Abstract
Immunoglobulin (Ig) G is formed by two antigen-binding moieties termed Fabs and a conserved Fc portion, which interacts with components of the immune systems. Within the Fc, N-linked carbohydrates are attached to each conserved asparagine residue at position 297 within the CH2 domain. These oligosaccharide moieties introduce a higher degree of heterogeneity within the molecule, by influencing stability of the antibody and its mediated effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC).
The carbohydrate moieties can vary strongly depending on the production host and can be manipulated by different fermentation conditions, thereby influencing the function of the antibody. Therefore it is necessary to carefully monitor changes in the carbohydrate composition during cell line development and production processes. This chapter describes two different mass spectrometry based methods used for analyses of the carbohydrate moieties attached to the Fc-part of human IgG1. In the first approach, the glycans are released from the antibody by endoglycosidase (Peptide N Glycosidase F) digestion and monitored by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS), whereas in the second method the carbohydrate structures, still attached to an enzymatically produced Fc-fragment, are analyzed by electrospray ionization mass spectrometry.
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References
Wright A, Morrison SL (1997) Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol 15: 26–32
Krapp S et al (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Mol Biol 325:979–989
Raju TS (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20:471–478
Jefferis R (2009) Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. Trends Pharmacol Sci 30: 356–362
Huhn C et al (2009) IgG glycosylation analysis. Proteomics 9:882–913
Jefferis R (2007) Antibody therapeutics: isotype and glycoform selection. Expert Opin Biol Ther 9:1401–1413
Hodoniczky J, Zheng YZ, James DC (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21:1644–1652
Malhotra R et al (1995) Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat Med 3:237–243
Shields RL et al (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–26740
Umana P et al (1999) Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17:176–180
Ferrara C et al (2006) Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II. Biotechnol Bioeng 93:851–861
Kanda Y et al (2006) Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology 17:104–118
Kunkel JP et al (1998) Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody. J Biotechnol 62:55–71
Baker KN et al (2001) Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol Bioeng 73: 188–202
Hills A et al (2001) Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnol Bioeng 75:239–251
Weithandler M et al (1994) Analysis of carbohydrates on IgG preparations. J Pharm Sci 83: 1670–1675
Mock KK, Davey M, Cottrell JS (1991) The analysis of underivatized oligosaccharides by matrix-assisted laser desorption mass spectrometry. Biochem Biophys Res Commun 177: 644–651
Huberty MC et al (1993) Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS. Anal Chem 65: 2791–2800
Papac DI, Wong A, Jones AJ (1996) Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem 68:3215–3223
Papac DI et al (1998) A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Glycobiology 8:445–454
Sinha S et al (2008) Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs. J Am Soc Mass Spectrom 19:1643–1654
Karg SR et al (2009) A small-scale method for the preparation of plant N-linked glycans from soluble proteins for analysis by MALDI-TOF mass spectrometry. Plant Physiol Biochem 47: 160–166
Schiller J et al (2004) Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research. Prog Lipid Res 43:449–488
Dillon TM et al (2004) Development of an analytical reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry method for characterization of recombinant antibodies. J Chromatogr A 1053:299–305
Yan B et al (2007) Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry. J Chromatogr A 1164:153–161
Lim A et al (2008) Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer. Anal Biochem 375:163–172
Von Pawel-Rammingen U et al (2002) IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 21:1607–1615
Chevreux G et al (2011) Fast analysis of recombinant monoclonal antibodies using IdeS proteoloytic digestion and electrospray mass spectrometry. Anal Biochem 415:212–214
Boyd PN, Lines AC, Patel AK (1995) The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol Immunol 32:1311–1318
Scallon BJ et al (2007) Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol Immunol 44:1524–1534
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Jäger, C., Ferrara, C., Umaña, P., Zeck, A., Regula, J.T., Koll, H. (2012). Determination of Antibody Glycosylation by Mass Spectrometry. In: Proetzel, G., Ebersbach, H. (eds) Antibody Methods and Protocols. Methods in Molecular Biology, vol 901. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-931-0_13
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DOI: https://doi.org/10.1007/978-1-61779-931-0_13
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