Cytotechnology

, Volume 34, Issue 1–2, pp 27–37 | Cite as

The gel microdrop secretion assay: Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells

  • Linda Hammill
  • Jacqueline Welles
  • Gerald R. Carson
Article

Abstract

The long-term stability of high-level expression is the mostimportant factor to consider when choosing cell lines for the expression of recombinant proteins. Declining volumetricyields in large-scale fermentation can be caused by changes affecting the cell population as a whole such as loss in viability, depletion of nutrients or accumulation of metabolites affecting cell growth. Alternatively, geneticinstability may lead to the outgrowth of a less productive,metabolically favored sub-population. Currently a variety ofparameters are measured to monitor the condition of cells infermenters including glucose uptake, lactate accumulation andoxygen consumption; in addition, periodic viable cell countsallow the determination of the growth rate and viability of the population. All of these methods measure the condition ofthe cell population as a whole and changes must involve a significantly large proportion of the total culture in orderto be detectable. Here we report on a method that allows theevaluation of the productivity of individual cells. Using the gel microdrop secretion assay, we detected the appearance ofa sub-population of cells with lower productivity. Subsequentanalysis of the culture confirmed the existence of lower productivity cells with a lower vector copy number. Therefore,the single cell secretion assay proved to be a rapid method todetect and isolate a low productivity variant of the producer cell line.

CHO cells gel microdrops human antibody population parameters productivity stability 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Albano CR, Randers-Eichhorn L, Bentlet WE and Rao G (1998) Green fluorescent protein as a real time quantitative reporter of heterologous protein production. Biotechnol Prog 14: 351–354.Google Scholar
  2. Al-Rubeai M and Emery N (1993) Flow cytometry in animal cell culture. Bio/Technology 11: 572–579.Google Scholar
  3. Ausubel FM, Brent R, Moore DM, Kingston RE, Seidman JG, Smith JA and Struhl K (1990) Current Protocols in Molecular Biology, Wiley Interscience, New York.Google Scholar
  4. Chuck AS and Palsson BO (1992) Population balance between producing and nonproducing hybridoma clones is very sensitive to serum level, state of inoculum, and medium composition. Biotechnol Bioeng 39: 354–360.Google Scholar
  5. Gandor C, Leist C, Fiechter A and Asselbergs FAM (1995) Amplification and expression of recombinant genes in serumindependent Chinese hamster ovary cells. FEBS Letters 377: 290–294.Google Scholar
  6. Kaufman RJ, Wasley LC, Spiliotes AJ, Gossels SD, Latt SA, Larsen GR and Kay RM (1985) Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol Cell Biol 5: 1750–1759.Google Scholar
  7. Lubiniecki AS, Anumula K, Callaway J, L'Italien J, Oka M, Okita B, Wasserman G, Zabriske D, Arathoon R and Builder S (1992) Effects of fermentation on product consistency. Dev Biol Stand 76: 105–115.Google Scholar
  8. McKinney KL, Dilwirth R and Belfort G (1991) Manipulation of heterogeneous hybridoma cultures for overproduction of monoclonal antibodies. Biotechnol Prog 7: 445–454.Google Scholar
  9. Powell KT and Weaver JC (1990) Gel microdroplets and flow cytometry: Rapid determination of antibody secretion by individual cells with a cell population. Bio/Technology 8: 333–337.Google Scholar
  10. Rhoads DD, Dixit A and Roufa D (1986) Primary structure of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol 6: 2774–2783.Google Scholar
  11. Swift RJ, Wiebe MG, Robson GD and Trinci APJ (1998) Recombinant glucoamylase production byAspergillus niger B1 in chemostat and pH auxostat cultures. Fungal Genet Biol 25: 100–109.Google Scholar
  12. Sugiura T and Kakuzaki M (1998) Dynamics of recombinant protein production by mammalian cells in immobilized perfusion culture. Enzyme Microb Technol 22: 699–704.Google Scholar
  13. Toledo F, Le Roscouet D, Buttin G and Debatisse M (1992) Coamplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification. EMBO J 11: 2665–2673.Google Scholar
  14. Urlaub G and Chasin LA (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 77: 4216–4220.Google Scholar
  15. Withers JM, Swift RJ, Wiebe MG, Robson GD, Punt PJ, van der Hondel CAM and Trinci APJ (1998) Optimization and stability of glucoamylase production by recombinant strains ofAspergillus niger in chemostat culture. Biotechnol Bioeng 59: 407–418.Google Scholar
  16. Weaver JC, McGrath P and Adams S (1997) Gel microdrop technology for rapid isolation of rare and high producer cells. Nat Med 3: 583–585.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Linda Hammill
    • 1
  • Jacqueline Welles
    • 1
  • Gerald R. Carson
    • 2
  1. 1.Department of Molecular BiologyBASF Bioresearch CorporationWorcesterU.S.A.
  2. 2.Department of Molecular BiologyBASF Bioresearch CorporationWorcesterU.S.A.

Personalised recommendations