Skip to main content
SpringerLink
Log in

An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

  • Originals
  • Published:
Bioprocess Engineering Aims and scope Submit manuscript

Abstract

In this work, metabolite and antibody production kinetics of hybridoma cultures were investigated as a function of cell density and growth rate in a homogeneous perfusion reactor. Hydrophilized hollow fiber polypropylene membranes with a pore size of 0.2 μm were used for medium perfusion. Oxygen was supplied to the cells through thin walled silicone tubing. The mouse-mouse hybridoma cells were grown in three identical bioreactors at perfusion rates of 1.1, 2.0, and 3.2/day for a period of eight days during which the viable cell concentrations reached stable values of 2.6×106, 3.5×106, and 5.2×106 cells/ml, respectively. Total cell densities reached values ranging from 8×106 to 1×106 cells/ml. Specific substrate consumption and product formation rates responded differently to changes in cell density and apparent specific growth rate, which were not varied independently. Using multiple regression analysis, the specific glucose consumption rate was found to vary with viable cell density while the specific glutamine uptake and lactate production rates varied with both viable cell density and apparent specific growth rate. These results suggest that cell density dictates the rate of glucose consumption while the cell growth rate influences how glucose is metabolized, i.e., through glycolysis or the TCA cycle. The specific antibody production rate was found to be a strong function of cell density, increasing as cell density increased, but was essentially independent of the specific growth rate for the cell line under study.

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

Abbreviations

MAb:

monoclonal antibody

X v :

viable cell density (cells/ml)

X d :

nonviable cell density (cells/ml)

μ:

specific growth rate (1/day)

k d :

specific death rate (1/day)

D :

dilution rate (1/day)

S f :

substrate concentration in feed (g/l or mM)

S :

substrate concentration (g/l or mM)

P f :

product concentration in feed (g/l or μg/ml)

P :

product concentration (g/l or ug/ml)

q s :

specific consumption rate of substrate (g/hr/cell or mmol/hr/cell)

q p :

specific production rate of product (g/hr/cell)

q MAb :

specific production rate of monoclonal antibody (μg/hr/cell)

References

  1. Kohler, G.; Milstein, C.: Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256 (1975) 495

    PubMed  Google Scholar 

  2. Klausner, A.: Taking aim at cancer with monoclonal antibodies. Bio/Technol. 4 (1986) 185–205

    Article  Google Scholar 

  3. Thorpe, R.: Monoclonal antibodies: clinical and regulatory issues. TIBTech. 11 (1993) 40–42

    Google Scholar 

  4. Shawler, D. L.; Bartholomew, R. M.; Smith, L. M.; Dillman, R. O.: Human immune response to multiple injections of murine monoclonal IgG. J. Immunol. 135 (1985) 1530–1535

    PubMed  Google Scholar 

  5. Werner, R. G.; Walz, F.; Wolfgang, N.; Konrad, A.: Safety and economic aspects of continuous mammalian cell culture. J. Biotechnol. 22 (1992) 51–68

    Article  PubMed  Google Scholar 

  6. Smith, C. G.; Guillaume, J. M.; Greenfield, P. F.; Randerson, F. H.: Experience in scale-up of homogeneous perfusion culture for hybridomas. Bioproc. Eng. 6 (1991) 213–219

    Google Scholar 

  7. Evans, T. L.; Miller, R. A.: Large scale production of murine monoclonal antibodies using hollow fiber bioreactors. Biotechniques 6 (1988) 762–767

    PubMed  Google Scholar 

  8. Munster, M. J.; Kearns, M. J.; Steegmans, U.; Behrendt, U.; Comer, M. J.: A high density culture system for the in-vitro production of human and mouse monoclonal antibodies. Bioproc. Eng. 6 (1991) 123–125

    Google Scholar 

  9. Oh, D. J.; Chang, H. N.: High density culture of hybridoma cells in a dual hollow fiber bioreactor. Biotechnol. Lett. 6 (1992) 77–82

    Google Scholar 

  10. Cadic, C.; Dupuy, B.; Pianet, I.; Merle, M.; Margerin, C.; Bezian, J.: In vitro cultivation of hybridoma cells in agarose beads producing antibody secretion for two weeks. Biotechnol. Bioeng. 39 (1992) 108–112

