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. 2000 Oct;34(1-2):83–99. doi: 10.1023/A:1008137712611

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

M Yang 1, M Butler 2,✉
PMCID: PMC3449730  PMID: 19003383

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.

Keywords: CHO cells, erythropoietin, glycosylation, glutamine

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References

  1. Andersen DC, Goochee CF. The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by Chinese hamster ovary cells. Biotechnol Bioeng. 1995;47:96–105. doi: 10.1002/bit.260470112. [DOI] [PubMed] [Google Scholar]
  2. Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB. Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem. 1995;230:229–238. doi: 10.1006/abio.1995.1468. [DOI] [PubMed] [Google Scholar]
  3. Binnie C, Cossar JD, Stewart DI. Heterologous biopharmaceutical protein expression inStreptomyces. Trends in Biotechnol. 1997;15:315–320. doi: 10.1016/s0167-7799(97)01062-7. [DOI] [PubMed] [Google Scholar]
  4. Borys MC, Linzer DIH, Papoutsakis ET. Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Biol Technol. 1993;11:720–724. doi: 10.1038/nbt0693-720. [DOI] [PubMed] [Google Scholar]
  5. Borys MC, Linzer DIH, Papoutsakis ET. Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by Chinese hamster ovary cells in a pH-dependent manner. Biotechnol Bioeng. 1994;43:505–514. doi: 10.1002/bit.260430611. [DOI] [PubMed] [Google Scholar]
  6. Butler M, Hassell T, Doyle C, Gleave S, Jennings P. The effect on metabolic by products on animal cells in culture. In: Spier RE, Griffiths JB, Meigner B, editors. Production of Biologicals from Animal Cells in Culture. Oxford, UK: Butterworth-Heinemann; 1991. pp. 226–228. [Google Scholar]
  7. Cartwright T. Animal Cells as Bioreactors. Cambridge, UK: Cambridge University Press; 1994. Adjusting cellular metabolism for optimum product yield; pp. 96–111. [Google Scholar]
  8. Castro PML, Ison AP, Hayter PM, Bull AT. 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. 1995;21:87–100. [PubMed] [Google Scholar]
  9. Christie A, Butler M. Glutamine-based dipeptides are utilized in mammalian cell culture by extracellular hydrolysis catalyzed by a specific peptidase. J Biotechnol. 1994;37:277–290. doi: 10.1016/0168-1656(94)90134-1. [DOI] [PubMed] [Google Scholar]
  10. Curling EMA, Hayter PM, Baines AJ, Bull AT, Gull K, Strange PG, Jenkins N. Recombinant human interferon-γ. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture. Biochem J. 1990;272:333–337. doi: 10.1042/bj2720333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Delorme E, Lorenzini T, Giffin J, Martin F, Jacobsen F, Boone T, Elliott S. Role of glycosylation on the secretion and biological activity of erythropoietin. Biochem. 1992;31:9871–9876. doi: 10.1021/bi00156a003. [DOI] [PubMed] [Google Scholar]
  12. Dordal MS, Wang FF, Goldwasser E. The role of carbohydrate in erythropoietin action. Endocrinology. 1985;116:2293–2299. doi: 10.1210/endo-116-6-2293. [DOI] [PubMed] [Google Scholar]
  13. Dubé S, Fisher JW, Powell JS. Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function. J Biol Chem. 1988;263:17516–17521. [PubMed] [Google Scholar]
  14. Eridani S. Erythropoietin. In: Spier RE, Griffiths JB, editors. Animal Cell Biotechnology, Vol. 4. London, UK: Academic Press; 1990. pp. 475–491. [Google Scholar]
  15. Ferrari J, Gunson J, Lofgren J, Krummen LL, Warner TG. Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid. Biotechnol Bioeng. 1998;60:589–595. [PubMed] [Google Scholar]
