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. 1999 Jul;30(1-3):85–93. doi: 10.1023/A:1008012518961

Modification of glucose and glutamine metabolism in hybridoma cells through metabolic engineering

C Paredes 1, E Prats 2, JJ Cairó 1, F Azorín 2, Ll Cornudella 2, F Gòdia 1
PMCID: PMC3449941  PMID: 19003358

Abstract

The present work describes the genetic modification of a hybridoma cell line with the aim to change its metabolic behaviour, particularly reducing the amounts of ammonia and lactate produced by the cells. The cellular excretion of ammonia was eliminated by transfection of a cloned glutamine synthetase gene. The metabolic characterisation of the transformed cell line includes the analysis of the changes introduced in its intracellular metabolic fluxes by means of a stoichiometric model. Furthermore, the reduction of lactate accumulation was attempted through an antisense mRNA approach, aiming to generate a rate limiting step in the glycolytic pathway, thus lowering the glucose consumption rate. The physiological results obtained with the transformed cells are discussed. A maximum reduction of about 47% in the glucose consumption rate was obtained for one of the transformations. However a main drawback was the lack of stability of the transformed cells

Keywords: antisense mRNA, glutamine synthetase, metabolic engineering

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References

  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, Albright LM, Coen DM, Varki A, editors. Current protocols in molecular biology. New York: John Wiley & Sons Inc.; 1997. [Google Scholar]
  2. Bebbington CR, Renner G, Thompson S, King D, Abrams D, Yarranton GT. High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology. 1990;10:169–175. doi: 10.1038/nbt0292-169. [DOI] [PubMed] [Google Scholar]
  3. Bell SL, Bebbington CR, Scott MF, Wardell JN, Spier RE, Bushell ME, Sanders PG. Genetic Engineering of Hybridoma Glutamine Metabolism. Enzyme Microb Technol. 1995;17:98–106. doi: 10.1016/0141-0229(94)00056-W. [DOI] [PubMed] [Google Scholar]
  4. Birch JR, Boraston RC, Metcalfe H, Brown ME, Bebbington CR, Field RP. Selecting and Designing Cell Lines for Improved Physiological Characteristics. Cytotechnol. 1994;15:11–16. doi: 10.1007/BF00762375. [DOI] [PubMed] [Google Scholar]
  5. Birnbaum MJ. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989;57:305–315. doi: 10.1016/0092-8674(89)90968-9. [DOI] [PubMed] [Google Scholar]
  6. Birnbaum MJ, Haspel HC, Rosen OM. Cloning and characterization of a cDNA encoding the rat-brain glucose transporter. Proc Natl Acad Sci USA. 1986;83:5784–5788. doi: 10.1073/pnas.83.16.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Butler M, Christie A. Adaptation of mammalian cells to non-ammoniagenic media. Cytotechnol. 1994;15:87–94. doi: 10.1007/BF00762382. [DOI] [PubMed] [Google Scholar]
  8. Cameron FH, Jennings PA. Inhibition of gene expression by a short sense fragment. Nucleic Acids Res. 1991;19:469–475. doi: 10.1093/nar/19.3.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Campmajó C, Cairó JJ, Sanfeliu A, Martínez E, Alegret S, Gòdia F. Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis. Cytotechnol. 1994;14:177–182. doi: 10.1007/BF00749614. [DOI] [PubMed] [Google Scholar]
  10. Carruthers A. Facilitated diffusion of glucose. Physiol Rev. 1990;70:1135–1176. doi: 10.1152/physrev.1990.70.4.1135. [DOI] [PubMed] [Google Scholar]
