Advertisement

Glycoconjugate Journal

, Volume 12, Issue 3, pp 318–330 | Cite as

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones

  • Aldert A. Bergwerff
  • Corné J. M. Stroop
  • Brendan Murray
  • Anke-Peggy Holtorf
  • Gerd Pluschke
  • Jan Van Oostrum
  • Johannis P. Kamerling
  • Johannes F. G. Vliegenthart
Glycoconjagate Journal

Abstract

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human γ1 and κ constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (α1 →6)-flucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAcβ1→2, Galβ1 → 4GlcNAcβ1 → 2 or Galα1 → 3Gα1β1 → 4GlcNAcβ1 → 2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (β1 → 2)-linked GlcNAc residues. Galactosylation of the GlcNAcβ1 → 2Manα1 → 6 branch occurs four times more frequently than that of the GlcNAcβ1 → 2Manα1 → 3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carryingN-acetylneuraminic acid (Neu5Ac) orN-glycolylneuraminic acid (Neu5Gc), exclusively (α2 → 6)-linked to βGal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.

Keywords

biotechnological antibody chimeric IgG1 N-glycosylation cell subclones glycoprotein 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Burton D (1985)Mol Immunol 22:161–206.Google Scholar
  2. 2.
    Kobata A (1990)Glycobiology 1:5–8.Google Scholar
  3. 3.
    Rademacher TW, Homans SW, Parekh RB, Dwek RA (1986)Biochem Soc Symp 51:131–48.Google Scholar
  4. 4.
    Wright A, Morrison SL (1993)Springer Semin Immunopathol 15:259–73.Google Scholar
  5. 5.
    Coco-Martin JM, Brunink F, Van der Velden-De Groot TAM, Beuvery EC (1992)J Immunol Meth 155:241–48.Google Scholar
  6. 6.
    Rademacher TW, Dwek RA (1983)Prog Immunol 5:95–112.Google Scholar
  7. 7.
    Savvidou G, Klein M, Horne C, Hofmann T, Dorrington KJ (1981)Mol Immunol 18:793–805.Google Scholar
  8. 8.
    Kim H, Yamaguchi Y, Masuda K, Matsunaga C, Yamamoto K, Irimura T, Takahashi N, Kato K, Arata Y (1994)J Biol Chem 269:12345–50.Google Scholar
  9. 9.
    Duncan AR, Winter G (1988)Nature 332:738–40.Google Scholar
  10. 10.
    Leatherbarrow RJ, Rademacher TW, Dwek RA, Woof JM, Clark A, Burton DR, Richardson N, Feinstein A (1985)Mol Immunol 22:407–15.Google Scholar
  11. 11.
    Nose M, Wigzell H (1983)Proc Natl Acad Sci USA 80:6632–36.Google Scholar
  12. 12.
    Koide N, Nose M, Muramatsu T (1977)Biochem Biophys Res Commun 75:838–44.Google Scholar
  13. 13.
    Matsuda H, Nakamura S, Ichikawa Y, Kozai K, Tanko R, Nose M, Endo S, Nishimura Y, Arata Y (1990)Mol Immunol 27:571–79.Google Scholar
  14. 14.
    Waldmann TA (1991)Science 252:1657–62.Google Scholar
  15. 15.
    Sandhu JS (1992)Crit Rev Biotech 12:437–62.Google Scholar
  16. 16.
    Boulianne GL, Hozumi N, Shulman MJ (1984)Nature 312:643–46.Google Scholar
  17. 17.
    Morrison SL, Johnson MJ, Herzenberg LA, Oi V (1984)Proc Natl Acad Sci USA 81:6851–55.Google Scholar
  18. 18.
    Lund J, Tanaka T, Takahashi N, Sarmay G, Arata Y, Jefferis R (1990)Mol Immunol 27:1145–53.Google Scholar
  19. 19.
    Lund J, Takahashi N, Nakagawa H, Goodall M, Bentley T, Hindley SA, Tyler R, Jefferis R (1993)Mol Immunol 30:741–48.Google Scholar
  20. 20.
    Tandai M, Endo T, Sasaki S, Masuho Y, Kochibe N, Kobata A (1991)Arch Biochem Biophys 291:339–48.Google Scholar
  21. 21.
    Rothman RJ, Warren L, Vliegenthart JFG, Hård KJ (1989)Biochemistry 28:1377–84.Google Scholar
  22. 22.
    Krotkiewski H, Grönberg G, Krotkiewska B, Nilsson B, Svensson S (1990)J Biol Chem 265:20195–201.Google Scholar
  23. 23.
    Matsui T, Hamako J, Kameyama K-z, Kurosawa Y, Titani K, Mizuochi T (1989)Biochem Biophys Res Commun 164:245–50.Google Scholar
  24. 24.
