Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 1999 Jul;30(1-3):107–120. doi: 10.1023/A:1008038515285

On-line heat flux measurements improve the culture medium for the growth and productivity of genetically engineered CHO cells

Yue H Guan 1, Richard B Kemp 1
PMCID: PMC3449938  PMID: 19003360

Abstract

With the increasingly competitive commercial production of target proteins by hybridoma and genetically engineered cells, there is an urgent requirement for biosensors to monitor and control on-line and in real time the growth of cultured cells. Since growth is accompanied by an enthalpy change, heat dissipation measured by calorimetry could act as an index for metabolic flow rate. Recombinant CHO cell suspensions producing interferon-γ were pumped to an on-line flow calorimeter. The results showed that an early reflection of metabolic change is size-specific heat flux obtained from dividing heat flow rate by the capacitance change of the cell suspension, using the on-line probe of a dielectric spectroscope. Comparison of heat flux with glucose and glutamine fluxes indicated that the former most accurately reflected decreased metabolic activity. Possibly this was due to accumulation of lactate and ammonia resulting from catabolic substrates being used as biosynthetic precursors. Thus, the heat flux probe is an ideal on-line biosensor for fed-batch culture. A stoichiometric growth reaction was formulated and data for material and heat fluxes incorporated into it. This showed that cell demand for glucose and glutamine was in the stoichiometric ratio of ∼3:1 rather than the ∼5:1 in the medium. It was demonstrated that the set of stoichiometric coefficients in the reaction were related through the extent of reaction (advancement) to overall metabolic activity (flux). The fact that this approach can be used for medium optimisation is the basis for an amino-acid-enriched medium which improved cell growth while decreasing catabolic fluxes.

Keywords: calorimetry, capacitance, heat flux, medium optimisation, metabolic activity, on-line biosensor

Full Text

The Full Text of this article is available as a PDF (142.3 KB).