  11. Chirgwin JM, Przybyla AE, McDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem. 1979;18:5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  12. Christie A, Butler M. Growth and metabolism of a murine hybridoma in cultures containing glutamine-based dipeptides. Focus. 1994;16:9–13. [Google Scholar]
  13. Chuppa S, Tsai Y-S, Yoon S, Shackleford S, Rozales C, Bhat R, Tsay G, Matanguihan C, Konstantinov K, Naveh D. Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells. Biotechnol Bioeng. 1997;55:328–338. doi: 10.1002/(SICI)1097-0290(19970720)55:2<328::AID-BIT10>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  14. Cockett MI, Bebbington CR, Yarranton GT. High-level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology. 1990;8:662–667. doi: 10.1038/nbt0790-662. [DOI] [PubMed] [Google Scholar]
  15. DiStefano DJ, Mark GE, Robinson DK. Feeding of nutrients delays apoptotic death in fed-batch cultures of recombinat NS0 myeloma cells. Biotechnol Lett. 1996;18:1067–1072. doi: 10.1007/BF00129733. [DOI] [Google Scholar]
  16. Duval D, Demangel C, Miossec S, Geahel I. Role of metabolic wastes in the control of cell proliferation and antibody production by mouse hybridoma cells. Hybridoma. 1992;11:311–322. doi: 10.1089/hyb.1992.11.311. [DOI] [PubMed] [Google Scholar]
  17. Glacken MW, Adema E, Sinskey AJ. Mathematical Descriptions of Hybridoma Culture Kinetics: I. Initial Metabolic Rates. Biotechnol Bioeng. 1988;32:491–506. doi: 10.1002/bit.260320412. [DOI] [PubMed] [Google Scholar]
  18. Glacken MW, Fleischaker RJ, Sinskey AJ. Reduction of waste product excretion via nutrient control: possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells. Biotechnol Bioeng. 1986;28:1376–1389. doi: 10.1002/bit.260280912. [DOI] [PubMed] [Google Scholar]
  19. Glacken MW, Huang C, Sinskey AJ. Mathemathical descriptions of hybridoma culture kinetics III. Simulation of fed-batch bioreactors. J Biotech. 1989;10:39–66. doi: 10.1016/0168-1656(89)90091-6. [DOI] [Google Scholar]
  20. Gould GW, Bell GI. Facilitative glucose transporters: an expanding family. TIBS. 1990;15:18–23. doi: 10.1016/0968-0004(90)90125-u. [DOI] [PubMed] [Google Scholar]
  21. Gould GW, Thomas HM, Jess TJ, Bell GI. Expression of human glucose transporters in Xenopus oocytes. Kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms. Biochem. 1991;30:5139–5145. doi: 10.1021/bi00235a004. [DOI] [PubMed] [Google Scholar]
  22. Holmlund A, Chatzisavido N, Bell SL, Lindner-Olsson E. Growth and metabolism of recombinant CHO cell-lines in serum-free medium containing derivatives of glutamine. In: Spier RE, Griffiths JB, MacDonald C, editors. Animal cell technology developments, processes and products, ESACT 11. Oxford: Butterworth-Heinemann; 1992. pp. 176–179. [Google Scholar]
  23. Jayme D. Nutrient optimization for high density biological production applications. Cytotechnol. 1991;5:15–30. doi: 10.1007/BF00365531. [DOI] [PubMed] [Google Scholar]
  24. Kaghad M, Dumont X, Chalon P, Lelias JM, Lamande N, Lucas M, Lazar M, Caput D. Nucleotide sequences of cDNA alpha and gamma enolase mRNAs from mouse brain. Nucleic Acids Res. 1990;18:3638. doi: 10.1093/nar/18.12.3638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena. Cell. 1982;31:147–157. doi: 10.1016/0092-8674(82)90414-7. [DOI] [PubMed] [Google Scholar]
  26. Ljunggren J, Haggstrom L. Glutamine limited fed-batch culture reduces ammonium ion production in animal cells. Biotechnol Lett. 1990;12:705–710. doi: 10.1007/BF01024725. [DOI] [Google Scholar]
  27. Minamoto Y, Ogawa K, Abe H, Iochi Y, Mitsugi K. Development of a serum-free and heat-sterilizable medium and continuous high-density culture. Cytotechnol. 1991;5:35–51. doi: 10.1007/BF00573879. [DOI] [PubMed] [Google Scholar]
