open Access   (Followers: 2)
International Journal of Biotechnology for Wellness Industries     Partially Free   (Followers: 1)
International Journal of Environment, Agriculture and Biotechnology     Open Access   (Followers: 5)
International Journal of Functional Informatics and Personalised Medicine     Hybrid Journal   (Followers: 4)
International Journal of Medicine and Biomedical Research     Open Access   (Followers: 1)
International Journal of Nanotechnology and Molecular Computation     Full-text available via subscription   (Followers: 3)
International Journal of Radiation Biology     Hybrid Journal   (Followers: 4)
Iranian Journal of Biotechnology     Open Access  
ISABB Journal of Biotechnology and Bioinformatics     Open Access  
Italian Journal of Food Science     Open Access   (Followers: 1)
Journal of Biometrics & Biostatistics     Open Access   (Followers: 3)
Journal of Bioterrorism & Biodefense     Open Access   (Followers: 6)
Journal of Petroleum & Environmental Biotechnology     Open Access   (Followers: 2)
Journal of Advanced Therapies and Medical Innovation Sciences     Open Access  
Journal of Advances in Biotechnology     Open Access   (Followers: 5)
Journal Of Agrobiotechnology     Open Access  
Journal of Analytical & Bioanalytical Techniques     Open Access   (Followers: 7)
Journal of Animal Science and Biotechnology     Open Access   (Followers: 6)
Journal of Applied Biomedicine     Open Access   (Followers: 3)
Journal of Applied Biotechnology     Open Access   (Followers: 2)
Journal of Applied Biotechnology Reports     Open Access   (Followers: 2)
Journal of Applied Mathematics & Bioinformatics     Open Access   (Followers: 5)
Journal of Biologically Active Products from Nature     Hybrid Journal   (Followers: 1)
Journal of Biomaterials and Nanobiotechnology     Open Access   (Followers: 6)
Journal of Biomedical Photonics & Engineering     Open Access  
Journal of Biomedical Practitioners     Open Access  
Journal of Bioprocess Engineering and Biorefinery     Full-text available via subscription  
Journal of Bioprocessing & Biotechniques     Open Access  
Journal of Biosecurity, Biosafety and Biodefense Law     Hybrid Journal   (Followers: 3)
Journal of Biotechnology     Hybrid Journal   (Followers: 68)
Journal of Chemical and Biological Interfaces     Full-text available via subscription   (Followers: 1)
Journal of Chemical Technology & Biotechnology     Hybrid Journal   (Followers: 10)
Journal of Chitin and Chitosan Science     Full-text available via subscription  
Journal of Colloid Science and Biotechnology     Full-text available via subscription  
Journal of Commercial Biotechnology     Full-text available via subscription   (Followers: 6)
Journal of Crop Science and Biotechnology     Hybrid Journal   (Followers: 7)
Journal of Essential Oil Research     Hybrid Journal   (Followers: 3)
Journal of Experimental Biology     Full-text available via subscription   (Followers: 25)
Journal of Genetic Engineering and Biotechnology     Open Access   (Followers: 5)
Journal of Ginseng Research     Open Access  
Journal of Industrial Microbiology and Biotechnology     Hybrid Journal   (Followers: 16)
Journal of Integrative Bioinformatics     Open Access  
Journal of International Biotechnology Law     Hybrid Journal   (Followers: 3)
Journal of Medical Imaging and Health Informatics     Full-text available via subscription  
Journal of Molecular Microbiology and Biotechnology     Full-text available via subscription   (Followers: 14)
Journal of Nano Education     Full-text available via subscription  
Journal of Nanobiotechnology     Open Access   (Followers: 4)
Journal of Nanofluids     Full-text available via subscription   (Followers: 2)
Journal of Organic and Biomolecular Simulations     Open Access  
Journal of Plant Biochemistry and Biotechnology     Hybrid Journal   (Followers: 6)
Journal of Science and Applications : Biomedicine     Open Access  
Journal of the Mechanical Behavior of Biomedical Materials     Hybrid Journal   (Followers: 11)
Journal of Trace Elements in Medicine and Biology     Hybrid Journal   (Followers: 1)
Journal of Tropical Microbiology and Biotechnology     Full-text available via subscription  
Journal of Yeast and Fungal Research     Open Access   (Followers: 1)
Marine Biotechnology     Hybrid Journal   (Followers: 5)
Messenger     Full-text available via subscription  
Metabolic Engineering Communications     Open Access   (Followers: 4)
Metalloproteinases In Medicine     Open Access  
Microalgae Biotechnology     Open Access   (Followers: 2)
Microbial Biotechnology     Open Access   (Followers: 9)
MicroMedicine     Open Access   (Followers: 3)
Molecular and Cellular Biomedical Sciences     Open Access  
Molecular Biotechnology     Hybrid Journal   (Followers: 16)
Molecular Genetics and Metabolism Reports     Open Access   (Followers: 3)
Nanobiomedicine     Open Access  