  16. Flickinger MC, Goebel NK, Bibila T, Boyce-Jacino S. Evidence for posttranscriptional stimulation of monoclonal antibody secretion by L-glutamine during slow hybridoma growth. J Biotechnol. 1992;22:201–226. doi: 10.1016/0168-1656(92)90142-v. [DOI] [PubMed] [Google Scholar]
  17. Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Stephens PE, Thompson S, Yarranton GT. Development of a process for the production of HIV 1 gp120 from recombinant cell lines. In: Spier RE, Griffiths JB, Meignier B, editors. Production of Biologicals from Animal Cells in Culture. London: Butterworth-Heinemann; 1991. pp. 110–115. [Google Scholar]
  18. Gawlitzek M, Conradt HS, Wagner R. Effect of different cell culture conditions on the polypeptide integrity and Nglycosylation of a recombinant model glycoprotein. Biotechnol Bioeng. 1995;46:536–544. doi: 10.1002/bit.260460606. [DOI] [PubMed] [Google Scholar]
  19. Gawlitzek M, Valley U, Wagner R. Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol Bioeng. 1998;57:518–528. [PubMed] [Google Scholar]
  20. Gershman H, Robbins PW. Transitory effects of glucose starvation on the synthesis of dolichol-linked oligosaccharides in mammalian cells. J Biol Chem. 1981;256:7774–7780. [PubMed] [Google Scholar]
  21. Goldman MH, James DC, Ison AP, Bull AT. Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells. Cytotechnology. 1997;23:103–111. doi: 10.1023/A:1007947130709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gramer MJ, Goochee CF. Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant. Biotechnol Prog. 1993;9:366–373. doi: 10.1021/bp00022a003. [DOI] [PubMed] [Google Scholar]
  23. Gramer MJ, Goochee CF, Chock VY, Brousseau DT, Sliwkowski MB. Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase. Biol. Technol. 1995;13:692–698. doi: 10.1038/nbt0795-692. [DOI] [PubMed] [Google Scholar]
  24. Gutmann I, Wahlefeld AW. L-(+)-Lactate-determination with lactate dehydrogenase and NAD. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. 2nd ed. Weinheim: Verlag Chemie; 1974. pp. 1464–1468. [Google Scholar]
  25. Hassell T, Butler M. Adaptation to non-ammoniagenic medium and selective substrate feeding lead to enhanced yields in animal cell cultures. J Cell Sci. 1990;96:501–508. doi: 10.1242/jcs.96.3.501. [DOI] [PubMed] [Google Scholar]
  26. Hassell T, Gleave S, Butler M. Growth inhibition in animal cell culture: The effect of lactate and ammonia. Appl Biochem Biotech. 1991;30:29–41. doi: 10.1007/BF02922022. [DOI] [PubMed] [Google Scholar]
  27. Hayter PM, Curling EMA, Gould ML, Baines AJ, Jenkins N, Salmon I, Strange PG, Bull AT. The effect of the dilution rate on CHO cell physiology and recombinant interferon-γ production in glucose-limited chemostat culture. Biotechnol Bioeng. 1993;42:1077–1085. doi: 10.1002/bit.260420909. [DOI] [PubMed] [Google Scholar]
  28. Hogrefe HH, McPhie P, Bekisz JB, Enterline JC, Dyer D, Webb DSA, Gerrard TL, Zoon KC. Amino terminus is essential to the structural integrity of recombinant human interferon-gamma. J Biol Chem. 1989;264:12179–12186. [PubMed] [Google Scholar]
  29. Hooker AD, Goldman MH, Markham NH, James DC, Ison AP, Bull AT, Strange PG, Salmon I, Baines AJ, Jenkins N. Nglycans of recombinant human interferon-γ change during batch culture of Chinese hamster ovary cells. Biotechnol Bioeng. 1995;48:639–648. doi: 10.1002/bit.260480612. [DOI] [PubMed] [Google Scholar]
  30. Hu GF. Fluorophore-assisted carbohydrate electrophoresis technology and applications. J Chromatography. 1995;705:89–103. doi: 10.1016/0021-9673(95)93203-8. [DOI] [PubMed] [Google Scholar]
  31. Ichimori Y, Suzuki N, Kitada C, Tsukamoto K. Monoclonal antibodies to human interferon-gamma. II: antibodies with neutralizing activity. Hybridoma. 1987;6:173–181. doi: 10.1089/hyb.1987.6.173. [DOI] [PubMed] [Google Scholar]