    Patel TP, Parekh RB, Moellering BJ, Prior CP (1992)Biochem J 285:839–45.Google Scholar
  25. 25.
    Yu Ip CC, Miller WJ, Silberklang M, Mark GE, Ellis RW, Huang L, Glushka J, Van Halbeek H, Zhu J, Alhadeff JA (1994)Arch Biochem Biophys 308:387–99.Google Scholar
  26. 26.
    Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR (1991)Bio/Technology 9:1347–55.Google Scholar
  27. 27.
    Kornfeld R, Kornfeld S (1985)Ann Rev Biochem 54:631–64.Google Scholar
  28. 28.
    Bergwerff AA, Van Oostrum J, Asselbergs FAM, Bürgi R, Hokke CH, Kamerling JP, Vliegenthart JFG (1993)Eur J Biochem 212:639–56.Google Scholar
  29. 29.
    Basa LJ, Spellman MW (1990)J Chromatogr 499:205–20.Google Scholar
  30. 30.
    Chen T-F, Yu H, Barofsky DF (1992)Anal Chem 64:2014–19.Google Scholar
  31. 31.
    Hara S, Yamaguchi M, Takemori Y, Furuhata K, Ogura H, Nakamura M (1989)Anal Biochem 179:162–66.Google Scholar
  32. 32.
    Hokke CH, Bergwerff AA, Van Dedem GWK, Van Oostrum J, Kamerling JP, Vliegenthart JFG (1990)FEBS Lett 275:9–14.Google Scholar
  33. 33.
    Kamerling JP, Vliegenthart JFG (1989) InClinical Biochemistry; Principles, Methods, Applications, Vol 1,Mass Spectrometry (Lawson AM, ed.) pp 175–263. Berlin: Walter de Gruyter & CoGoogle Scholar
  34. 34.
    Vliegenthart JFG, Dorland L, Van Halbeek H (1983)Adv Carbohydr Chem Biochem 41:209–374.Google Scholar
  35. 35.
    Hård K, Van Zadelhoff G, Moonen P, Kamerling JP, Vliegenthart JFG (1992)Eur J Biochem 209:895–915.Google Scholar
  36. 36.
    Brockhausen I, Grey AA, Pang H, Schachter H, Carver JP (1988)Glycoconjugate J 5:419–48.Google Scholar
  37. 37.
    Fujii S, Nishiura T, Nishikawa A, Miura R, Taniguchi N (1990)J Biol Chem 265:6009–18.Google Scholar
  38. 38.
    De Waard P, Koorevaar A, Kamerling JP, Vliegenthart JFG (1991)J Biol Chem 266:4237–43.Google Scholar
  39. 39.
    Debray H, Dus D, Wieruszeski J-M, Strecker G, Montreuil J (1991)Glycoconjugate J 8:29–37.Google Scholar
  40. 40.
    Damm JBL, Bergwerff AA, Hård K, Kamerling JP, Vliegenthart JFG (1989)Recl Trav Chim Pays-Bas 108:351–59.Google Scholar
  41. 41.
    Shaw L, Schauer R (1988)Biol Chem Hoppe-Seyler 369:477–86.Google Scholar
  42. 42.
    Blanken WM, Van Vliet A, Van den Eijnden DH (1984)J Biol Chem 259:15131–35.Google Scholar
  43. 43.
    Narasimhan S, Freed JC, Schachter H (1985)Biochemistry 24:1694–700.Google Scholar
  44. 44.
    Schröder C, Nöhle U, Shukla AK, Schauer R (1983) InGlycoconjugates, Proc 7th Int Symp (Chester MA, Heinegard D, Lundblad A, Svensson S, eds) pp. 162–63. Rahms: Lund.Google Scholar
  45. 45.
    Galili U (1993)Springer Semin Immunopathol 15:155–71.Google Scholar
  46. 46.
    Mizuochi T, Hamako J, Titani K (1987)Arch Biochem Biophys 257:387–94.Google Scholar
  47. 47.
    Fujii Y, Higashi H, Ikuta K, Kato S, Naiki M (1982)Mol Immunol 19:87–94.Google Scholar
  48. 48.
    Schauer R (1988)Adv Exp Med Biol 228:47–72.Google Scholar
  49. 49.
    Deisenhofer J (1981)Biochemistry 20:2361–70.Google Scholar
  50. 50.
    Mohan SB, Chohan SR, Eade J, Lyddiatt A (1993)Biotechnol Bioeng 42:974–86.Google Scholar

Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • Aldert A. Bergwerff
    • 1
  • Corné J. M. Stroop
    • 1
  • Brendan Murray
    • 2
  • Anke-Peggy Holtorf
    • 3
  • Gerd Pluschke
    • 2
  • Jan Van Oostrum
    • 2
  • Johannis P. Kamerling
    • 1
  • Johannes F. G. Vliegenthart
    • 1
  1. 1.Bijvoet Center, Department of Bio-Organic ChemistryUtrecht UniversityUtrechtThe Netherlands
  2. 2.Core Drug Discovery TechnologiesCiba-Geigy Ltd.BaselSwitzerland
  3. 3.Biopharmaceutical DevelopmentCiba-Geigy Ltd.BaselSwitzerland

Personalised recommendations