Abstract
Decoy receptor 3 (DcR3) is a novel member of the tumor necrosis factor (TNF) receptor superfamily that plays an important role in regulating apoptosis in normal physiology (1–5). The engineered (Arg218Gln) soluble form of DcR3 was termed Fas ligand inhibitory protein (FLINT; LY498919) in an effort to create a molecule that was less susceptible to proteolysis (5). FLINT, expressed in Chinese ovary (CHO) cells, is a therapeutic glycoprotein with 271 amino acid residues, 10 disulfide bonds, 1 potential N-glycosylation site, and 2 potential O-glycosylation sites. Oligosaccharide structures strongly influence FLINT pharmacokentics (6). Hence, it is essential to characterize carbohydrate structures of FLINT in routine testing to monitor batch-to-batch consistency. There are many ways to characterize the carbohydrate structures of a glycoprotein. In this chapter, we will describe routine carbohydrate characterization of FLINT to monitor its batch-to-batch consistency. The methods include (1) neutral and amino monosaccharide composition analysis; (2) sialic acid content assay; and (3) oligosaccharide profiling by weak-anion exchange (WAX) high performance liquid chromatography (HPLC) with fluorescence detection, and additional glycosylation characterization, including site mapping and oligosaccharide structural elucidation.
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References
Kramer, P. H., Behrmann, I., Dhein, J., and Debatin, K. M. (1994) Regulation of apoptosis in the immune system. Curr. Opin. Immunol. 64, 279–289.
Yu, K.-Y., Kwon, B., Ni, J., Zhai, Y., Ebner, R., and Kwon, B. S. (1999) A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHTmediated apoptosis. J. Biol. Chem. 274, 13733–13736.
Zhang, J., Salcedo, T. W., Wan, X., Ullrich, S., Hu, B., Gregorio, T., et al. (2001) Modulation of T-cell responses to alloantigens by TR6/DcR3. J. Clin. Investig. 107, 1459–1468.
Wroblewski, V. J., Witcher, D. R., Becker, G. W., Davis, K. A., Dou, S., Micanovic, R., et al. (2003) Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT. Biochem. Pharmacol. 65, 657–677.
Wroblewski, V. J., McCloud, C., Davis, K. A., Manetta, J., Micanovic, R., and Witcher, D. R. (2003) Pharmacokinetics, metabolic stability, and subcutaneous bioavailability of a genetically engineered analog of DcR3, FLINT [DcR3(R218Q)], in cynomolgus monkeys and mice. Drug Metab. Disp. 31, 502–507.
Jenkins, N., Witcher, D. R., and Wroblewski, V. J. (2002) Enhancing sialic acid content of FLINT (Fas ligand inhibitory protein) R218Q with improved protease-resistance by having additional glycosylation sites engineered into the native FLINT sequence. WO 2002060949. 42 pp.
Rocklin, R. D., Clarke, A. P., and Weitzhandler, M. (1998) Improved long-term reproducibility for pulsed amperometric detection of carbohydrates via a new quadruple-potential waveform. Anal. Chem. 70, 1496–1501.
Carr, S. A., Huddleston, M. J., and Bean, M. F. (1993) Selective identification and differentiation of N-and O-linked oligosaccharides in glycoproteins by liquid chromatographymass spectrometry. Protein Sci. 2, 183–196.
Huddleston, M. J., Bean, M. F., and Carr, S. A. (1993) Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in progress digests. Anal. Chem. 65, 877–884.
Spellman, M. W., Leonard, C. K., Basa, L. J., Gelineo, I., and van Halbeek, H. (1991) Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells. Biochemistry 30, 2395–2406.
Spellman, M. W., Basa, L. J., Leonard, C. K., et al. (1989) Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells. J. Biol. Chem. 264, 14,100–14,111.
Watson, E., Bhide, A., and van Halbeek, H. (1994) Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Glycobiology 4, 227–237.
Amoresano, A., Siciliano, R., Orru, S., Napoleoni, R., Altarocca, V., De Luca, E., et al. (1996) Structural characterisation of human recombinant glycohormones follitropin, lutropin and choriogonadotropin expressed in Chinese hamster ovary cells. Eur. J. Biochem. 242, 608–618.
Fan, J. Q., Yamamoto, K., Kumagai, H., and Tochikura., T. (1990) Induction and efficient purification of endo-alpha-N-acetyl-D-galactosaminidase from Alcaligenes sp. Agric. Biol. Chem. 54, 233–234.
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Huang, L., Mitchell, C.E., Yu, L., Gough, P.C., Riggin, A. (2005). Carbohydrate Structural Characterization of Fas Ligand Inhibitory Protein. In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:421
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DOI: https://doi.org/10.1385/1-59259-922-2:421
Publisher Name: Humana Press
Print ISBN: 978-1-58829-390-9
Online ISBN: 978-1-59259-922-6
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