  32. Jackson P. High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Methods Enzymol. 1994;230:250–256. doi: 10.1016/0076-6879(94)30017-8. [DOI] [PubMed] [Google Scholar]
  33. Jacobs K, Shoemaler C, Rudersdorf R. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature. 1985;313:806–810. doi: 10.1038/313806a0. [DOI] [PubMed] [Google Scholar]
  34. Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature. 1957;170:633–634. doi: 10.1038/179633a0. [DOI] [PubMed] [Google Scholar]
  35. Jelkmann W. Erythropoietin: Structure, control of production, and function. Physiol Rev. 1992;72:449–489. doi: 10.1152/physrev.1992.72.2.449. [DOI] [PubMed] [Google Scholar]
  36. Jenkins N, Curling EMA. Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol. 1994;16:354–364. doi: 10.1016/0141-0229(94)90149-x. [DOI] [PubMed] [Google Scholar]
  37. Jenkins N, Castro P, Menon S, Ison A, Bull A. Effect of lipid supplements on the production and glycosylation of recombinant interferon-gamma expressed in CHO cells. Cytotechnology. 1994;15:209–215. doi: 10.1007/BF00762395. [DOI] [PubMed] [Google Scholar]
  38. Kratje RB, Lind W, Wagner R. Evaluation of the proteolytic potential of in vitro-cultivated hybridoma and recombinant mammalian cells. J Biotechnol. 1994;32:107–125. doi: 10.1016/0168-1656(94)90174-0. [DOI] [PubMed] [Google Scholar]
  39. Kunkel JP, Jan DCH, Jamieson JC, Butler M. Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody. J Biotechnol. 1998;62:55–71. doi: 10.1016/s0168-1656(98)00044-3. [DOI] [PubMed] [Google Scholar]
  40. Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, Chen KK, Fox M, Matin F, Stabinsky Z. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci USA. 1985;82:7580–7584. doi: 10.1073/pnas.82.22.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lind W, Lietz M, Jager V, Wagner R. Proteolytic activities in serum-free supernatants of mammalian cell lines. In: Sasaki R, Ikura K, editors. Animal cell culture and production of biologicals. Dordrecht: Kluwer Academic Publishers; 1991. pp. 319–327. [Google Scholar]
  42. Lund P. L-glutamine and L-glutamate. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. 3rd ed. Weinheim: VCH Verlagsgesellschaft; 1985. pp. 357–363. [Google Scholar]
  43. McKeehan WL. Glycolysis, glutaminolysis and cell proliferation. Cell Biol Int Rep. 1982;6:635–650. doi: 10.1016/0309-1651(82)90125-4. [DOI] [PubMed] [Google Scholar]
  44. Morimoto K, Maeda M, Abdel-Alim A-AF, Toyoshima S, Hayakawa T. Structure characterization of recombinant human erythropoietin by fluorophore-assisted carbohydrate electrophoresis. Biol Pharm Bull. 1999;22:5–10. doi: 10.1248/bpb.22.5. [DOI] [PubMed] [Google Scholar]
  45. Munzert E, Müthing J, Buntemeyer H, Lehmann J. Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. Biotechnol Prog. 1996;12:559–563. doi: 10.1021/bp9600086. [DOI] [PubMed] [Google Scholar]
  46. Narhi LO, Arakawa T, Aoki KH, Elmore R, Rohde MF, Boone T, Strickland TW. The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem. 1991;266:23022–23026. [PubMed] [Google Scholar]
  47. Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DIC. Metabolic effects on recombinant interferon-γ glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng. 1999;62:336–347. doi: 10.1002/(sici)1097-0290(19990205)62:3<336::aid-bit10>3.0.co;2-n. [DOI] [PubMed] [Google Scholar]
  48. Omasa T, Ishimoto M, Higashiyama K, Shioya S, Suga K. The enhancement of specific antibody production rate in glucose-and glutamine-controlled fed-batch culture. Cytotechnology. 1992;8:75–84. doi: 10.1007/BF02540032. [DOI] [PubMed] [Google Scholar]
