Biotechnology and Bioengineering

Cover image for Vol. 109 Issue 6

Special Issue: CHO Cell Genomics Special Section

June 2012

Volume 109, Issue 6

Pages C1–C1, fmi–fmvi, 1353–1599

Issue edited by: Michael J. Betenbaugh, Nicole Borth, Kelvin H. Lee

  1. Cover

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. You have free access to this content
      Biotechnology and Bioengineering: Volume 109, Number 6, June 2012 (page C1)

      Version of Record online: 18 APR 2012 | DOI: 10.1002/bit.24299

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      Cover Picture: A word cloud was generated using key words from the abstracts of the CHO Cell Genomics Special Section and arranged around a chromosome spread. Top panels represent important aspects of CHOmics (left to right): transcriptomics, CHO cells; genomics; bioprocessing; microRNAs. Image courtesy of Vaibhav Jadhav, Matthias Hackl and Nicole Borth (BOKU University Vienna). CHO cell image (stained red with anti-cofi lin and green with phalloidin) from Stephanie Hammond and Kelvin H. Lee (University of Delaware).

  2. Contents

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
  3. Spotlights

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
  4. Communication to the Editor

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. Chinese hamster genome database: An online resource for the CHO community at www.CHOgenome.org (pages 1353–1356)

      Stephanie Hammond, Mihailo Kaplarevic, Nicole Borth, Michael J. Betenbaugh and Kelvin H. Lee

      Version of Record online: 22 NOV 2011 | DOI: 10.1002/bit.24374

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      The Chinese hamster genome database (www.chogenome.org) is an online tool for the CHO cell community. The aim of this effort is to provide a common resource to host CHO genomic data and facilitate the development of genome-scale tools and technologies for CHO cell engineering.

  5. Articles

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. Construction of BAC-based physical map and analysis of chromosome rearrangement in chinese hamster ovary cell lines (pages 1357–1367)

      Yihua Cao, Shuichi Kimura, Takayuki Itoi, Kohsuke Honda, Hisao Ohtake and Takeshi Omasa

      Version of Record online: 6 NOV 2011 | DOI: 10.1002/bit.24347

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      Karyotypic comparison between Chinese hamster ovary (CHO) DG44, K1, and primary Chinese hamster cells based on BAC-FISH results is described. The homologous regions of CHO DG44 and K1 to that of Chinese hamster are colored according to the Chinese hamster chromosomes. The scale bar corresponds to 2 µm. Background photo: scanning electron microscope image of the CHO cell chromosome.

  6. Communication to the Editors

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. CGCDB: A web-based resource for the investigation of gene coexpression in CHO cell culture (pages 1368–1370)

      Colin Clarke, Padraig Doolan, Niall Barron, Paula Meleady, Stephen F. Madden, Dana DiNino, Mark Leonard and Martin Clynes

      Version of Record online: 4 JAN 2012 | DOI: 10.1002/bit.24416

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      The CGCDB is an online system developed to provide a straightforward user friendly means of exploring gene coexpression in CHO cell culture across a large-scale microarray profiling dataset. It is envisaged that the database will facilitate the prioritization of cell line engineering and/or biomarker candidates to enhance CHO based cell culture for the production of biotherapeutics.

    2. Profiling conserved microRNA expression in recombinant CHO cell lines using illumina sequencing (pages 1371–1375)

      Stephanie Hammond, Jeffrey C. Swanberg, Shawn W. Polson and Kelvin H. Lee

      Version of Record online: 23 JAN 2012 | DOI: 10.1002/bit.24415

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      Illumina sequencing was used to profile the differential expression of conserved miRNAs in recombinant CHO cell lines relative to expression in CHO-K1 cells. The majority of differentially expressed miRNAs were found to be up-regulated in CHO-SEAP cells but down-regulated in CHO-tPA cells.

  7. Articles

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells (pages 1376–1385)

      Vaibhav Jadhav, Matthias Hackl, Juan A. Hernandez Bort, Matthias Wieser, Eva Harreither, Renate Kunert, Nicole Borth and Johannes Grillari

      Version of Record online: 22 MAR 2012 | DOI: 10.1002/bit.24490

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      This work presents the screening method for overexpression and functional analysis of miRNAs in CHO cells, which can be rapidly customized to test large numbers of miRNAs for cell engineering applications. The same procedure can be applied to different production cell lines for assaying specific responses. Thus the presented system represents a general platform to functionally screen candidates for rational cell factory design using miRNA, miR-sponges, siRNAs or mRNA overexpression along with detailed functional characterization.

    2. Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics (pages 1386–1394)

      Paula Meleady, Raimund Hoffrogge, Michael Henry, Oliver Rupp, Juan Hernandez Bort, Colin Clarke, Karina Brinkrolf, Shane Kelly, Benjamin Müller, Padraig Doolan, Matthias Hackl, Tim Frederik Beckmann, Thomas Noll, Johannes Grillari, Niall Barron, Alf Pühler, Martin Clynes and Nicole Borth

      Version of Record online: 2 MAR 2012 | DOI: 10.1002/bit.24476

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      The availability of CHO-specific genomic and cDNA sequence information has greatly enhanced protein identification by MS based proteomics of CHO cells. Both the number of proteins that could be identified and the confidence of identification are now significantly increased. Many of the newly identified proteins are related to important cellular processes such as energy metabolism and translation, stressing the importance of these new databases in bioprocess related research.

    3. Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: Osmoprotective effect of glycine betaine addition (pages 1395–1403)

      Jee Yon Kim, Yeon-Gu Kim and Gyun Min Lee

      Version of Record online: 23 JAN 2012 | DOI: 10.1002/bit.24442

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      The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. Here, the authors apply the two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis to find differentially expressed key proteins in this condition. The proteins identified can be applied to a genetic modulation strategy for maximizing the efficacy in the use of glycine betaine combined with hyperosmolality in rCHO cell culture.

    4. Metabolite profiling of CHO cells with different growth characteristics (pages 1404–1414)

      Stefanie Dietmair, Mark P. Hodson, Lake-Ee Quek, Nicholas E. Timmins, Panagiotis Chrysanthopoulos, Shana S. Jacob, Peter Gray and Lars K. Nielsen

      Version of Record online: 30 MAR 2012 | DOI: 10.1002/bit.24496

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      The authors employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra- and extracellular metabolite concentrations detected medium specific and time dependent changes. Using multivariate data analysis, the authors identified a range of metabolites correlating with growth rate (e.g., CTP, UDP-glucuronic acid), illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism.

    5. Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture (pages 1415–1429)

      Suresh Selvarasu, Ying Swan Ho, William P. K. Chong, Niki S. C. Wong, Faraaz N. K. Yusufi, Yih Yean Lee, Miranda G. S. Yap and Dong-Yup Lee

      Version of Record online: 31 JAN 2012 | DOI: 10.1002/bit.24445

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      The current study exploited a combined metabolomics and in silico modeling approach to characterize the cellular mechanisms of Chinese hamster ovary (CHO) fed-batch cultures. The results identified major growth-limiting factors including the oxidative stress and depletion of lipid metabolites. Such key information on growth-related mechanisms derived from the current approach can potentially guide the development of new strategies to enhance CHO culture performance.

  8. Review Articles

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. The use of high-solids loadings in biomass pretreatment—a review (pages 1430–1442)

      Alicia A. Modenbach and Sue E. Nokes

      Version of Record online: 22 FEB 2012 | DOI: 10.1002/bit.24464

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      The use of high-solids loadings (≥15% solids, w/w) in biomass pretreatment processes potentially offers many advantages over lower-solids loadings, including increased sugar and ethanol concentrations and decreased production and capital costs. Limitations like the lack of available water to promote mass transfer, increased substrate viscosity, and increased production of inhibitors must be overcome for the use of high-solids loadings in pretreatments to reach full potential. This paper showcases the advances made in the use of high-solids loadings in biomass pretreatment.

    2. Vaccine process technology (pages 1443–1460)

      Jessica O. Josefsberg and Barry Buckland

      Version of Record online: 30 MAR 2012 | DOI: 10.1002/bit.24493

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      Throughout history, as vaccine technology has advanced, the methods to produce the vaccine have advanced in parallel. This review paper will illustrate this with special emphasis on the advances made possible using recombinant DNA technology and the resulting impact on the discovery of new vaccines and on process development.

  9. Articles

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. Biocatalysis, Protein Engineering, and Nanobiotechnology

      Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation (pages 1461–1470)

      Mariusz P. Madej, Gregory Coia, Charlotte C. Williams, Joanne M. Caine, Lesley A. Pearce, Rebecca Attwood, Nick A. Bartone, Olan Dolezal, Rebecca M. Nisbet, Stewart D. Nuttall and Timothy E. Adams

      Version of Record online: 26 DEC 2011 | DOI: 10.1002/bit.24407

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      Sortase A enzyme (SrtA) from Staphylococcus aureus site-specifically conjugates poly-glycine constructs to LPETG-tagged acceptor molecules. Here, an anti-Epidermal Growth Factor Receptor scFv antibody fragment is functionalized with biotin or fluorescein, and used to specifically label an A431 EGFR over-expressing cell line with Streptavidin Alexa 488. This technology has further application in directed immobilization of antibody fragments to biosensor surfaces for rapid generation of high quality kinetic data sets.

