Skip to main content
SpringerLink
Log in

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

  • Published:
Cytotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adachi S, Cross AR, Babior BM and Gottlieb RA (1997) Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis. J Biol Chem 272: 21878-21882.

    Article  PubMed  CAS  Google Scholar 

  • Bailey JE and Ollis DF (1986) Biochemical engineering fundamentals, 2nd ed., McGraw-Hill, London, pp. 373-456.

    Google Scholar 

  • Bonarius HPJ, Hatzimanikatis V, Meesters KPH, De Gooijer CD, Schmid G and Tramper J (1996) Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol Bioeng 43: 505-514.

    Google Scholar 

  • Borys MC, Linzer DIH and Papoutsakis ET (1994) Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-1 by Chinese hamster ovary cells in a pH-dependent manner. Biotechnol Bioeng 50: 299-318.

    Google Scholar 

  • Castro PML, Hayter PM, Ison AP and Bull AT (1992) Application of a statistical deign to the optimisation of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 38: 84-90.

    Article  PubMed  CAS  Google Scholar 

  • Castro PML, Ison AP, Hayter PM and Bull AT (1995) 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 21: 87-100.

    PubMed  CAS  Google Scholar 

  • Davey CL, Guan Y, Kemp RB and Kell DB (1997) Real-time monitoring of the biomass content of animal cell cultures using dielectric spectroscopy. In: Funatsu K, Shirai Y and Matsushita T (eds) Animal Cell Technology: Basic and Applied Aspects. Vol. 8, Kluwer Academic Publ., Dordrecht, The Netherlands. (pp. 61-65).

    Google Scholar 

  • Funatsu K, Shirai Y and Matsushita T (eds) (1997) Animal Cell Technology: Basic and Applied Aspects. Vol. 8 Kluwer Academic Publ., Dordrecht, The Netherlands.

    Google Scholar 

  • Gnaiger E and Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: Information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332.

    Article  PubMed  CAS  Google Scholar 

  • Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T and Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27: 583-596.

    Article  PubMed  CAS  Google Scholar 

  • Gu MB, Todd P and Kompala DS (1993) Foreign gene expression (β-galactosidase) during the cell cycle phases in recombinant CHO cells. Biotechnol Bioeng 42: 1113-1123.

    Article  CAS  PubMed  Google Scholar 

  • Guan Y, Evans PM and Kemp RB (1998) 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 58: 464-477.

    Article  PubMed  CAS  Google Scholar 

  • Guan Y and Kemp RB (1996) Medium design with the aid of heat flux measurement in mammalian cell culture. In: Westerhoff HV, Snoep JL, Wijker JE, Sluse FE and Kholodenko BN (eds) BioThermoKinetics of the living cell, (pp. 387-397), BTK, Amsterdam.

    Google Scholar 

  • Guan Y and Kemp RB (1998a) The viable cell monitor: a dielectric spectroscope for growth and metabolic studies of animal cells on macroporous carriers. In: Merten O-W, Perrin P and Griffiths B (eds) New Developments and New Applications in Animal Cell Technology, (pp. 321-326) Kluwer, Dordrecht, The Netherlands.

    Google Scholar 

  • Guan Y and Kemp RB (1998b) Dynamic medium optimisation by on-line heat flux measurement and stoichiometric modelling in animal cell culture. In: Merten O-W, Perrin, P and Griffiths B (eds) New Developments and New Applications in Animal Cell Technology, (pp. 355-357) Kluwer, Dordrecht, The Netherlands.

    Google Scholar 

  • Guan Y and Kemp RB (1998c) Detection of dynamic substrate requirements of animal cells by stoichiometric growth equations and enthalpy balance. Biotechnol. In press.

  • Guan Y, Lloyd PC, Evans PM and Kemp RB (1997) A modified continuous flow microcalorimeter for measuring heat dissipation by mammalian cells in batch culture J Thermal Anal 49: 785-794.

    Article  CAS  Google Scholar 

  • Harris CM, Todd RW, Bungard SJ, Lovitt RW, Morris JG and Kell DB (1987) The dielectric permittivity of microbial suspensions at radio-frequencies: a novel method for the real-time estimation of microbial biomass. Enzyme Microb Technol 9: 181-186.

    Article  CAS  Google Scholar 

  • Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG and Bull AT (1991) Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Appl Microbiol Biotechnol 34: 327-335.

    Article  Google Scholar 

  • Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Tong JM and Bull AT (1992) Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-γ. Biotechnol Bioeng 39: 327-335.

    Article  CAS  PubMed  Google Scholar 

  • Hooker AD, Goldman NH, Markham, NH, James DC, Ison AP, Bull AT, Strange PG, Salmon I, Baines AJ and Jenkins N (1995) N-Glycans of recombinant human interferon-γ change during batch culture of Chinese hamster ovary cells. Biotechnol Bioeng 48: 327-337.

    Article  Google Scholar 

  • Jenkins N, Castro P, Menon A and Bull A (1994) Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells. Cytotechnol 15: 209-215.

    Article  CAS  Google Scholar 

  • Jenkins N (1995) Monitoring and control of recombinant glycoprotein heterogeneity in animal cell cultures. Biochem Soc Trans 23: 171-175.

    PubMed  CAS  Google Scholar 

  • Jeong Y-H and Wang SS (1995) Role of glutamine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism. Enzyme Microb Technol 17: 47-55.