Nanobiotechnology     Hybrid Journal   (Followers: 3)
Nanomaterials and Nanotechnology     Open Access  
Nanomaterials and Tissue Regeneration     Open Access  
Nanomedicine and Nanobiology     Full-text available via subscription  
Nanomedicine Research Journal     Open Access  
Nanotechnology Reviews     Hybrid Journal   (Followers: 5)
Nature Biotechnology     Full-text available via subscription   (Followers: 520)
Network Modeling and Analysis in Health Informatics and Bioinformatics     Hybrid Journal   (Followers: 3)
New Biotechnology     Hybrid Journal   (Followers: 4)
Nigerian Journal of Biotechnology     Open Access  
Nova Biotechnologica et Chimica     Open Access  
NPG Asia Materials     Open Access  
npj Biofilms and Microbiomes     Open Access  
OA Biotechnology     Open Access  
Plant Biotechnology Journal     Open Access   (Followers: 10)
Plant Biotechnology Reports     Hybrid Journal   (Followers: 4)
Preparative Biochemistry and Biotechnology     Hybrid Journal   (Followers: 4)

        1 2 | 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  [1597 journals]
  • Development of a novel affinity chromatography resin for platform
           purification of bispecific antibodies with modified protein a binding
    • Authors: Andrew D. Tustian; Linus Laurin, Henrik Ihre, Travis Tran, Robert Stairs, Hanne Bak
      Abstract: There is strong interest in the production of bispecific monoclonal antibodies that can simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Regeneron's bispecific technology, based upon a standard IgG, consists of a heterodimer of two different heavy chains, and a common light chain. Co-expression of two heavy chains leads to the formation of two parental IgG impurities, the removal of which is facilitated by a dipeptide substitution in the Fc portion of one of the heavy chains that ablates Fc Protein A binding. Therefore the affinity capture (Protein A) step of the purification process must perform both bulk capture and high resolution of these mAb impurities, a task current commercially available resins are not designed for. Resolution can be further impaired by the ability of Protein A to bind some antibodies in the variable region of the heavy chain (VH). This paper details development of a novel Protein A resin. This resin combines an alkali stable ligand with a base matrix exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies with high yields. The developed resin, named MabSelect SuRe™ pcc, is implemented in GMP production processes for several bispecific antibodies. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:45:48.28025-05:0
      DOI: 10.1002/btpr.2622
  • Evaluation of cellular adhesion and organization in different microporous
           polymeric scaffolds
    • Authors: Amish Asthana; Charles McRae White, Megan Douglass, William S. Kisaalita
      Abstract: The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-L Lysine (PLL) and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:42:11.277419-05:
      DOI: 10.1002/btpr.2627
  • Microbial lipid extraction from Lipomyces starkeyi using irreversible
    • Authors: Ahasanul Karim; Abu Yousuf, M Amirul Islam, Yasir H Naif, Che Ku Mohammad Faizal, Md. Zahangir Alam, Domenico Pirozzi
      Abstract: The aim of the study was to investigate the feasibility of using irreversible electroporation (EP) as a microbial cell disruption technique to extract intracellular lipid within short time and in an eco—friendly manner. An EP circuit was designed and fabricated to obtain 4 kV with frequency of 100 Hz of square waves. The yeast cells of Lipomyces starkeyi (L. starkeyi) were treated by EP for 2-10 min where the distance between electrodes was maintained at 2, 4 and 6 cm. Colony forming units (CFU) were counted to observe the cell viability under the high voltage electric field. The forces of the pulsing electric field caused significant damage to the cell wall of L. starkeyi and the disruption of microbial cells was visualized by field emission scanning electron microscopic (FESEM) image. After breaking the cell wall, lipid was extracted and measured to assess the efficiency of EP over other techniques. The extent of cell inactivation was up to 95% when the electrodes were placed at the distance of 2 cm, which provided high treatment intensity (36.7 kWh/m3). At this condition, maximum lipid (63 mg/g) was extracted when the biomass was treated for 10 min. During the comparison, EP could extract 31.88% lipid while the amount was 11.89% for ultrasonic and 16.8% for Fenton's reagent. The results recommend that the EP is a promising technique for lowering the time and solvent usage for lipid extraction from microbial biomass. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:41:55.643522-05:
      DOI: 10.1002/btpr.2625
  • Metabolic responses and pathway changes of mammalian cells under different
           culture conditions with media supplementations