References

  1. Adachi S, Cross AR, Babior BM, Gottlieb RA. Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis. J Biol Chem. 1997;272:21878–21882. doi: 10.1074/jbc.272.35.21878. [DOI] [PubMed] [Google Scholar]
  2. Bailey JE, Ollis DF. Biochemical engineering fundamentals. 2nd ed. London: McGraw-Hill; 1986. pp. 373–456. [Google Scholar]
  3. Bonarius HPJ, Hatzimanikatis V, Meesters KPH, De Gooijer CD, Schmid G, Tramper J. Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol Bioeng. 1996;43:505–514. doi: 10.1002/(SICI)1097-0290(19960505)50:3<299::AID-BIT9>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  4. Borys MC, Linzer DIH, Papoutsakis ET. Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-1 by Chinese hamster ovary cells in a pH-dependent manner. Biotechnol Bioeng. 1994;50:299–318. doi: 10.1002/bit.260430611. [DOI] [PubMed] [Google Scholar]
  5. Castro PML, Hayter PM, Ison AP, Bull AT. Application of a statistical deign to the optimisation of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol. 1992;38:84–90. doi: 10.1007/BF00169424. [DOI] [PubMed] [Google Scholar]
  6. Castro PML, Ison AP, Hayter PM, Bull AT. The microheterogeneity of recombinant human interferon-γ produced by Chinese-hamster ovary cells is affected by the protein and lipid content of the culture medium. Biotechnol Appl Biochem. 1995;21:87–100. [PubMed] [Google Scholar]
  7. Davey CL, Guan Y, Kemp RB, Kell DB. Real-time monitoring of the biomass content of animal cell cultures using dielectric spectroscopy. In: Funatsu K, Shirai Y, Matsushita T, editors. Animal Cell Technology: Basic and Applied Aspects. Dordrecht, The Netherlands: Kluwer Academic Publ.; 1997. pp. 61–65. [Google Scholar]
  8. Funatsu K, Shirai Y, Matsushita T, editors. Animal Cell Technology: Basic and Applied Aspects. Dordrecht, The Netherlands: Kluwer Academic Publ.; 1997. [Google Scholar]
  9. Gnaiger E, Kemp RB. Anaerobic metabolism in aerobic mammalian cells: Information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta. 1990;1016:328–332. doi: 10.1016/0005-2728(90)90164-Y. [DOI] [PubMed] [Google Scholar]
  10. Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R. Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr. 1995;27:583–596. doi: 10.1007/BF02111656. [DOI] [PubMed] [Google Scholar]
  11. Gu MB, Todd P, Kompala DS. Foreign gene expression (β-galactosidase) during the cell cycle phases in recombinant CHO cells. Biotechnol Bioeng. 1993;42:1113–1123. doi: 10.1002/bit.260420914. [DOI] [PubMed] [Google Scholar]
  12. Guan Y, Evans PM, Kemp RB. Specific heat flow rate: an on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy. Biotechnol Bioeng. 1998;58:464–477. doi: 10.1002/(SICI)1097-0290(19980605)58:5&#x0003c;464::AID-BIT2&#x0003e;3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  13. Guan Y, Kemp RB. Medium design with the aid of heat flux measurement in mammalian cell culture. In: Westerhoff HV, Snoep JL, Wijker JE, Sluse FE, Kholodenko BN, editors. BioThermoKinetics of the living cell. Amsterdam: BTK; 1996. pp. 387–397. [Google Scholar]
  14. Guan Y, Kemp RB. The viable cell monitor: a dielectric spectroscope for growth and metabolic studies of animal cells on macroporous carriers. In: Merten O-W, Perrin P, Griffiths B, editors. New Developments and New Applications in Animal Cell Technology. Dordrecht, The Netherlands: Kluwer; 1998. pp. 321–326. [Google Scholar]
  15. Guan Y, Kemp RB. Dynamic medium optimisation by on-line heat flux measurement and stoichiometric modelling in animal cell culture. In: Merten O-W, Perrin P, Griffiths B, editors. New Developments and New Applications in Animal Cell Technology. Dordrecht, The Netherlands: Kluwer; 1998. pp. 355–357. [Google Scholar]
  16. Guan Y and Kemp RB (1998c) Detection of dynamic substrate requirements of animal cells by stoichiometric growth equations and enthalpy balance. Biotechnol. In press. [DOI] [PubMed]
  17. Guan Y, Lloyd PC, Evans PM, Kemp RB. A modified continuous flow microcalorimeter for measuring heat dissipation by mammalian cells in batch culture. J Thermal Anal. 1997;49:785–794. doi: 10.1007/BF01996761. [DOI] [Google Scholar]
  18. Harris CM, Todd RW, Bungard SJ, Lovitt RW, Morris JG, Kell DB. The dielectric permittivity of microbial suspensions at radio-frequencies: a novel method for the real-time estimation of microbial biomass. Enzyme Microb Technol. 1987;9:181–186. doi: 10.1016/0141-0229(87)90075-5. [DOI] [Google Scholar]
  19. Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Bull AT. Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Appl Microbiol Biotechnol. 1991;34:327–335. doi: 10.1007/BF00167898. [DOI] [PubMed] [Google Scholar]
  20. Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Tong JM, Bull AT. Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-γ. Biotechnol Bioeng. 1992;39:327–335. doi: 10.1002/bit.260390311. [DOI] [PubMed] [Google Scholar]
  21. Hooker AD, Goldman NH, Markham NH, James DC, Ison AP, Bull AT, Strange PG, Salmon I, Baines AJ, Jenkins N. N-Glycans of recombinant human interferon-γ change during batch culture of Chinese hamster ovary cells. Biotechnol Bioeng. 1995;48:327–337. doi: 10.1002/bit.260480612. [DOI] [PubMed] [Google Scholar]
  22. Jenkins N, Castro P, Menon A, Bull A. Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells. Cytotechnol. 1994;15:209–215. doi: 10.1007/BF00762395. [DOI] [PubMed] [Google Scholar]
  23. Jenkins N. Monitoring and control of recombinant glycoprotein heterogeneity in animal cell cultures. Biochem Soc Trans. 1995;23:171–175. doi: 10.1042/bst0230171. [DOI] [PubMed] [Google Scholar]