  49. Rearick JI, Chapman A, Kornfeld S. Glucose starvation alters lipid-linked oligosaccharide biosynthesis in Chinese hamster ovary cells. J Biol Chem. 1981;256:6255–6261. [PubMed] [Google Scholar]
  50. Reitzer LJ, Wice BM, Kennell D. Evidence that glutamine, not sugar, is the major energy source for cultures Hela cells. J Biol Chem. 1979;254:2669–2676. [PubMed] [Google Scholar]
  51. Renard JM, Spagnoli R, Mazier C, Salles MF, Mandine E. Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotech Lett. 1988;10:91–96. [Google Scholar]
  52. Ridley DM, Dawkins F, Perlin E. Erythropoietin: A review. J Natl Med Assoc. 1994;86:129–135. [PMC free article] [PubMed] [Google Scholar]
  53. Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in CHO cells by a human erythropoietin cDNA. J Biol Chem. 1987;262:12059–12076. [PubMed] [Google Scholar]
  54. Satoh M, Hosol S, Sato S. Chinese hamster ovary cells continuously secrete a cysteine endopeptidase. In Vitro Cell Dev Biol. 1990;26:1101–1104. doi: 10.1007/BF02624447. [DOI] [PubMed] [Google Scholar]
  55. Schlaeger EJ, Eggimann B, Gast A. Proteolytic activity in the culture supernatants of mouse hybridoma cells. Dev Biol Standard. 1987;66:403–408. [PubMed] [Google Scholar]
  56. Singh RP, AL-Rubeai M, Gregory CD, Emery AN. Cell death in bioreactors: A role for apoptosis. Biotechnol Bioeng. 1994;44:720–726. doi: 10.1002/bit.260440608. [DOI] [PubMed] [Google Scholar]
  57. Sugimoto S, Lind W, Wagner R. Activation of a specific proteolytic activity in suspension cultures of recombinant adherent cells. In: Spier RE, Griffiths JB, MacDonald C, editors. Animal Cell Technology: Developments, Processes and Products'. Oxford, UK: Butterworth-Heinemann; 1992. pp. 547–551. [Google Scholar]
  58. Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kocibe N, Kubota A. 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. 1988;263:3657–3663. [PubMed] [Google Scholar]
  59. Takeuchi M, Takasaki S, Shimada M, Kobata A. Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J Biol Chem. 1990;265:12127–12130. [PubMed] [Google Scholar]
  60. Teige M, Weidemann R, Kretzmer G. Problems with serum-free production of antithrombin III regarding proteolytic activity and product quality. J Biotechnol. 1994;34:101–105. doi: 10.1016/0168-1656(94)90171-6. [DOI] [PubMed] [Google Scholar]
  61. Thorens B, Vassalli P. Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion. Nature. 1986;321:618–620. doi: 10.1038/321618a0. [DOI] [PubMed] [Google Scholar]
  62. Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem. 1983;52:655–710. doi: 10.1146/annurev.bi.52.070183.003255. [DOI] [PubMed] [Google Scholar]
  63. Tsuda E, Kawanishi G, Ueda M, Masuda S, Sasaki R. The role of carbohydrate in recombinant human erythropoietin. Eur J Biochem. 1990;188:405–411. doi: 10.1111/j.1432-1033.1990.tb15417.x. [DOI] [PubMed] [Google Scholar]
  64. Wang FF, Kung CKH, Goldwasser E. Some chemical properties of human erythropoietin. Endocrinology. 1985;116:2286–2292. doi: 10.1210/endo-116-6-2286. [DOI] [PubMed] [Google Scholar]
  65. Yang M and Butler M (2000) Effects of ammonia on CHO cell growth, erythropoietin production and glycosylation. Biotechnol Bioeng (in press). [DOI] [PubMed]
  66. Zeilke HR, Ozand PT, Tildon JT, Sevdalian DA, Cornblath M. Reciprocal regulation of glucose and glutamine utilization by cultures human diploid fibroblasts. J Cell Physiol. 1978;95:41–48. doi: 10.1002/jcp.1040950106. [DOI] [PubMed] [Google Scholar]

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