    Google Scholar 

  11. Murdin, A. D.; Thorpe, J. S.; Groves, D. J.; Spier, R. E.: Growth and metabolism of hybridomas immobilized in packed beds: comparison with static and suspension cultures. Enz. Microb. Technol. 11 (1988) 341–346

    Article  Google Scholar 

  12. Tyo, M. A.; Spier, R. E.: Dense cultures of animal cells at the industrial scale. Enz. Microb. Technol. 9 (1987) 514–520

    Article  Google Scholar 

  13. Avgerinos, G. C.; Drapeau, D.; Socolow, J. S.; Mao, J.; Hsaio, K.; Broeze, R. J.: Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells. Bio/Technol. 8 (1990) 54–58

    Article  Google Scholar 

  14. Schmialek, P.; Geyer, A.; Miosga, V.; Nundel, M.; Zapf, B.: An automatic apparatus for continuous suspension culture with a concentration device. Biotechnol. Bioeng. 18 (1976) 1497–1506

    PubMed  Google Scholar 

  15. Tokashiki, M.; Arai, T.; Hamamoto, K.; Ishimaru, K.: High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge. Cytotechnol. 3 (1990) 239–244

    Google Scholar 

  16. Jager, V.; Eichner, W.; Lehmann, J.: Production of human PDGF-A in a stirred tank perfusion reactor and in a hollow fiber reactor. In: Spier, R. E.; Griffiths, J. B.; Stephenne, J.; Crooy, P. J. (Eds.): Advances in animal cell biology and technology for bioprocesses, pp. 323–326. London: Butterworths Publishing 1989

    Google Scholar 

  17. Batt, B. C.; Davis, R. H.; Kompala, D. S.: Inclined sedimentation for selective retention of viable hybridomas in continuous suspension bioreactor. Biotechnol. Prog. 6 (1990) 458–464

    PubMed  Google Scholar 

  18. Buser, C. W.; Thilly, W. G.: Molecular dynamics of antibody production in high cell density perfused fermenters. Presented at: AIChE National Mtg., Los Angeles, CA, November 1991

  19. Ozturk, S. S.; Palsson, B. O.; Midgeley, A. R.; Halberstadt, C. R.: Transtubular bioreactor: a perfusion device for mammalian cell cultivation. Biotechnol. Tech. 3 (1989) 55–60

    Google Scholar 

  20. Al-Rubeai, M.; Emery, A. N.: Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell culture. J. Biotechnol. 16 (1990) 67–86

    Article  PubMed  Google Scholar 

  21. Miller, W. M.; Blanch, H. W.; Wilke, C. R.: 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 (1987) 947–965

    Google Scholar 

  22. Ramirez, O. T.; Mutharasan, R.: Cell cycle- and growth phase-dependent variations in size distributions, antibody productivity, and oxygen demand in hybridoma cultures. Biotechnol. Bioeng. 36 (1990) 839–848

    Google Scholar 

  23. Richieri, R. A.; Williams, L. S.; Chau, P. C.: Cell cycle dependency of monoclonal antibody production in asynchronous serum-free hybridoma cultures. Cytotechnol. 5 (1991) 243–254

    Google Scholar 

  24. Suzuki, E.; Ollis, D. F.: Cell cycle model for antibody production kinetics. Biotechnol. Bioeng. 34 (1989) 1398–1402

    Google Scholar 

  25. Suzuki, E.; Ollis, D. F.: Enhanced antibody production at slowed growth rates: experimental demonstration and a simple structured model. Biotechnol. Prog. 6 (1990) 231–236

    PubMed  Google Scholar 

  26. Kasehagen, C.; Linz, F.; Kretzmer, G.; Scheper, T.; Schugerl, K.: Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing. Enz. Microb. Technol. 13 (1991) 873–880

    Article  Google Scholar 

  27. Shirai, Y.; Hashimoto, K.; Yamaji, H.; Kawahara, H.: Oxygen uptake rate of immobilized growing cells. Appl. Microbiol. Biotechnol. 29 (1988) 113–118

    Article  Google Scholar 

  28. Shirai, Y.; Hashimoto, K.; Takamatsu, H.: Growth kinetics of hybridoma cells in high density culture. J.Ferment. Bioeng. 73 (1992) 159–165

