Skip to main content
SpringerLink
Log in

Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level

  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16–20 mM) in the culturedecreased the apparent degradation of the EPO.

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

  • Andersen DC and Goochee CF (1995) The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by Chinese hamster ovary cells. Biotechnol Bioeng 47: 96–105.

    Google Scholar 

  • Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM and Parekh RB (1995) Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem 230: 229–238.

    Google Scholar 

  • Binnie C, Cossar JD and Stewart DI (1997) Heterologous biopharmaceutical protein expression inStreptomyces. Trends in Biotechnol 15: 315–320.

    Google Scholar 

  • Borys MC, Linzer DIH and Papoutsakis ET (1993) Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Biol Technol 11: 720–724.

    Google Scholar 

  • Borys MC, Linzer DIH and Papoutsakis ET (1994) Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by Chinese hamster ovary cells in a pH-dependent manner. Biotechnol Bioeng 43: 505–514.

    Google Scholar 

  • Butler M, Hassell T, Doyle C, Gleave S and Jennings P (1991) The effect on metabolic by products on animal cells in culture. In: Spier RE, Griffiths JB and Meigner B (eds), Production of Biologicals from Animal Cells in Culture, Butterworth-Heinemann, Oxford, UK, pp. 226–228.

    Google Scholar 

  • Cartwright T (1994) Adjusting cellular metabolism for optimum product yield. In: Animal Cells as Bioreactors. Cambridge Studies in Biotechnology 11, Cambridge University Press, Cambridge, UK, pp. 96–111.

    Google Scholar 

  • Castro PML, Ison AP, Hayter PM and Bull AT (1995) The macro-heterogeneity 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.

    Google Scholar 

  • Christie A and Butler M (1994) Glutamine-based dipeptides are utilized in mammalian cell culture by extracellular hydrolysis catalyzed by a specific peptidase. J Biotechnol 37: 277–290.

    Google Scholar 

  • Curling EMA, Hayter PM, Baines AJ, Bull AT, Gull K, Strange PG and Jenkins N (1990) Recombinant human interferon-γ. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture. Biochem J 272: 333–337.

    Google Scholar 

  • Delorme E, Lorenzini T, Giffin J, Martin F, Jacobsen F, Boone T and Elliott S (1992) Role of glycosylation on the secretion and biological activity of erythropoietin. Biochem 31: 9871–9876.

    Google Scholar 

  • Dordal MS, Wang FF and Goldwasser E (1985) The role of carbohydrate in erythropoietin action. Endocrinology 116: 2293–2299.

    Google Scholar 

  • Dubé S, Fisher JW and Powell JS (1988) Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function. J Biol Chem 263: 17516–17521.

    Google Scholar 

  • Eridani S (1990) Erythropoietin. In: Spier RE and Griffiths JB (eds), Animal Cell Biotechnology, Vol. 4, Academic Press, London, UK, pp. 475–491.

    Google Scholar 

  • Ferrari J, Gunson J, Lofgren J, Krummen LL and Warner TG (1998) Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid. Biotechnol Bioeng 60: 589–595.

    Google Scholar 

  • Flickinger MC, Goebel NK, Bibila T and Boyce-Jacino S (1992) Evidence for posttranscriptional stimulation of monoclonal antibody secretion by L-glutamine during slow hybridoma growth. J Biotechnol 22: 201–226.

    Google Scholar 

  • Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Stephens PE, Thompson S and Yarranton GT (1991) Development of a process for the production of HIV 1 gp120 from recombinant cell lines. In: Spier RE, Griffiths JB and Meignier B (eds), Production of Biologicals from Animal Cells in Culture, London, Butterworth-Heinemann, pp. 110–115.

    Google Scholar 

  • Gawlitzek M, Conradt HS and Wagner R (1995) Effect of different cell culture conditions on the polypeptide integrity and Nglycosylation of a recombinant model glycoprotein. Biotechnol Bioeng 46: 536–544.

    Google Scholar 

  • Gawlitzek M, Valley U and Wagner R (1998) Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol Bioeng 57: 518–528.

    Google Scholar 

  • Gershman H and Robbins PW (1981) Transitory effects of glucose starvation on the synthesis of dolichol-linked oligosaccharides in mammalian cells. J Biol Chem 256: 7774–7780.

    Google Scholar 

  • Goldman MH, James DC, Ison AP and Bull AT (1997) Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells. Cytotechnology 23: 103–111.

    Google Scholar 

  • Gramer MJ and Goochee CF (1993) Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant. Biotechnol Prog 9: 366–373.