    Article  CAS  Google Scholar 

  • Kemp RB (1993) Developments in cellular microcalorimetry with particular emphasis on the valuable role of the energy (enthalpy) balance method. Thermochim Acta 219: 17-41.

    Article  Google Scholar 

  • Kemp RB (1995) Cytotoxicity of an anchorage-independent fibroblast cell line measured by a combination of fluorescent dyes. In: O'Hare SD and Atterwill CK (eds) Methods in Molecular Biology, Vol. 43. In vitro Toxicity Testing Protocols. Humana Press, (pp. 211-218) Totowa, New Jersey, U.S.A.

    Google Scholar 

  • Kemp RB, Evans PM and Guan Y (1997) An enthalpy balance approach to studies of metabolic activity in mammalian cells. J Thermal Anal 49: 755-770.

    Article  CAS  Google Scholar 

  • Kemp RB and Guan Y (1997) Heat flux and the calorimetric-respirometric ratio as measures of catabolic flux in mammalian cells. Thermochim Acta 300: 199-211.

    Article  CAS  Google Scholar 

  • Kemp RB and Guan Y (1998) Probing the metabolism of genetically-engineered mammalian cells by heat flux. Thermochim Acta 309: 63-78.

    Article  CAS  Google Scholar 

  • Kim CN, Wang X, Huang Y, Ibrado AM, Liu L, Fang G and Bhalla K (1997) 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 57: 3115-3120.

    PubMed  CAS  Google Scholar 

  • Kubbies M and Stockinger H (1990) Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry. Exp Cell Res 188: 267-271.

    Article  PubMed  CAS  Google Scholar 

  • Liu X, Kim CN, Yang J, Jemmerson R and Wang X (1996) Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 86: 147-157.

    Article  PubMed  CAS  Google Scholar 

  • Mastrangelo AJ and Betenbaugh MJ (1998) Overcoming apoptosis: new methods for improving protein-expression systems. Trends Biotechnol 16: 88-94.

    Article  PubMed  CAS  Google Scholar 

  • Omasa T, Higashiyama K-I, Shioya S and Suga K-I (1992) Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng 39: 556-564.

    Article  CAS  PubMed  Google Scholar 

  • Prigogine I (1967) Introduction to the thermodynamics of irreversible processes. 3rd ed. Interscience, New York.

    Google Scholar 

  • Roels JA (1983). Energetics and Kinetics in Biotechnology. Elsevier, Amsterdam.

    Google Scholar 

  • Simpson NH, Milner AE and Al-Rubeai M (1997) Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions. Biotechnol Bioeng 54: 1-16.

    Article  CAS  PubMed  Google Scholar 

  • Simpson NH, Singh RP, Perani A, Goldenzon C and Al-Rubeai M (1998) 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 59: 90-98.

    Article  PubMed  CAS  Google Scholar 

  • Singh RP, Emery AN and Al-Rubeai M (1996) Enhancement of survivability of mammalian cells by overexpression of apoptosis-suppressor gene bcl-2. Biotechnol Bioeng 52: 166-175.

    Article  CAS  PubMed  Google Scholar 

  • Spier RE, Griffiths JB and Berthold W (eds) (1994) Animal Cell Technology. Butterworth-Heinemann, London.

    Google Scholar 

  • Von Stockar U, Gustafsson L, Larsson L, Marison I, Tissot P and Gnaiger E (1993) Thermodynamic considerations in constructing energy balances for cellular growth. Biochim Biophys Acta 1183: 221-240.

    Article  CAS  Google Scholar 

  • Wadsö I (1994) Microcalorimetry of aqueous and biological systems. In: Experimental Thermodynamics. Vol. IV Solution Calorimetry, Blackwell Scientific Publ., Oxford, pp. 267-301.

    Google Scholar 

  • Wu P, Ozturk SS, Blackie JD, Thrift JC, Figueroa C and Navah D (1995) Evaluation and applications of optical-cell density probes in mammalian-cell bioreactors. Biotechnol Bioeng 45: 495-502.

    Article  CAS  PubMed  Google Scholar 

  • Xie L and Wang DIC (1994) Stoichiometric analysis of animal cell growth and its application in medium design. Biotechnol Bioeng 43: 1175-1189.

    Article  CAS  PubMed  Google Scholar 

  • Xie L and Wang DIC (1996a) High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor. Biotechnol Bioeng 51: 725-729.

    Article  CAS  PubMed  Google Scholar 

  • Xie L and Wang DIC (1996b) Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network. Biotechnol Bioeng 52: 591-601.

    Article  CAS  PubMed  Google Scholar 

  • Xie L, Nyberg G, Gu X, Li H, Möllborn F and Wang DIC (1997) Gamma-interferon production and quality in stoichiometric fed-batch cultures of Chinese hamster ovary cells (CHO). Biotechnol Bioeng 56: 577-582.

    Article  CAS  PubMed  Google Scholar 

  • Zhou W and Mulchandani A (1995) Recent advances in bioprocess monitoring and control. ACS Symp Ser 613: 88-98.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biological Sciences, University of Wales, Edward Llwyd Building, Aberystwyth, SY23 3DA, UK

    Yue H. Guan & Richard B. Kemp

Authors
  1. Yue H. Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard B. Kemp
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guan, Y.H., Kemp, R.B. On-line heat flux measurements improve the culture medium for the growth and productivity of genetically engineered CHO cells. Cytotechnology 30, 107–120 (1999). https://doi.org/10.1023/A:1008038515285

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008038515285

Search

Navigation