    2. Plasma-activated carbon nanotube-based high sensitivity immunosensors for monitoring Legionella pneumophila by direct detection of maltose binding protein peptidoglycan-associated lipoprotein (MBP-PAL) (pages 1471–1478)

      Jun-Yong Lee, Joon-Hyung Jin, Joon Hyub Kim, Min Ja Kim, Cheol Jin Lee and Nam Ki Min

      Version of Record online: 6 JAN 2012 | DOI: 10.1002/bit.24418

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      The authors introduce an immunobiosensor based on a plasma-functionalized MWCNT-transferred working electrode. Although Legionella PAL is an excellent target antigen for monitoring Legionnaires' disease by the ELISA-based diagnostic test, the PAL antigen requires complicated separation and purification steps before use. On the other hand, electrochemical direct detection of MBP-PAL, that is basically unavailable for ELISA, is cost-effective and much faster for monitoring pathogenic L. pneumophila because no such separation or purification processes are required in this case.

    3. Kinetic investigation of a solvent-free, chemoenzymatic reaction sequence towards enantioselective synthesis of a β-amino acid ester (pages 1479–1489)

      Simon Strompen, Markus Weiß, Thomas Ingram, Irina Smirnova, Harald Gröger, Lutz Hilterhaus and Andreas Liese

      Version of Record online: 23 JAN 2012 | DOI: 10.1002/bit.24422

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      A solvent-free, chemoenzymatic reaction sequence comprising a thermal aza-Michael addition and a lipase catalyzed aminolysis for the enantioselective synthesis of β-amino acid esters has been kinetically and thermodynamically characterized. Due to the added complexity of the enzyme catalyzed reaction in a solvent-free system kinetic parameters were initially determined in an organic solvent and subsequently transferred to the solvent free system by applying a kinetic model based on thermodynamic activities.

    4. Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Zbasic2: Design of a heterogeneous D-amino acid oxidase catalyst on porous glass (pages 1490–1498)

      Juan M. Bolivar and Bernd Nidetzky

      Version of Record online: 17 JAN 2012 | DOI: 10.1002/bit.24423

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      A generally applicable concept of enzyme immobilization by design is proposed and applied in the development of a heterogeneous D-amino acid oxidase biocatalyst. Tailored electrostatic complementarity between the positively charged binding module Zbasic2 and mesoporous silica carrier is introduced by functionalization of the carrier surface with negatively charged sulfonate groups (see the Scheme). The Zbasic2 fusion of the enzyme is immobilized with almost absolute selectivity and excellent affinity, exploiting multisubunit oriented interactions of the binding module with the carrier.

    5. Biofuels and Environmental Biotechnology

      Two-temperature stage biphasic CO2–H2O pretreatment of lignocellulosic biomass at high solid loadings (pages 1499–1507)

      Jeremy S. Luterbacher, Jefferson W. Tester and Larry P. Walker

      Version of Record online: 7 JAN 2012 | DOI: 10.1002/bit.24417

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      Biphasic CO2–H2O mixtures can be used as a pretreatment medium for mixed hardwood and switchgrass to obtain glucan to glucose yields of about 80% using a novel two-stage temperature approach.

    6. Bioprocess Engineering and Supporting Technologies

      On enhancing productivity of bioethanol with multiple species (pages 1508–1517)

      Jun Geng, Hyun-Seob Song, Jingqi Yuan and Doraiswami Ramkrishna

      Version of Record online: 7 JAN 2012 | DOI: 10.1002/bit.24419

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      In the comparison of various reactor configurations including single or mixed cultures, the authors' modeling study shows that the highest performance is achieved by two-stage fermentation sequentially using two different species i.e., Kluyveromyces marxianus (KM) and Pichia stipitis (PS) switched at a suitable sugar conversion. In all cases, it is assumed that ethanol is continually removed from the reactor throughout fermentation.

    7. Cellular and Metabolic Engineering

      Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain (pages 1518–1527)

      J. Tyler Youngquist, Rebecca M. Lennen, Don R. Ranatunga, William H. Bothfeld, Wesley D. Marner II and Brian F. Pfleger

      Version of Record online: 6 JAN 2012 | DOI: 10.1002/bit.24420

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      A plasmid-free strain of Escherichia coli was engineered to produce free fatty acids (FFAs) by integrating three copies of a thioesterase gene under the control of an inducible promoter onto the chromosome. FFA production was studied in continuous cultivation under carbon limitation. The highest yield and specific productivity were observed at dilution rates of 0.05 h−1 and 0.2 h−1, respectively. The observed yields under the lowest dilution rate were 15% higher than that observed in batch cultures.

