, Volume 15, Issue 1–3, pp 95–102 | Cite as

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

  • Patrick J. Farrell
  • Nicolas Kalogerakis
  • Leo A. Behie


It has been shown that some B-cell hybridomas secrete autocrine factorsin vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G1 phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.

Key Words

Cell cycle cytokine flow cytometry hybridoma cells ultrafiltration 



fetal bovine serum


monoclonal antibody


molecular weight cut off


sodium dodecyl-sulphate-polyacrylamide gel electrophoresis


exponential phase death rate, h−1


exponential phase specific monoclonal antibody productivity, pg/(cell·h)


time, h


dead cell density, cells/mL


viable cell density, cells/mL


specific growth rate, h−1

μmax app

apparent maximum specific growth rate, h−1


maximum specific growth rate, h−1 μ max = μ max app + Kd


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Baserga R (1985) The biology of cell reproduction. Harvard University Press, London.Google Scholar
  2. Darzynkiewics Z, Crissman H, Traganos F and Steinkamp J (1982) Cell heterogeneity during the cell cycle. J. Cell. Physiol. 113: 465–474.Google Scholar
  3. Hagedorn J and Kargi F (1990) Production of monoclonal antibodies by hybridoma cells in a flat sheet membrane bioreactor. Biotechnol. Prog. 6: 220–224.Google Scholar
  4. Jochems GJ, Klein MR, Jordens R, Pascual-Salcedo D, van Boxtel-Oosterhof F, van Lier RA and Zeijlemaker WP (1991) Heterogeneity in both cytokine production and responsiveness of a panel of monoclonal human Epstein-Barr virus-transformed B-cell lines. Hum. Antibodies Hybridomas 2(2): 57–64.Google Scholar
  5. Kidwell WR (1989) Filtering out inhibition. Bio Technol. 7: 462–463.Google Scholar
  6. Kidwell W, Knazek R and Wu Y (1990) Effect of fiber pore size on performance of cells in hollow fibre bioreactors. In: Murakami H (ed.) Trends in Animal Cell Culture Technology. Kodansha Ltd., Tokyo, pp. 29–33.Google Scholar
  7. Lee GM, Chuck AS and Palsson BO (1993) Cell culture conditions determine the enhancement of specific monoclonal antibody productivity of calcium alginate-entrapped S3H5/y2bA2 hybridoma cells. Biotechnol. Bioeng. 41: 330–340.Google Scholar
  8. Linardos TI, Kalogerakis N and Behie LA (1992) Monoclonal antibody production in dialyzed continuous suspension culture. Biotechnol. Bioeng. 39: 504–510.Google Scholar
  9. Linardos TI, Kalogerakis N, Behie LA and Lamontagne LR (1991). The effect of specific growth rate and death rate on monoclonal antibody production in hybridoam chemostat cultures. Can. J. Chem. Eng. 69: 429–438.Google Scholar
  10. Makishima F, Mikami T and Terada S (1990) Effect of some cytokines on antibody productivity of a murine B-cell hybridoma. In: Murakami H (ed.) Trends in Animal Cell Culture Technology. Kodansha Ltd., Tokyo, pp. 299–302.Google Scholar
  11. Martens DE, de Gooijer CD, van der Velden-de Groot CAM, Beuvery EC and Tramper J (1993) Effect of dilution rate on growth, productivity, cell cycle and size, and shear sensitivity of a hybridoma cell in continuous culture. Biotechnol. Bioeng. 41: 429–439.Google Scholar
  12. Miller WM, Blanch HW and Wilke CR (1988) A kinetic analysis of hybridoma growth and metabolism in batch and continuous culture: effect of nutrient concentration, dilution rate and pH. Biotechnol. Bioeng. 32: 947–965.Google Scholar
  13. Negri C, Chiesa R, Giulia CB and Ricotti A (1991) Factor(s) required by EBV transformed lymphocytes to grow under limiting dilution conditions. Cytotech. 7: 173–178.Google Scholar
  14. Ozturk OS and Pallson BO (1991) Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors. Biotechnol. Prog. 7: 471–480.Google Scholar
  15. Pendse GJ and Bailey JE (1990) Effects of growth factors on cell proliferation and monoclonal antibody production of batch hybridoma cultures. Biotechnol. Lett. 7(12): 487–492.Google Scholar
  16. Ramirez OT and Mutharason R (1990) Cell cycle and growth phase dependant variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures. Biotechnol. Bioeng. 36: 839–848.Google Scholar
  17. Smith JA and Martin L (1973) Do cells cycle? Proc. Natl. Acad. Sci. USA 70: 1263–1267.Google Scholar
  18. Suzuki E and Ollis DF (1989) Cell cycle model for antibody production kinetics. Biotechnol. Bioeng. 34: 1398–1402.Google Scholar
  19. Suzuki E and Ollis D (1990) Enhanced antibody productivity at low growth rates: Experimental demonstration and a simple structured model. Biotechnol. Prog. 6: 231–236.Google Scholar
  20. Yamada K, Ikeda I, Sugahara T, Shirahata and Murakami H (1990) Enhancement of immunoglobuiin production of human-human hybridomas by immunoglobulin production stimulating factors. In: Murakami H (ed.) Trends in Animal Cell Culture Technology. Kodansha Ltd., Tokyo, pp. 291–297.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Patrick J. Farrell
    • 1
  • Nicolas Kalogerakis
    • 1
  • Leo A. Behie
    • 1
  1. 1.Pharmaceutical Production Research Facility (PPRF) Faculty of EngineeringUniversity of CalgaryCanada

Personalised recommendations