    Google Scholar 

  • Gramer MJ, Goochee CF, Chock VY, Brousseau DT and Sliwkowski MB (1995) Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase. Biol. Technol 13: 692–698.

    Google Scholar 

  • Gutmann I and Wahlefeld AW (1974) L-(+)-Lactate-determination with lactate dehydrogenase and NAD. In: Bergmeyer HU (ed), Methods of Enzymatic Analysis, 2nd ed., Verlag Chemie, Weinheim, pp. 1464–1468.

    Google Scholar 

  • Hassell T and Butler M (1990) Adaptation to non-ammoniagenic medium and selective substrate feeding lead to enhanced yields in animal cell cultures. J Cell Sci 96: 501–508.

    Google Scholar 

  • Hassell T, Gleave S and Butler M (1991) Growth inhibition in animal cell culture: The effect of lactate and ammonia. Appl Biochem Biotech 30: 29–41.

    Google Scholar 

  • Hayter PM, Curling EMA, Gould ML, Baines AJ, Jenkins N, Salmon I, Strange PG and Bull AT (1993) The effect of the dilution rate on CHO cell physiology and recombinant interferon-γ production in glucose-limited chemostat culture. Biotechnol Bioeng 42: 1077–1085.

    Google Scholar 

  • Hogrefe HH, McPhie P, Bekisz JB, Enterline JC, Dyer D, Webb DSA, Gerrard TL and Zoon KC (1989) Amino terminus is essential to the structural integrity of recombinant human interferon-gamma. J Biol Chem 264: 12179–12186.

    Google Scholar 

  • Hooker AD, Goldman MH, Markham NH, James DC, Ison AP, Bull AT, Strange PG, Salmon I, Baines AJ and Jenkins N (1995) Nglycans of recombinant human interferon-γ change during batch culture of Chinese hamster ovary cells. Biotechnol Bioeng 48: 639–648.

    Google Scholar 

  • Hu GF (1995) Fluorophore-assisted carbohydrate electrophoresis technology and applications. J Chromatography 705: 89–103.

    Google Scholar 

  • Ichimori Y, Suzuki N, Kitada C and Tsukamoto K (1987) Monoclonal antibodies to human interferon-gamma. II: antibodies with neutralizing activity. Hybridoma 6: 173–181.

    Google Scholar 

  • Jackson P (1994) High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Methods Enzymol 230: 250–256.

    Google Scholar 

  • Jacobs K, Shoemaler C and Rudersdorf R (1985) Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313: 806–810.

    Google Scholar 

  • Jacobson LO, Goldwasser E, Fried W and Plzak L (1957) Role of the kidney in erythropoiesis. Nature 170: 633–634.

    Google Scholar 

  • Jelkmann W (1992) Erythropoietin: Structure, control of production, and function. Physiol Rev 72: 449–489.

    Google Scholar 

  • Jenkins N and Curling EMA (1994) Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol 16: 354–364.

    Google Scholar 

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

    Google Scholar 

  • Kratje RB, Lind W and Wagner R (1994) Evaluation of the proteolytic potential of in vitro-cultivated hybridoma and recombinant mammalian cells. J Biotechnol 32: 107–125.

    Google Scholar 

  • Kunkel JP, Jan DCH, Jamieson JC and Butler M (1998) Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody. J Biotechnol 62: 55–71.

    Google Scholar 

  • Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, Chen KK, Fox M, Matin F and Stabinsky Z (1985) Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci USA 82: 7580–7584.

    Google Scholar 

  • Lind W, Lietz M, Jager V and Wagner R (1991) Proteolytic activities in serum-free supernatants of mammalian cell lines. In: Sasaki R and Ikura K (eds), Animal cell culture and production of biologicals. Kluwer Academic Publishers, Dordrecht, pp. 319–327.

    Google Scholar 

  • Lund P (1985) L-glutamine and L-glutamate. In: HU Bergmeyer (ed), Methods of Enzymatic Analysis, 3rd ed., VCH Verlagsgesellschaft, Weinheim, pp. 357–363.

    Google Scholar 

  • McKeehan WL (1982) Glycolysis, glutaminolysis and cell proliferation. Cell Biol Int Rep 6: 635–650.

    Google Scholar 

  • Morimoto K, Maeda M, Abdel-Alim A-AF, Toyoshima S and Hayakawa T (1999) Structure characterization of recombinant human erythropoietin by fluorophore-assisted carbohydrate electrophoresis. Biol Pharm Bull 22: 5–10.

