Springer Protocols: Abstract: Comparability and Monitoring Immunogenic N-linked Oligosaccharides from Recombinant Monoclonal Antibodies from Two Different Cell Lines using HPLC with Fluorescence Detection and Mass Spectrometry

Comparability and Monitoring Immunogenic N-linked Oligosaccharides from Recombinant Monoclonal Antibodies from Two Different Cell Lines using HPLC with Fluorescence Detection and Mass Spectrometry

By: Bruce R. Kilgore³, Adam W. Lucka³, Rekha Patel³, Bruce A. Andrien Jr.³, Shirish T. Dhume³

Abstract

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One of the most important structural features of recombinant monoclonal antibodies produced in mammalian cells is the N-linked oligosaccharide profile. These profiles impact recombinant therapeutics in a multitude of ways affecting distribution, efficacy, and immunogenicity. High mannose, α-gal and other oligosaccharide species are highly immunogenic and in most cases should be minimized during manufacturing. A recombinant monoclonal antibody, h5G1.1, was produced in NS0 and CHO cell lines and tested to identify changes in the N-linked oligosaccharide profiles caused from a change in cell line. Traditional peak analysis using HPLC with fluorescence detection was augmented by mass spectrometric analysis. Nano LC-MS following tryptic digestion corroborated HPLC findings of the presence of several α-gal oligosaccharide species in the recombinant IgG (rIgG) from NS0 cell line. Both cell lines possessed rIgGs with complex and small amounts of high mannose glycans.

Affiliation(s): (3) Alexion Pharmaceuticals, Cheshire, CT, USA

Book Title: Post-translational Modifi cations of Proteins: Tools for Functional Proteomics

Series: Methods in Molecular Biology | Volume: 446 | Pub. Date: Apr-04-2008 | Page Range: 333-346 | DOI: 10.1007/978-1-60327-084-7_23

Subject: Protein Science

Key Words: Mass Spectrometry - ESI-ToF - nano LC-MS - oligosaccharide - glycans - saccharides - carbohydrates - N-linked - glycoprotein - glycopeptides - HPLC - fluorescent - anthranilic acid

References

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1.	1. Humphreys, A. and Boersig, C. (2003) Cholesterol drugs dominate. MedAdNews. 22, 42–57.

2.	2. Glennie, M. J. and Johnson, P.W.M. (2000) Clinical Trials of Antibody Therapy. Immunol. Today. 21, 403–410.

3.	3. Gottschalk U. (2003) Biotech Manufacturing Is Coming of Age. BioProcess International. 1, 54–61.

4.	4. Jefferis, R. (2005) Glycosylation of Recombinant Antibody Therapeutics. Biotechnol. Prog. 21, 11–16.

5.	5. Lund J., Takahashi, N., Popplewell, A., Goodall, M., Pound, J. D., Tyler, R., King, D. J., and Jefferis, R. (2000) Expression and characterization of truncated glycoforms of humanized L243 IgG1: architectural features can influence synthesis of its oligosaccharide chains and affect superoxide production triggered through human FcγR1. Eur. J. Biochem. 267, 7246–7257.

6.	6. Stockert, R. J. (1995) The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. Rev. 75, 591–609.

7.	7. Taylor, P. R., Martinez-Pomares, L., Stacey, M., Lin, H-H., Brown, G.D., and Gordon S. (2005) Macrophage receptors and immune recognition. Ann Rev Immunol. 23, 901–944.

8.	8. Raju, T. S., Briggs, J. B., Borge, S. M., and Jones A. J. S. (2000) Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galac-tosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology. 10, 477–486.

9.	9. Noguchi, A., Mukuria C. J., Suzuki, E., and Naiki, M. (1995) Immunogenicity of N-glycolyl-neuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in chinese hamster ovary cells. Biochem. Japanese Biochem. Soc. 117, 59–62.

10.	10. Wilson, I. B. H., Zeleny, R., Kolarich, D., Staudacher, E., Stroop, C. J. M., Kamerling, J. P., and Altmann, F. (2001) Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core α1,3-linked fucose and xylose substitutions. Glycobiology. 11, 261–274.

11.	11. Garcia-Casado, G., Sanchez-Monge, R., Chrispeels, M. J., Armentia, A., Salcedo, G., and Gomez, L. (1996) Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins. Glycobiology. 6, 471–477.

12.	12. Galili, U., Rachmilewitz, E.A., Peleg, A., and Flechner, I. (1984) A unique natural human IgG antibody with anti-α-galactosyl specificity. J. Exp. Med. 160, 1519–1531.

13.	13. Galili U. and Matta, K. L. (1996) Inhibition of anti-Gal IgG binding to porcine endothelial cells by synthetic oligosaccharides. Transplantation. 62, 356–362.

14.	14. Wang, L., Anaraki, F., Henion, T. R., and Galili, U. (1995) Variations in activity of the human natural anti-Gal antibody in young and elderly populations. J. Gerontol. (Med. Sci.) 50A, M227–M233.

15.	15. Harvey, D. J. (2001) Identification of protein-bound carbohydrates by mass spectrometry. Proteomics. 1, 311–328.

16.	16. Anumula, K. R. and Dhume, S. T. (1998) High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chro-matography following derivatization with highly fluorescent anthranilic acid. Glycobiology. 8, 685–694.

17.	17. Dhume, S. T., Ebert, M. B., Saddic, G. N., and Anumula, K. R. (2002) Monitoring glycosyla-tion of therapeutic glycoproteins for consistency using highly fluorescent anthranilic acid in Methods in Molecular Biology, Posttranslational Modifications of Proteins. (Kannicht, C., ed), Humana, Totowa, NJ, pp. 127–142.

18.	18. Anumula, K. R. (1995) Novel fluorescent methods for quantitative determination of monosac-charides and sialic acids in glycoproteins by reversed phase high performance liquid chromatography in Methods in Protein Structure Analysis (Atassi, M. Z. and Apella, E., eds.), Plenum, New York, NY, pp. 195–206.

19.	19. Anumula, K. R. (1997) Highly sensitive pre-column derivatization procedures for quantitative determination of monosaccharides, sialic acids and amino sugar alcohols of glycoproteins by reversed phase HPLC in Techniques in Glycobiology (Townsend, R. R. and Hotchkiss, A. T., eds.), Marcel Dekker, New York, NY, pp. 349–357.

20.	20. Anumula, K.R. (2006) Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal. Biochem. 350, 1–23.

21.	21. Neville, D. C. A., Coquard, V., Priestman, D. A., te Vruchte, D. J. M., Sillence, D. J., Dwek, R. A., Platt, F. M., and Butters, T. D. (2004) Analysis of fluorescently labeled glycosphingoli-pid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling. Anal. Biochem. 331, 275–282.

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