Abstract
Conditions for the enzymatic release, chemical derivatization, and analysis of oligosaccharides from the consensus glycosylation sites on antibodies are described. Release of the oligosaccharides is from the native protein. The APTS derivatives of the released oligosaccharides are then analyzed by capillary electrophoresis (CE) using a free solution separation in a bare fused silica capillary. An example of the application of the method to the analysis of the oligosaccharide population from antibodies obtained from different cell lines is provided. The separation conditions provide for resolution of various galactose positional isomers, including those derived from different linkage configurations.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Patrick, J. S. (2002) Advances in analytical chemistry for biotechnology—mass spectrometry of peptides, proteins and glycoproteins, in Handbook of Industrial Cell Culture: Mammalian, Microbial and Plant Cells, (Vinci, V. A. and Parekh, S. R., eds.), Humana, Totowa, NJ.
Chapman, J. R., ed. (2000) Mass Spectrometry of Proteins and Peptides—Methods in Molecular Biology, vol. 146. Humana, Totowa, NJ.
Snyder, A. P., ed. (1995) Biochemical and biotechnological applications of electrospray ionization mass spectrometry, ACS Symposium Series, vol. 619, American Chemical Society, Washington, DC.
Patrick, J. S. and Lagu, A. L. (2001) Review: applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins. Electrophoresis 22, 4179–4196.
Taverna, M., Tran, N. T., Merry, T., Horvath, E., and Ferrier, D. (1998) Electrophoretic methods for process monitoring and the quality assessment of recombinant glycoproteins. Electrophoresis 19, 2572–2594.
Kakehi, K., Kinoshita, M., and Nakano, M. (2002) Analysis of glycoproteins and the oligosaccharides thereof by high-performance capillary electrophoresis-significance in regulatory studies on biopharmaceutical products. Biomed. Chrom. 16, 103–115.
Patel, T., Parekh R., Moellering, B., and Prior, C. (1992) Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody. Biochem. J. 285, 839–845.
Tao, M.-H. and Morrison, S.L. (1989), Studies of aglycosylated chimeric mousehuman IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J. Immunol. 143, 2595–2601.
Dwek, R. (1995) Glycobiology: Towards understanding the function of sugars. Biochem. Soc. Trans. 23, 1–25.
Townsend, R. R., Hardy, M. R. and Lee, Y. C. (1989) Separation of oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection. Methods in Enzymology 179, 66–76.
Basa, L. J. and Spellman, M. W. (1990) Analysis of glycoprotein-derived oligosaccharides by high-pH anion exchange chromatography, J. Chromatogr. 499, 205–220.
Maness, N. O., Miranda, E. T., and Mort, A. J. (1991) Recovery of sugar derivatives from 2-aminopyridine labeling mixtures for high-performance liquid chromatography using UV or fluorescence detection. J. Chromatogr. 587, 177–183.
Ma, S. and Nashabeh W. (1999) Carbohydrate analysis of a chimeric recombinant monoclonal antibody by capillary electrophoresis. Anal. Chem. 71, 5185–5192.
http://www.beckman.com/products/instrument/analytical/ce/pacemdq._carbohydratesystem._inst._dcr.asp
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Patrick, J.S., Rener, B.P., Clanton, G.S., Lagu, A.L. (2004). Analysis of Neutral N-Linked Oligosaccharides From Antibodies Using Free-Solution Capillary Electrophoresis in Bare Fused-Silica Capillaries. In: Strege, M.A., Lagu, A.L. (eds) Capillary Electrophoresis of Proteins and Peptides. Methods in Molecular Biology™, vol 276. Humana Press. https://doi.org/10.1385/1-59259-798-X:137
Download citation
DOI: https://doi.org/10.1385/1-59259-798-X:137
Publisher Name: Humana Press
Print ISBN: 978-1-58829-017-5
Online ISBN: 978-1-59259-798-7
eBook Packages: Springer Protocols