    • Authors: Seo-Young Park; Thomas M. Reimonn, Cyrus Agarabi, Kurt Brorson, Seongkyu Yoon
      Abstract: Amino acids and glucose consumption, cell growth and monoclonal antibody (mAb) production in mammalian cell culture are key considerations during upstream process and particularly media optimization. Understanding the interrelations and the relevant cellular physiology will provide insight for setting strategy of robust and effective mAb production. The aim of this study was to further our understanding of nutrient consumption metabolism, since this could have significant impact on enhancing mAb titer, cell proliferation, designing feeding strategies, and development of feed media. The nutrient consumption pattern, mAb concentration, and cell growth were analyzed in three sets of cell cultures with media supplementation of glucose, methionine, threonine, tryptophan, and tyrosine. The amino acids metabolism and its impact on cell growth and mAb production during the batch and fed-batch culture were closely analyzed. It was shown that the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered under different culture conditions with different media. These changes were more apparent in the fed-batch process in which higher mAb titer was observed due to the metabolic changes than mAb titer in the batch process. The pathway analysis approach was well utilized for evaluating the impact on the relevant pathways involved under different cell culture conditions to improve cell growth and mAb titer. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:41:44.855678-05:
      DOI: 10.1002/btpr.2623
  • Evaluation of differences between dual salt-pH gradient elution and mono
           gradient elution using a thermodynamic model: Simultaneous separation of
           six monoclonal antibody charge and size variants on preparative-scale ion
           exchange chromatographic resin
    • Authors: Yi Feng Lee; Matthias Jöhnck, Christian Frech
      Abstract: The efficiencies of mono gradient elution and dual salt-pH gradient elution for separation of six mAb charge and size variants on a preparative-scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt-pH gradient elution with increasing pH gradient and simultaneously decreasing salt gradient is best suited for the separation of these mAb charge and size variants on Eshmuno® CPX. Besides giving high binding capacity, this type of opposite dual salt-pH gradient also provides better resolved mAb variant peaks and lower conductivity in the elution pools compared to single pH or salt gradients. To have a mechanistic understanding of the differences in mAb variants retention behaviors of mono pH gradient, parallel dual salt-pH gradient, and opposite dual salt-pH gradient, a linear gradient elution model was used. After determining the model parameters using the linear gradient elution model, 2-D plots were used to show the pH and salt dependencies of the reciprocals of distribution coefficient, equilibrium constant, and effective ionic capacity of the mAb variants in these gradient elution systems. Comparison of the 2-D plots indicated that the advantage of opposite dual salt-pH gradient system with increasing pH gradient and simultaneously decreasing salt gradient is the non-continuous increased acceleration of protein migration. Furthermore, the fitted model parameters can be used for the prediction and optimization of mAb variants separation in dual salt-pH gradient and step elution. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:41:33.715414-05:
      DOI: 10.1002/btpr.2626
  • Selective extraction of lipase and amylase from enzyme mixture by
           employing liquid emulsion membrane
    • Authors: B.S. Priyanka; Navin K. Rastogi
      Abstract: The present work deals with the extraction of lipase and amylase from enzyme mixture by employing liquid emulsion membranes (LEM). The electrostatic interaction between enzymes and reverse micellar surfactant polar head group plays an important role for selective extraction of two different enzymes having different isoelectric points. The optimised conditions for lipase extraction (pH 7.0) resulted in the purification fold and activity recovery of 5.43 fold and 89.53%, respectively, whereas, in case of amylase (pH 9.0) the purification fold and activity recovery were 6.58 and 94.32%, respectively. The results were compared with the control sample (containing individual enzymes) and mixture of enzymes lipase and amylase and it was shown that for optimum conditions the activity recovery and purification fold was higher for the individual enzymes as compared to their mixture. Downstream processing involving LEM was shown to be a feasible method for selective extraction of enzymes. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:41:15.850412-05:
      DOI: 10.1002/btpr.2624
  • Corrigendum to Biotechnology Progress 2003, 19, 928-935 by Y.O. Shin, M.E.
