Skip to main content
SpringerLink
Log in

Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells

  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.

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.

Similar content being viewed by others

References

  • Al-Rubeai M, Oh SKW, Musaheb R & Emery AN (1990) Modified cellular metabolism in hybridomas subjected to hydrodynamic and other stresses. Biotechnol Lett 12: 323-328.

    Google Scholar 

  • Barford JP, Phillips PJ, Marquis CP & Harbour C (1996) Biosynthesis of protein products by animal cells. Are growth and non-growth associated concepts valid or useful? Cytotechnology 21: 133-148.

    Google Scholar 

  • Bibila TA & Robinson DK (1995) In pursuit of the optimal fedbatch process for monoclonal antibody production. Biotech Prog 11: 1-13.

    Google Scholar 

  • Bonarius HPJ, Hatzumanikatis V, Meesters KPH, de Gooijer CD, Schmid G & Tramper J (1996) Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol Bioeng 50: 299-318.

    Google Scholar 

  • Boraston R, Thompson PW, Garland S & Birch JR (1984) Growth and oxygen requirements of antibody-producing mouse hybridoma cells in suspension culture. Devel Biol Stand 55: 103-111.

    Google Scholar 

  • Bradford MM (1976) A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-254.

    Google Scholar 

  • Brown AH (1946) Determination of pentose in the presence of large quantities of glucose. Arch Biochem 11: 269-278.

    Google Scholar 

  • Bushell ME, Bell SL, Scott MF, Spier RE, Wardell JN & Sanders PG (1994) Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population. Biotechnol Bioeng 44: 1099-1106.

    Google Scholar 

  • Dische Z (1955) Color reactions of nucleic acid components. In: Chargaff E & Davison JN (eds) Nucleic Acids, Vol. 1 (p. 287) Academic Press, New York.

    Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK, Rebers PA & Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356.

    Google Scholar 

  • Fermo I, Ruboni FM, Bolzacchini E, Arcelloni C & Bonini PA (1988) Pre-column derivatization of amino acids with N,N,-diethyl-2,4-dinitro-5-fluoroaniline and reversed-phase liquid chromatographic separation. J Chromatogr 433: 53-62.

    Google Scholar 

  • Fermo I & Vecchi ED (1990) Serum amino acid analysis with pre-column derivatization: comparison of the o-phthaldialdehyde and N,N,-diethyl-2,4-dinitro-5-fluoroaniline method. J Chromatogr 534: 23-25.

    Google Scholar 

  • Folch JL & Sloane SG (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 97-509.

    Google Scholar 

  • Frame KK & Hu W-S (1991) Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cells. Biotechnol Bioeng 37: 55-64.

    Google Scholar 

  • Garatun-Tjeldstø O, Pryme IF, Weltman JK & Dowben RM (1976) Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells. J Cell Biol 68: 232-239.

    Google Scholar 

  • Glacken MW, Fleischaker RJ & Sinsky AJ (1986) 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 28: 1376-1389.

    Google Scholar 

  • Golding B, Pillemer SR, Roussou P, Peter EA, Tsokos GC, Ballow JE & Hoffman T (1988) Inverse relationship between proliferation and differentiation in a Human TNP-specific B cell line. Cell cycle dependence of antibody secretion. J Immunol 131: 2564-2568.

    Google Scholar 

  • Halliburton IW & Thompson RY (1965) Chemical aspects of compensatory renal hypertrophy. Cancer Res 25: 1882-1887.

    Google Scholar 

  • Hayter PM, Kirkby NF & Spier RE (1992) Relationship between hybridoma growth and monoclonal antibody production. Enzyme Microb Technol 14: 454-461.

    Google Scholar 

  • King KL & Cidlowski JA (1998) Cell cycle regulation and apoptosis. Ann Rev Physiol 60: 601-617.

    Google Scholar 

  • Köhler G & Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497.

    Google Scholar 

  • Leno M, Merten O-W & Hache J (1992) Kinetic analysis of hybridoma growth and monoclonal antibody production in semicontinuous culture. Biotechnol Bioeng 39: 596-606.

    Google Scholar 

  • Linardos TI, Kalogerakis N & Behie LA (1992) Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures. Biotechnol Bioeng 40: 359-368.