    Google Scholar 

  29. Wohlpart, D.; Gainer, J.; Kirwan, D.: Oxygen uptake by entrapped hybridoma cells. Biotechnol. Bioeng. 37 (1991) 1050–1053

    Google Scholar 

  30. Lee, G. M.; Kaminski, M. S.; Palsson, B. O.: Observations consistent with autocrine simulation of hybridoma cell growth and implications for large-scale antibody production. Biotechnol. Lett. 14 (1990) 257–262

    Google Scholar 

  31. Lee, G. M.; Palsson, B. O.: Immobilization can improve the stability of hybridoma antibody productivity in serum-free media. Biotechnol. Bioeng. 36 (1990) 1049–1055

    Google Scholar 

  32. Takazawa, Y.; Tokashiki, M.; Murakami, H.; Yamada, K.; Omura, H.: High-density culture of mouse-human hybridoma in serum-free defined medium. Biotechnol. Bioeng. 31 (1988) 168–172

    Google Scholar 

  33. Glacken, M. W.; Huang, C.; Sinskey, A. J.: Mathematical descriptions of hybridoma culture kinetics. III. Simulation of fed-batch bioreactors. J. Biotechnol. 10 (1989) 39–66

    Article  Google Scholar 

  34. Hiller, G. W.; Clark, D. S.; Blanch, H. W.: Cell retention —chemostat studies of hybridoma cells — analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium. Biotechnol. Bioeng. 42 (1993) 185–195

    Google Scholar 

  35. Nikolai, T. J.; Hu, W. S.: Cultivation of mammalian cells on macroporous microcarriers. Enz. Microb. Technol. 14 (1992) 203–208

    Article  Google Scholar 

  36. Seamans, T. C.; Hu, W. S.: Kinetics of growth and antibody production by a hybridoma cell line in perfusion culture. J. Ferment. Biotechnol. 70 (1990) 241–245

    Google Scholar 

  37. Zhang, S.; Handa-Corrigan, A.; Spier, R. E.: A comparison of oxygenation methods for high-density perfusion cultures of animal cells. Biotechnol. Bioeng. 41 (1993) 685–692

    Google Scholar 

  38. Wohlpart, D.; Kirwan, D.; Gainer, J.: Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells. Biotechnol. Bioeng. 36 (1990) 630–635

    Google Scholar 

  39. de la Broise, D.; Noiseux, M.; Lemieux, R.; Massie, B.: Long-term perfusion culture of hybridoma: a “grow or die” cell cycle system. Biotechnol. Bioeng. 38 (1991) 781–787

    Google Scholar 

  40. Schmid, G.; Wilke, C. R.; Blanch, H. W.: Continuous hybridoma suspension cultures with and without cell retention: kinetics of growth, metabolism and product formation. J. Biotechnol. 22 (1992) 31–40

    Article  PubMed  Google Scholar 

  41. Reddy, S.; Miller, W. M.: Hybridoma antibody content and production rate in continuous culture: effect of dilution rate. Biotechnol. Lett. 14 (1992) 1007–1012

    Google Scholar 

  42. Muir, D.; Varon, S.; Manthorpe, M.: Schwann cell proliferation in vitro is under negative autocrine control. J. Cell Biol. 111 (1990) 2663–2671

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Thayer School of Engineering, Dartmouth College, 03755-8000, Hanover, NH, USA

    G. G. Banik

  2. Department of Chemical Engineering, Iowa State University, 50011-2230, Ames, IA, USA

    C. A. Heath

Authors
  1. G. G. Banik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. A. Heath
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported in part by a grant for the National Science Foundation (BCS-9157851) and by matching funds from Merck and Monsanto. We sincerely thank Mr. Roland Buchele of Akzo Inc. (Germany) for donation of the polypropylene membranes, Dr. Michael Fanger (Dartmouth Medical School) for the hybridoma cell line, Dr. Sadettin Ozturk (Verax Corp., Lebanon, NH) for technical discussions regarding reactor design, and Dr. Derrick Rollins (Iowa State University) for advice on statistical methods.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Banik, G.G., Heath, C.A. An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor. Bioprocess Engineering 11, 229–237 (1994). https://doi.org/10.1007/BF00387697

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00387697

Keywords

Search

Navigation