Abstract
Antibodies are the first line of defense of the adaptive immune response and are found in blood plasma and extracellular fluids. Many monoclonal antibodies (MAbs) have been marketed and are being tested in clinical trials as therapeutics in immune and inflammatory diseases, oncology, and other therapeutic areas (1,2). Development of these therapeutic protein-based drugs is a multistep process typically involving the selection of an appropriate production cell line, evaluation of a library of monoclonal clones, and optimization of bioreactor growth conditions. Each step requires a careful evaluation of the quantity and quality of the antibodies produced. Of particular interest are the carbohydrate chains attached to all immunoglobulin G (IgG) heavy chains. One such structure is Galactose (Gal)α(1→3)Galβ(1→4)GlcNAc, which is an epitope of one of the natural human antibodies (3). Traditionally, only titer is considered as a criterion for clone selection of therapeutic proteins for development. For certain cell lines, aside from the clone titer, it may also be necessary to consider oligosaccharide structures as a criterion of clone selection.
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References
Andreakos, E., Taylor, P. C., and Feldmann, M. (2002) Monoclonal antibodies in immune and inflammatory diseases. Curr. Opin. Biotech. 13, 615–620.
Trikha, M., Yan, L., and Nakada, M. T. (2002) Monoclonal antibodies as therapeutics in oncology. Curr. Opin. Biotech. 13, 609–614.
Galili, U., Buehler, J., Shohet, S. B., and Macher, B. A. (1987) The human natural anti-Gal IgG III. The subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies. J. Exp. Med. 165, 693–704.
Davies, M. J. and Hounsell, E. F. HPLC and HPAEC of oligosaccharides and glycopeptides, in Methods in Molecular Biology, vol 76: Glycoanalysis Protocols (Hounsell, E. F., ed.), Humana, Totowa, NJ, 1998, pp. 79–100.
Papac, D. I., Wong, A., and Jones, A. J. S. (1996) Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 68, 3215–3223.
Hardy, M. R. Glycan labeling with the fluorophores 2-aminobenzamide and anthranilic acid, in Techniques in Glycobiology (Townsend, R. R. and Hotchkiss, A. T., ed.), Marcel Dekker, Inc., New York, NY, 1997, pp. 359–375.
Honda, S. and Kakehi, K. (1996) Analysis of glycoproteins, glycopeptides and glycoprotein-derived oligosaccharides by high-performance capillary electrophoresis. J. Chromatogr. A. 720, 377–393.
Lund, J., Takahashi, N., Pound, J. D., Goodall, M., and Jefferis, R. (1996) Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fcγreceptor I and influence the synthesis of its oligosaccharide chains. J. Immunol. 157, 4963–4969.
Jefferis, R., Lund, J., and Pound, J. D. (1998) IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation. Immunol. Rev. 163, 59–76.
Zhu, A. and Goldstein, J. (1994) Cloning and functional expression of a cDNA encoding coffee bean α-galactosidase. Gene 140, 227–231.
Aubert, M., Crotte, C., Bernard, J.-P., Lombardo, D., Sadoulet, M.-O., and Mas, E. (2003) Decrease of human pancreatic cancer cell tumorigenicity by α1,3galactosyltransferase gene transfer. Int. J. Cancer 107, 910–918.
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Huang, L., Mitchell, C.E. (2005). High-Throughput LC/MS Methodology for α(1→3)Gal Determination of Recombinant Monoclonal Antibodies. In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:411
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DOI: https://doi.org/10.1385/1-59259-922-2:411
Publisher Name: Humana Press
Print ISBN: 978-1-58829-390-9
Online ISBN: 978-1-59259-922-6
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