  24. Jeong Y-H, Wang SS. Role of glutamine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism. Enzyme Microb Technol. 1995;17:47–55. doi: 10.1016/0141-0229(94)00041-O. [DOI] [Google Scholar]
  25. Kemp RB. Developments in cellular microcalorimetry with particular emphasis on the valuable role of the energy (enthalpy) balance method. Thermochim Acta. 1993;219:17–41. doi: 10.1016/0040-6031(93)80480-X. [DOI] [Google Scholar]
  26. Kemp RB. Cytotoxicity of an anchorage-independent fibroblast cell line measured by a combination of fluorescent dyes. In: O'Hare SD, Atterwill CK, editors. In vitro Toxicity Testing Protocols. Totowa, New Jersey, U.S.A.: Humana Press; 1995. pp. 211–218. [Google Scholar]
  27. Kemp RB, Evans PM, Guan Y. An enthalpy balance approach to studies of metabolic activity in mammalian cells. J Thermal Anal. 1997;49:755–770. doi: 10.1007/BF01996759. [DOI] [Google Scholar]
  28. Kemp RB, Guan Y. Heat flux and the calorimetric-respirometric ratio as measures of catabolic flux in mammalian cells. Thermochim Acta. 1997;300:199–211. doi: 10.1016/S0040-6031(96)03125-5. [DOI] [Google Scholar]
  29. Kemp RB, Guan Y. Probing the metabolism of genetically-engineered mammalian cells by heat flux. Thermochim Acta. 1998;309:63–78. doi: 10.1016/S0040-6031(97)00426-7. [DOI] [Google Scholar]
  30. Kim CN, Wang X, Huang Y, Ibrado AM, Liu L, Fang G, Bhalla K. Overexpression of Bcl-xL inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis. Cancer Res. 1997;57:3115–3120. [PubMed] [Google Scholar]
  31. Kubbies M, Stockinger H. Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry. Exp Cell Res. 1990;188:267–271. doi: 10.1016/0014-4827(90)90169-B. [DOI] [PubMed] [Google Scholar]
  32. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell. 1996;86:147–157. doi: 10.1016/S0092-8674(00)80085-9. [DOI] [PubMed] [Google Scholar]
  33. Mastrangelo AJ, Betenbaugh MJ. Overcoming apoptosis: new methods for improving protein-expression systems. Trends Biotechnol. 1998;16:88–94. doi: 10.1016/S0167-7799(97)01159-1. [DOI] [PubMed] [Google Scholar]
  34. Omasa T, Higashiyama K-I, Shioya S, Suga K-I. Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng. 1992;39:556–564. doi: 10.1002/bit.260390511. [DOI] [PubMed] [Google Scholar]
  35. Prigogine I. Introduction to the thermodynamics of irreversible processes. 3rd ed. New York: Interscience; 1967. [Google Scholar]
  36. Roels JA. Energetics and Kinetics in Biotechnology. Amsterdam: Elsevier; 1983. [Google Scholar]
  37. Simpson NH, Milner AE, Al-Rubeai M. Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions. Biotechnol Bioeng. 1997;54:1–16. doi: 10.1002/(SICI)1097-0290(19970405)54:1&#x0003c;1::AID-BIT1&#x0003e;3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  38. Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M. Deprivation of any single amino acid leads to the induction of apoptosis in hybridoma cultures, which is suppressed by the bcl-2 gene. Biotechnol Bioeng. 1998;59:90–98. doi: 10.1002/(SICI)1097-0290(19980705)59:1&#x0003c;90::AID-BIT12&#x0003e;3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  39. Singh RP, Emery AN, Al-Rubeai M. Enhancement of survivability of mammalian cells by overexpression of apoptosis-suppressor gene bcl-2. Biotechnol Bioeng. 1996;52:166–175. doi: 10.1002/(SICI)1097-0290(19961005)52:1&#x0003c;166::AID-BIT17&#x0003e;3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  40. Spier RE, Griffiths JB, Berthold W, editors. Animal Cell Technology. London: Butterworth-Heinemann; 1994. [Google Scholar]
  41. Von Stockar U, Gustafsson L, Larsson L, Marison I, Tissot P, Gnaiger E. Thermodynamic considerations in constructing energy balances for cellular growth. Biochim Biophys Acta. 1993;1183:221–240. doi: 10.1016/0005-2728(93)90225-5. [DOI] [Google Scholar]
  42. Wadsö I. Experimental Thermodynamics. Oxford: Solution Calorimetry, Blackwell Scientific Publ.; 1994. Microcalorimetry of aqueous and biological systems; pp. 267–301. [Google Scholar]
  43. Wu P, Ozturk SS, Blackie JD, Thrift JC, Figueroa C, Navah D. Evaluation and applications of optical-cell density probes in mammalian-cell bioreactors. Biotechnol Bioeng. 1995;45:495–502. doi: 10.1002/bit.260450606. [DOI] [PubMed] [Google Scholar]
  44. Xie L, Wang DIC. Stoichiometric analysis of animal cell growth and its application in medium design. Biotechnol Bioeng. 1994;43:1175–1189. doi: 10.1002/bit.260431123. [DOI] [PubMed] [Google Scholar]
  45. Xie L, Wang DIC. High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor. Biotechnol Bioeng. 1996;51:725–729. doi: 10.1002/(SICI)1097-0290(19960920)51:6&#x0003c;725::AID-BIT12&#x0003e;3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  46. Xie L, Wang DIC. Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network. Biotechnol Bioeng. 1996;52:591–601. doi: 10.1002/(SICI)1097-0290(19961205)52:5&#x0003c;591::AID-BIT6&#x0003e;3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  47. Xie L, Nyberg G, Gu X, Li H, Möllborn F, Wang DIC. Gamma-interferon production and quality in stoichiometric fed-batch cultures of Chinese hamster ovary cells (CHO) Biotechnol Bioeng. 1997;56:577–582. doi: 10.1002/(SICI)1097-0290(19971205)56:5&#x0003c;577::AID-BIT11&#x0003e;3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  48. Zhou W, Mulchandani A. Recent advances in bioprocess monitoring and control. ACS Symp Ser. 1995;613:88–98. doi: 10.1021/bk-1995-0613.ch009. [DOI] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES