Carbohydrate Structural Characterization of Fas Ligand Inhibitory Protein
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, 2, 3, 4, 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.
KeywordsHigh Performance Liquid Chromatography Sialic Acid Internal Standard Solution High Performance Liquid Chromatography System Sialic Acid Content
- 4.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.PubMedCrossRefGoogle Scholar
- 5.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.CrossRefGoogle Scholar
- 6.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.Google Scholar
- 13.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.PubMedCrossRefGoogle Scholar