           Weber and J.H. Vera
    • Authors: Grazyna Wilczek-Vera; Younok O. Dumortier Shin, Eva Rodil, Juan H. Vera
      PubDate: 2018-02-21T03:41:13.677728-05:
      DOI: 10.1002/btpr.2621
  • Probing lactate metabolism variations in large-scale bioreactors
    • Authors: Sen Xu; Rubin Jiang, Roland Mueller, Nadja Hoesli, Thomas Kretz, John Bowers, Hao Chen
      Abstract: Lactate metabolism variations are frequently encountered in mammalian cell culture processes, especially during process scale-up. In this study, we took a multipronged approach to investigate the impact of pH, pCO2, osmolality, base addition, and mixing conditions on the observed lactate variations in a Chinese Hamster Ovary (CHO) fed-batch process at 2,000 L scale. Two cultivating methods, CO2-controlled and pH-controlled, were used to decouple the individual and synergistic effects from those factors. The individual effects from pH, pCO2, and osmolality on lactate consumption/reproduction in the stationary phase were insignificant in the ranges studied though the initial lactate production rates varied. In contrast, lactate metabolism was found to be impacted by an interaction between mixing conditions and CO2 accumulation. High CO2 accumulation and poor mixing led to lactate reproduction, whereas either low CO2 or improved mixing were sufficient to result in lactate consumption. Base addition was not required for pH control in the low CO2 conditions, and therefore lactate reproduction was correlated with base addition under poor mixing conditions. Under good mixing conditions, CO2-triggered base addition did not significantly impact lactate reproduction. It is reasonable to postulate that increased mixing times further promoted lactate production during base addition. As lactate reproduction results in more base addition to maintain pH, a cycle could be formed between lactate production and base addition. As a remediation, we showed that such lactate reproduction could be eliminated by improving CO2 removal at 2,000 L scale. This article is protected by copyright. All rights reserved.
