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  • 1
    Electronic Resource
    Electronic Resource
    A mathematical model of N-linked glycoform biosynthesis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 890-908 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; N-linked glycosylation ; mathematical model ; CHO cells ; glycoform ; oligosaccharides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic engineering of N-linked oligosaccharide biosynthesis to produce novel glycoforms or glycoform distributions of a recombinant glycoprotein can potentially lead to an improved therapeutic performance of the glycoprotein product. Effective engineering of this pathway to maximize the fractions of beneficial glycoforms within the glycoform population of a target glycoprotein can be aided by a mathematical model of the N-linked glycosylation process. A mathematical model is presented here, whose main function is to calculate the expected qualitative trends in the N-linked oligosaccharide distribution resulting from changes in the levels of one or more enzymes involved in the network of enzyme-catalyzed reactions that accomplish N-linked oligosaccharide biosynthesis. It consists of mass balances for 33 different oligosaccharide species N-linked to a specified protein that is being transported through the different compartments of the Golgi complex. Values of the model parameters describing Chinese hamster ovary (CHO) cells were estimated from literature information. A basal set of kinetic parameters for the enzyme-catalyzed reactions acting on free oligosaccharide substrates was also obtained from the literature. The solution of the system for this basal set of parameters gave a glycoform distribution consisting mainly of complex-galactosylated oligosaccharides distributed in structures with different numbers of antennae in a fashion similar to that observed for various recombinant proteins produced in CHO cells. Other simulations indicate that changes in the oligosaccharide distribution could easily result from alteration in glycoprotein productivity within the range currently attainable in industry. The overexpression of N-acetylglucosaminyltransferase III in CHO cells was simulated under different conditions to test the main function of the model. These simulations allow a comparison of different strategies, such as simultaneous overexpression of several enzymes or spatial relocation of enzymes, when trying to optimize a particular glycoform distribution. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:890-908, 1997.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    A novel cytostatic process enhances the productivity of Chinese hamster ovary cells (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 927-939 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; human secreted alkaline phosphatase ; tumor suppressor genes ; green fluorescent protein ; cell cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have established a novel production process which allows up to fourfold higher production of a model secreted protein, the human secreted alkaline phosphatase (SEAP), in Chinese hamster ovary (CHO) cells. A cytostatic production phase is established in which cell proliferation is inhibited or completely abolished. Such a cytostatic production phase is established by overexpression of the tumor suppressor genes p21, p27, or p53175P (a p53 mutant showing specific loss of apoptotic function) under transcriptional control of a tetracycline-repressible promoter (PhCMV*-1). In order to minimize complications due to possible clonal variation of selected, stable cell lines, our investigations are based on transiently transfected subpopulations, that have become a useful tool in industrial R&D. These subpopulations have been selected by flow cytometry for the expression of genes encoded on a dicistronic expression vector. These vectors contain a dicistronic expression unit consisting of the genes encoding the green fluorescent protein (GFP) or SEAP, followed by one of the cytostatic genes p21, p27 or p53175P encoded by the second cistron. p21, p27 as well as p53175P block the cell cycle of CHO cells in the G1-phase for a prolonged period. However, these G1-arrested cells remain viable and proliferation proficient upon repression of expression of the cytostatic gene. All three of the cytostatic genes studied provided similar regulation of proliferation, and also similar enhancements in SEAP production, suggesting that higher productivity may be a general and intrinsic feature of G1-phase arrested CHO cells. Overall productivity is most likely enhanced because growth-arrested cells do not need to devote cellular resources to biomass production. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:927-939, 1997.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 476-482 
    Details
    ISSN: 0006-3592
    Keywords: cyclin E expression ; CHO cells ; insulin ; fibroblast growth factor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. © 1995 John Wiley & Sons Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260470409
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  • 4
    Electronic Resource
    Electronic Resource
    Effect of vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant chinese hamster ovary cells (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 1367-1370 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; Cloned proteins ; VHb hemoglobin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture. © 1994 John Wiley & Sons, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260441114
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  • 5
    Electronic Resource
    Electronic Resource
    Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 119-129 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; methotrexate ; dhfr gene amplification ; HbsAg secretion ; flow cytometry ; gene copy number ; intracellular HbsAg degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster overy (CHO) cell line producing recombinant hepatits B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gane copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autroadiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse-chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5-h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260400117
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  • 6
    Electronic Resource
    Electronic Resource
    Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 445-450 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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