    8. Engineering Science of Biological Systems

      Activation of target signal transducers utilizing chimeric receptors with signaling-molecule binding motifs (pages 1528–1537)

      Koichiro Saka, Masahiro Kawahara, Hiroshi Ueda and Teruyuki Nagamune

      Version of Record online: 7 JAN 2012 | DOI: 10.1002/bit.24421

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      The authors regulated signal transduction artificially by locating a tyrosine motif of interest into the intracellular domain of specific receptors. Several known tyrosine motifs were selected from native cytokine receptors that strongly bind to their target molecule, and located them downstream of the JAK binding domain of a chimeric receptor. The results indicated that each chimeric receptor preferentially activated the corresponding signaling molecule even when the tyrosine motif was distant from the JAK binding domain.

    9. Systems Biotechnology

      Debottlenecking recombinant protein production in Bacillus megaterium under large-scale conditions—targeted precursor feeding designed from metabolomics (pages 1538–1550)

      Claudia Korneli, Christoph Josef Bolten, Thibault Godard, Ezequiel Franco-Lara and Christoph Wittmann

      Version of Record online: 31 JAN 2012 | DOI: 10.1002/bit.24434

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      The presented strategy displays a valuable tool for targeted large scale process optimization. By correlating culture performance and intracellular metabolite availability in a reactor configuration mimicking large scale, bottlenecks for production of recombinant proteins caused by limitation of specific precursors can be easily identified. This is demonstrated by targeted feeding of five identified precursors which increased protein production under large scale conditions by 100%.

    10. Tissue Engineering and Delivery Systems

      Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination (pages 1551–1560)

      Chi-Lin Lee, Michael Chou, Bingbing Dai, Liang Xiao and Pin Wang

      Version of Record online: 17 JAN 2012 | DOI: 10.1002/bit.24413

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      The method demonstrated in the article for generating robust producer cells is capable of making SIN-based LVs for DC-based vaccination application. This production system helps generate high quantity and quality vectors for future testing of this DC-LV as a vaccine carrier in large animal models. By removing the troublesome transient transfection step, this LVs production system can be easily utilized in a GMP condition to manufacture clinical grade materials for use in humans.

    11. Combining submerged electrospray and UV photopolymerization for production of synthetic hydrogel microspheres for cell encapsulation (pages 1561–1570)

      Cara J. Young, Laura A. Poole-Warren and Penny J. Martens

      Version of Record online: 11 JAN 2012 | DOI: 10.1002/bit.24430

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      This study presents a novel method for the production of covalently crosslinked synthetic hydrogel microspheres for cell encapsulation. Submerged electrospray was combined with UV photopolymerization to prepare smooth, spherical microspheres from 50–1500 µm by varying the flow rate and applied voltage. Importantly, L929 fibroblasts were encapsulated within PVA microspheres and showed viability >90% after 24 hours. This process could be used to produce synthetic hydrogel microspheres for many applications, and specifically supports encapsulation of cells.

    12. Biomimetic micropatterned multi-channel nerve guides by templated electrospinning (pages 1571–1582)

      Eric M. Jeffries and Yadong Wang

      Version of Record online: 2 JAN 2012 | DOI: 10.1002/bit.24412

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      Templated electrospinning was used to create aligned microchannels within a construct comprised exclusively of electrospun polycaprolactone (PCL) fibers. This process uses sutures as sacrificial templates to create a complex three-dimensional design. Shown here is a multi-channeled guide aimed at mimicking the channels and fibers of native nerves for peripheral nerve repair.

    13. Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor (pages 1583–1594)

      R.J. McCoy, C. Jungreuthmayer and F.J. O'Brien

      Version of Record online: 17 JAN 2012 | DOI: 10.1002/bit.24424

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      HRSEM image showing cells adapting bridging (centre) and flat (edges) attachment morphology types. In this study, the authors employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Bridging cells were shown to experience greater levels of deformation and a greater susceptibility to cell detachment.

  10. Communications to the Editor

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Communication to the Editor
    6. Articles
    7. Communication to the Editors
    8. Articles
    9. Review Articles
    10. Articles
    11. Communications to the Editor
    1. Biocatalysis, Protein Engineering, and Nanobiotechnology

      Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates (pages 1595–1599)

      Sneha Srikrishnan, Arlo Randall, Pierre Baldi and Nancy A. Da Silva

      Version of Record online: 2 JAN 2012 | DOI: 10.1002/bit.24414

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      Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via rational site-directed mutagenesis based on structural bioinformatics and evolutionary analysis. The single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in kcat and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, while maintaining thermostability and optimal activity at 70°C, pH 5.0. This work further demonstrates that non-catalytic residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.

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