    Google Scholar 

  • Ljunggren J & Häggström L (1994) Catabolic control of hybridoma cells by glucose and glutamine limited fed batch culture. Biotechnol Bioeng 44: 808-818.

    Google Scholar 

  • Merlo GR, Fiore L, Basolo F, Woods-Cook K & Hynes NE (1997) In mammary epithelial cells p53-mediated apoptosis in response to DNA damage is dependent on the agent and can be influenced by growth factors. Endoc Rel Cancer 4: 55-66.

    Google Scholar 

  • Miller WM, Blanch HW & Wilke CR (1988) A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH. Biotechnol Bioeng 32: 947-965.

    Google Scholar 

  • Newland M, Kamal MN, Greenfield PF & Nielsen LK (1994) Ammonia inhibition of hybridomas propagated in batch, fed-batch, and continuous culture. Biotechnol Bioeng 43: 434-438.

    Google Scholar 

  • Nielsen LK, Niloperbowo W, Reid S & Greenfield PF (1992) Avoiding rapid growth at high cell densities: A potentially important optimization criterion for hybridoma cultures. Cytotechnology 9: 21-27.

    Google Scholar 

  • Pentz M & Shott M (1989) Handling Experimental Data. Open University Press, Milton Keynes, U.K.

    Google Scholar 

  • Phillips PJ, Marquis CP, Barford JP & Harbour C (1991) An analysis of some batch and continuous kinetic data of specific monoclonal antibody production from hybridomas. Cytotechnology 6: 189-195.

    Google Scholar 

  • Pirt SJ (1957) Principles of microbe and cell cultivation. John Wiley & Sons, New York.

    Google Scholar 

  • Pörtner R & Schäfer T (1996) Modeling hybridoma cell growth and metabolism-a comparison of selected models and data. J Biotechnol 49: 119-135.

    Google Scholar 

  • Ramírez OT & Mutharasan R (1990) Cell cycle-and growth phasedependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures. Biotechnol Bioeng 36: 839-848.

    Google Scholar 

  • Ray NG, Karkare SB & Runstadler Jr, PW (1989) Cultivation of hybridoma cells in continuous cultures: Kinetics of growth and product formation. Biotechnol Bioeng 33: 724-730.

    Google Scholar 

  • Robinson DK & Memmert KW (1991) Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture. Biotechnol Bioeng 38: 972-976.

    Google Scholar 

  • Savinell JM & Palsson BO (1992) Network analysis of intermediary metabolism using linear optimization. I. Development of metabolism formulation. J Theor Biol 154: 421-454.

    Google Scholar 

  • Savonnière S, Zeghari N, Miccoli L, Muller S, Maugras M & Donner M (1996) Effect of lipid supplementation of culture media on cell growth, antibody production, membrane structure and dynamics in two hybridomas. J Biotechnol 48: 161-173.

    Google Scholar 

  • Schneider M, Marison IW & Stocker U (1996) The importance of ammonia in mammalian cell culture. J Biotechnol 46: 161-185.

    Google Scholar 

  • Suzuki E & Ollis DF (1989) Cell cycle model for antibody production kinetics. Biotechnol Bioeng 34: 1398-1402

    Google Scholar 

  • Takazawa Y, Tokashiki M, Hamamoto K & Murakami H (1988) High cell density perfusion culture of hybridoma cells recycling high molecular weight compounds. Cytotechnology 1: 171-178.

    Google Scholar 

  • Xie L & Wang DI (1994a) Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor. Cytotechnology 15(1-3): 17-29.

    Google Scholar 

  • Xie L & Wang DI (1994b) Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol Bioeng 43: 1175-1189.

    Google Scholar 

  • Zupke C & Stephanopoulos G (1995) Intracellular flux analysis in hybridomas using mass balances and in vitro 13C NMR. Biotechnol Bioeng 45: 292-303.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Sydney University, Sydney, NSW, 2006, Australia

    Jae Deog Jang

  2. Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, (SAR), Hong Kong China

    John P. Barford

Authors
  1. Jae Deog Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John P. Barford
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John P. Barford.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jang, J.D., Barford, J.P. Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells. Cytotechnology 32, 229–242 (2000). https://doi.org/10.1023/A:1008169417980

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008169417980

Search

Navigation