    Google Scholar 

  • Munzert E, Müthing J, Buntemeyer H and Lehmann J (1996) Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. Biotechnol Prog 12: 559–563.

    Google Scholar 

  • Narhi LO, Arakawa T, Aoki KH, Elmore R, Rohde MF, Boone T and Strickland TW (1991) The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem 266: 23022–23026.

    Google Scholar 

  • Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G and Wang DIC (1999) Metabolic effects on recombinant interferon-γ glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng 62: 336–347.

    Google Scholar 

  • Omasa T, Ishimoto M, Higashiyama K, Shioya S and Suga K (1992) The enhancement of specific antibody production rate in glucose-and glutamine-controlled fed-batch culture. Cytotechnology 8: 75–84.

    Google Scholar 

  • Rearick JI, Chapman A and Kornfeld S (1981) Glucose starvation alters lipid-linked oligosaccharide biosynthesis in Chinese hamster ovary cells. J Biol Chem 256: 6255–6261.

    Google Scholar 

  • Reitzer LJ, Wice BM and Kennell D (1979) Evidence that glutamine, not sugar, is the major energy source for cultures Hela cells. J Biol Chem 254: 2669–2676.

    Google Scholar 

  • Renard JM, Spagnoli R, Mazier C, Salles MF and Mandine E (1988) Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotech Lett 10: 91–96.

    Google Scholar 

  • Ridley DM, Dawkins F and Perlin E (1994) Erythropoietin: A review. J Natl Med Assoc 86: 129–135.

    Google Scholar 

  • Sasaki H, Bothner B, Dell A and Fukuda M (1987) Carbohydrate structure of erythropoietin expressed in CHO cells by a human erythropoietin cDNA. J Biol Chem 262: 12059–12076.

    Google Scholar 

  • Satoh M, Hosol S and Sato S (1990) Chinese hamster ovary cells continuously secrete a cysteine endopeptidase. In Vitro Cell Dev Biol 26: 1101–1104.

    Google Scholar 

  • Schlaeger EJ, Eggimann B and Gast A (1987) Proteolytic activity in the culture supernatants of mouse hybridoma cells. Dev Biol Standard 66: 403–408.

    Google Scholar 

  • Singh RP, AL-Rubeai M, Gregory CD and Emery AN (1994) Cell death in bioreactors: A role for apoptosis. Biotechnol Bioeng 44: 720–726.

    Google Scholar 

  • Sugimoto S, Lind W and Wagner R (1992) Activation of a specific proteolytic activity in suspension cultures of recombinant adherent cells. In: Spier RE, Griffiths JB and MacDonald C (eds), Animal Cell Technology: Developments, Processes and Products', Butterworth-Heinemann, Oxford, UK, pp. 547–551.

    Google Scholar 

  • Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kocibe N and Kubota A (1988) Comparative study of the asparagine-linked sugar chains of human erythropoietin purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Bio Chem 263: 3657–3663.

    Google Scholar 

  • Takeuchi M, Takasaki S, Shimada M and Kobata A (1990) Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J Biol Chem 265: 12127–12130.

    Google Scholar 

  • Teige M, Weidemann R and Kretzmer G (1994) Problems with serum-free production of antithrombin III regarding proteolytic activity and product quality. J Biotechnol 34: 101–105.

    Google Scholar 

  • Thorens B and Vassalli P (1986) Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion. Nature 321: 618–620.

    Google Scholar 

  • Travis J and Salvesen GS (1983) Human plasma proteinase inhibitors. Annu Rev Biochem 52: 655–710.

    Google Scholar 

  • Tsuda E, Kawanishi G, Ueda M, Masuda S and Sasaki R (1990) The role of carbohydrate in recombinant human erythropoietin. Eur J Biochem 188: 405–411.

    Google Scholar 

  • Wang FF, Kung CKH and Goldwasser E (1985) Some chemical properties of human erythropoietin. Endocrinology 116: 2286–2292.

    Google Scholar 

  • Yang M and Butler M (2000) Effects of ammonia on CHO cell growth, erythropoietin production and glycosylation. Biotechnol Bioeng (in press).

  • Zeilke HR, Ozand PT, Tildon JT, Sevdalian DA and Cornblath M (1978) Reciprocal regulation of glucose and glutamine utilization by cultures human diploid fibroblasts. J Cell Physiol 95: 41–48.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada

    M. Yang

  2. Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada

    M. Butler

Authors
  1. M. Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Butler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Butler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, M., Butler, M. Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level. Cytotechnology 34, 83–99 (2000). https://doi.org/10.1023/A:1008137712611

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008137712611

Search

Navigation