      PubDate: 2018-02-21T03:40:22.685537-05:
      DOI: 10.1002/btpr.2620
  • Surfactant selection for a liquid foam-bed photobioreactor
    • Authors: Agnes Janoska; María Vázquez, Marcel Janssen, René H. Wijffels, María Cuaresma, Carlos Vílchez
      Abstract: A novel liquid foam-bed photobioreactor has been shown to hold potential as an innovative technology for microalgae production. In this study, a foam stabilizing agent has been selected which fits the requirements of use in a liquid foam-bed photobioreactor. Four criteria were used for an optimal surfactant: the surfactant should have good foaming properties, should not be rapidly biodegradable, should drag up microalgae in the foam formed, and it should not be toxic for microalgae. Ten different surfactants (nonionic, cationic, and anionic) and two microalgae genera (Chlorella and Scenedesmus) were compared on the above-mentioned criteria. The comparison showed the following facts. Firstly, poloxameric surfactants (Pluronic F68 and Pluronic P84) have acceptable foaming properties described by intermediate foam stability and liquid holdup and small bubble size. Secondly, the natural surfactants (BSA and Saponin) and Tween 20 were easily biodegraded by bacteria within 3 days. Thirdly, for all surfactants tested the microalgae concentration is reduced in the foam phase compared to the liquid phase with exception of the cationic surfactant CTAB. Lastly, only BSA, Saponin, Tween 20, and the two Pluronics were not toxic at concentrations of 10 CMC or higher. The findings of this study indicate that the Pluronics (F68 and P84) are the best surfactants regarding the above-mentioned criteria. Since Pluronic F68 performed slightly better, this surfactant is recommended for application in a liquid foam-bed photobioreactor. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-02-16T12:01:45.600594-05:
      DOI: 10.1002/btpr.2614
  • Directed evolution of an endoinulinase from Talaromyces purpureogenus
           toward efficient production of inulooligosaccharides
    • Authors: Livnat Afriat-Jurnou; Rami Cohen, Irina Paluy, Ran Ben-Adiva, Itamar Yadid
      Abstract: Inulinases are fructofuranosyl hydrolases that target the β-2,1 linkage of inulin and hydrolyze it into fructose, glucose and inulooligosaccharides (IOS), the latter are of growing interest as dietary fibers. Inulinases from various microorganisms have been purified, characterized and produced for industrial applications. However, there remains a need for inulinases with increased catalytic activity and better production yields to improve the hydrolysis process and fulfill the growing industrial demands for specific fibers. In this study, we used directed enzyme evolution to increase the yield and activity of an endoinulinase enzyme originated from the filamentous fungus Talaromyces purpureogenus (Penicillium purpureogenum ATCC4713). Our directed evolution approach yielded variants showing up to fivefold improvements in soluble enzyme production compared to the starting point which enabled high-yield production of highly purified recombinant enzyme. The distribution of the enzymatic reaction products demonstrated that after 24 h of incubation, the main product (57%) had a degree of polymerization of 3 (DP3). To the best of our knowledge, this is the first application of directed enzyme evolution to improve inulooligosaccharide production. The approach enabled the screening of large genetic libraries within short time frames and facilitated screening for improved enzymatic activities and properties, such as substrate specificity, product range, thermostability and pH optimum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-02-14T04:30:44.779365-05:
      DOI: 10.1002/btpr.2618
  • Cryopreservation of chicken blastodermal cells and their quality
           assessment by flow cytometry and transmission electron microscopy
    • Authors: Andrea Svoradová; Lenka Kuželová, Jaromír Vašíček, Lucia Olexíková, Peter Chrenek
      Abstract: The goal of this study was to evaluate effect of slow freezing and vitrification methods on the viability of chicken blastodermal cells (BCs). Proper aliquot of isolated BCs were diluted in the freezing medium composed of 10% DMSO and frozen in the freezing vessel BICELL to reach desired temperature up to −80°C. Then samples were immersed in liquid nitrogen. Other cell aliquot was vitrified in solution containing 10% DMSO and samples were immediately immersed in the liquid nitrogen. The viability of fresh and frozen/thawed BCs was evaluated using Trypan blue method and flow cytometry. Flow cytometry analysis was provided by DRAQ5 dye in combination with Live-Dead kit. Overall, this technique provides both quantitative and qualitative information about BCs. Results obtained from Trypan blue method showed significant differences (P 
      PubDate: 2018-02-12T01:31:22.342823-05:
      DOI: 10.1002/btpr.2615
  • One-pot strategy for on-site enzyme production, biomass hydrolysis, and