  28. Moellering BJ, Tedesco JL, Townsend RR, Hardy MR, Scott RW and Prior CP (1990) Electrophoretic differences in a MAb expressed in three media. BioPharm: 30-38.
  29. Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell. 1990;2:279–289. doi: 10.1105/tpc.2.4.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ozturk SS, Riley MR, Palsson BO. Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production. Biotechnol Bioeng. 1992;39:418–431. doi: 10.1002/bit.260390408. [DOI] [PubMed] [Google Scholar]
  31. Paredes C, Sanfeliu A, Cárdenas F, Cairó JJ, Gòdia F. Estimation of the intracellular fluxes for a hybridoma cell line by material balances. Enzyme Microb Technol. 1998;23:187–198. doi: 10.1016/S0141-0229(98)00023-4. [DOI] [Google Scholar]
  32. Reuveny S, Velez D, Macmillan JD. Factors affecting cell growth and monoclonal antibody production in stirred reactors. J Immunol Methods. 1986;86:53–59. doi: 10.1016/0022-1759(86)90264-4. [DOI] [PubMed] [Google Scholar]
  33. Robinson DK, Chan CP, Yu Ip C, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J, Silberklang M. Characterization of a recombinant antibody produced in the course of a high yield fed-batch process. Biotechnol Bioeng. 1994;44:727–735. doi: 10.1002/bit.260440609. [DOI] [PubMed] [Google Scholar]
  34. Roth E, Ollenschlager G, Simmel A, Langer K, Fekyl W, Jakesz R. Influence of two glutamine containing dipeptides on growth of mammalian cells. In Vitro Cell Develop Biol. 1988;24:696. doi: 10.1007/BF02623607. [DOI] [PubMed] [Google Scholar]
  35. Sanfeliu A, Cairó JJ, Casas C, Solà C, Gòdia F. Analysis of nutritional factors and physical conditions affecting growth and monoclonal antibody production of the hybridoma KB-26.5 cell line. Biotechnol Progress. 1996;12:209–216. doi: 10.1021/bp950078x. [DOI] [PubMed] [Google Scholar]
  36. Sanfeliu A, Paredes C, Cairó JJ, Gòdia F. Identification of key patterns in the metabolism of hybridoma cells in culture. Enzyme Microb Technol. 1997;21:421–428. doi: 10.1016/S0141-0229(97)00015-X. [DOI] [Google Scholar]
  37. Scanlon KK, Giao L, Funato T, Wang WP, Ton T, Rossi JJ, Kashanisabat M. Ribozymic-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc Natl Acad Sci USA. 1991;88:10591–10595. doi: 10.1073/pnas.88.23.10591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Scherczinger CA, Yates AA, Knecht DA. Variables affecting antisense RNA inhibition of gene expression. In: Baserga R, Denhardt DT, editors. Antisense Strategies. New York: New York Academy of Sciences; 1992. pp. 45–56. [DOI] [PubMed] [Google Scholar]
  39. Smith CJ, Watson CF, Bird CR, Ray J, Schuch W, Grierson D. Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet. 1990;224:477–481. doi: 10.1007/BF00262443. [DOI] [PubMed] [Google Scholar]
  40. Sureshkumar G, Mutharasan R. The influence of temperature on a mouse-mouse hybridoma growth and monoclonal antibody production. Biotechnol Bioeng. 1991;37:292–295. doi: 10.1002/bit.260370313. [DOI] [PubMed] [Google Scholar]
  41. Van der Krol AR, Mur LA, Beld M, Mol JNM, Suite AR. Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to suppression of gene expression. Plant Cell. 1990;2:291–299. doi: 10.1105/tpc.2.4.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walder RY, Walder JA. Role of RNAse H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA. 1988;85:5011–5015. doi: 10.1073/pnas.85.14.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]

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