           ethanol production using the whole solid-state fermentation medium of
           mixed filamentous fungi
    • Authors: Larissa Maehara; Sandra C. Pereira, Adilson J. Silva, Cristiane S. Farinas
      Abstract: The efficient use of renewable lignocellulosic feedstocks to obtain biofuels and other bioproducts is a key requirement for a sustainable biobased economy. This requires novel and effective strategies to reduce the cost contribution of the cellulolytic enzymatic cocktails needed to convert the carbohydrates into simple sugars, in order to make large-scale commercial processes economically competitive. Here, we propose the use of the whole solid-state fermentation (SSF) medium of mixed filamentous fungi as an integrated one-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production. Ten different individual and mixed cultivations of commonly used industrial filamentous fungi (Aspergillus niger, Aspergillus oryzae, Trichoderma harzianum, and Trichoderma reesei) were performed under SSF and the whole media (without the extraction step) were used in the hydrolysis of pretreated sugarcane bagasse. The cocultivation of T. reesei with A. oryzae increased the amount of glucose released by around 50%, compared with individual cultivations. The release of glucose and reducing sugars achieved using the whole SSF medium was around 3-fold higher than obtained with the enzyme extract. The addition of soybean protein (0.5% w/w) during the hydrolysis reaction further significantly improved the saccharification performance by blocking the lignin and avoiding unproductive adsorption of enzymes. The results of the alcoholic fermentation validated the overall integrated process, with a volumetric ethanol productivity of 4.77 g/L.h, representing 83.5% of the theoretical yield. These findings demonstrate the feasibility of the proposed one-pot integrated strategy using the whole SSF medium of mixed filamentous fungi for on-site enzymes production, biomass hydrolysis, and ethanol production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-02-12T01:31:01.668542-05:
      DOI: 10.1002/btpr.2619
  • Covalent immobilization of halohydrin dehalogenase for efficient synthesis
           of epichlorohydrin in an integrated bioreactor
    • Authors: Shu-Ping Zou; Kai Gu, Yu-Guo Zheng
      Abstract: Halohydrin dehalogenase (HHDH)-mediated dehalogenation of 1,3-dichloro-2-propanol (1,3-DCP) is a key step in the chemoenzymatic synthesis of epichlorohydrin (ECH) from glycerol. In this study, a covalent immobilization strategy was employed to enhance the stability of Agrobacterium tumefaciens HHDH using epoxy resin ES-103B as a carrier. Under optimal conditions, the activity recovery of ES-103B-immobilized HHDH (HHDH@ES-103B) was 62.4% and the specific activity was 1604 U/g. The HHDH@ES-103B exhibited excellent thermostability, with a half-life of 68.6 days at 40°C, which is 8.0-times higher than that of the free HHDH. A semicontinuous biotransformation of 1,3-DCP to ECH was performed using HHDH@ES-103B as biocatalyst in a recirculating packed bed reactor (RPBR), resulting in an ECH yield of 94.2%, with an average productivity of 5.2 g/L/h. The RPBR system exhibited a high operational stability and even after 50 cycles of reaction, it retained > 90% of the initial conversion. Furthermore, an integrated bioprocess based on in situ product recovery (ISPR) was developed in RPBR to overcome product inhibition. The integrated bioreactor equipped with an external macroporous adsorption resin HZD-9 column led to another 1.6-fold increase in ECH productivity to 8.46 g/L/h. This improved stability and reusability of HHDH@ES-103B demonstrated its potential for the biotransformation of 1,3-DCP to ECH. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-02-12T01:30:57.460697-05:
      DOI: 10.1002/btpr.2617
  • Extraction of bioactive compounds against cardiovascular diseases from
           Lentinula edodes using a sequential extraction method
    • Authors: Diego Morales; Adriana J. Piris, Alejandro Ruiz-Rodriguez, Marin Prodanov, Cristina Soler-Rivas
      Abstract: Three extraction methods were sequentially combined to obtain fractions from Lentinula edodes (shiitake mushrooms) containing bioactive compounds against cardiovascular diseases (CVDs). Fruiting bodies were first extracted with plain water, obtained residue was then submitted to supercritical fluid extraction (SFE) and remaining residue submitted to hot water extraction. Sequential design allowed reutilization of the nonextracted material as raw material for the successive extractions increasing extraction yields and separating interesting compounds. Obtained fractions contained different amounts of ß-glucans, chitins, eritadenine, lenthionine, ergosterol, proteins/peptides and phenolic compounds conferring them different bioactivities. Water soluble fractions showed high antioxidant activities (ABTS+• and DPPH• scavenging capacity and reducing power), they were also able to inhibit one of the main enzymes involved in hypertension (angiotensin-I converting enzyme) and the key enzyme of cholesterol metabolism (3-hydroxy-3-methylglutaryl coenzyme A reductase). The latter inhibitory activity was also noticed in SFE extracts although ergosterol and other lipid-like molecules were isolated. Dietary fibers were separated in the third extraction. Therefore, with this sequential extraction procedure bioactive compounds against CVDs can be selectively separated from a single batch of shiitake powder. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-02-12T01:30:39.144911-05:
      DOI: 10.1002/btpr.2616
  • Investigation of the free heavy chain homodimers of a monoclonal antibody
    • Authors: Hyo (Helen) Chung; Lynette Buck, Kristi Daris, Brent Welborn, Quanzhou Luo, Jette Wypych
      Abstract: Monoclonal antibodies (mAbs) are composed of two heavy chain (HC) and two light chain (LC) polypeptides. The proper folding and assembly of HC and LC is critical for antibody production. Current dogma indicates that the free HCs are retained in the endoplasmic reticulum (ER) unless assembled with LCs into antibodies, while the LCs on the other hand can be secreted as free monomer or dimer molecules. In this study, high levels of extracellular HC homodimers (7%-45%) were observed in the cell culture media during cell line development for mAb1. Excellent correlation (R2 > 0.9) between the level of free HC homodimers and the percentage of high molecular weight species indicates that the free HC homodimers might be causative of unwanted aggregation. Due to the different surface charge of HC homodimer and fully assembled antibodies, the unwanted extracellular HC homodimers were successfully removed by downstream processing, through a cation exchange chromatography step. Reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) analysis of the cell culture media from different MTX-amplified pools indicated that insufficient expression of LC is one potential root cause for the high level of free HC homodimers. The level of free HC homodimers decreased significantly (3%-25%) after retransfecting the MTX amplified pools with additional LC gene. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-02-02T11:26:05.486134-05:
      DOI: 10.1002/btpr.2611
  • Rapid fabrication of detachable three-dimensional tissues by layering of
           cell sheets with heating centrifuge
    • Authors: Yuji Haraguchi; Yuki Kagawa, Akiyuki Hasegawa, Hirotsugu Kubo, Tatsuya Shimizu
      Abstract: Confluent cultured cells on a temperature-responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three-dimensional (3-D) tissue can be fabricated by the layering of cell sheets. A resulting 3-D multilayered cell sheet-tissue on a temperature-responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3-D multilayered constructs, we attempted to layer cell sheets on a temperature-responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22-23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36-37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet-layering process. The heating centrifugation shortened the fabrication time by one-fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue-fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-01-31T12:36:24.55754-05:0
      DOI: 10.1002/btpr.2612
  • Effects of ultrasonication variables on the activity and properties of
           alpha amylase preparation
    • Authors: Thi Thu Tra Tran; Khanh Tien Nguyen, Van Viet Man Le
      Abstract: Recently, ultrasound was demonstrated to increase enzyme activity in food industry. In most studies, an enzyme–substrate mixture was ultrasonicated. Very little is reported on the ultrasonication of enzyme preparation; the effects of ultrasonication variables on the enzyme activity and properties remained unclear. In this study, an α-amylase preparation was ultrasonicated under different conditions. At the ultrasonic frequency, power, temperature, and time of 20 kHz, 25 W/mL, 30°C, and 75 s, respectively, the secondary structure of the α-amylase was changed and that improved the catalytic activity by 47% over the control. The optimal temperature and pH of the α-amylase preparation did not change under the ultrasonic treatment. In addition, ultrasonic treatment increased kinetic parameters of the α-amylase including maximal velocity Vmax, Michaelis constant Km, turnover number Kcat, and catalytic specificity constant Kcat/Km. Nevertheless, the ultrasonicated α-amylase had lower thermal inactivation rate constant and half-life value than the nonultrasonicated enzyme. Ultrasonic treatment can be considered as a novel solution for improvement in enzyme activity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
      PubDate: 2018-01-31T12:36:15.465461-05:
      DOI: 10.1002/btpr.2613
  • 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
  • Issue Information
    • Pages: 1 - 4
      PubDate: 2018-02-20T22:54:19.972961-05:
      DOI: 10.1002/btpr.2549
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
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