Analytical characterization of IgG Fc subclass variants through high-resolution separation combined with multiple LC-MS identification
- 573 Downloads
With the rapid growth of recombinant monoclonal antibodies and intravenous immunoglobulin (IVIg) medicines, the understanding of human immunoglobulin G (IgG) subclasses becomes more necessary. It is essential to develop effective techniques and methodologies which have the capability for deep characterization. We have created an approach by applying LC and liquid chromatography-mass spectrometry (LC-MS) methods to thoroughly characterize Fc/2 sequence variants for human IgG subclasses in complex samples. Identification and relative quantitation of sequence variants have been provided. Unique glycan information of each IgG subclass can also be obtained by this method. The approach was based on high-resolution HPLC separation followed by intact LC-MS. Peptide mapping was performed following sample fractionation to identify sequence variants. IVIg, a purified IgG mixture from pooled human plasma of thousands of blood donors, was selected as an example for method development. The amino acid sequence variants in IgG Fc/2 constant region were fully investigated for all subclasses by these methods. A total of 19 sequence variants were identified, and their relative abundances were quantitated, which included six variants in IgG1, eight in IgG2, three in IgG3, and two in IgG4. Unique glycan data was also provided for each Fc subclass, which is particularly important for IgG3; glycans from this subclass have only previously been reported together with IgG2 or IgG4. The method described in this paper has been proved to be an effective approach for deep characterization of IgG Fc/2 for complex samples. The findings of IVIg from these studies are also valuable for better understanding of human IgGs.
KeywordsSerum IgGFc LC-MS Sequence variants Glycopeptide Quantitation
We would like to thank Carlos Bosques for helpful comments and a thorough review of the manuscript.
- 2.Dam TK, Torres M, Brewer CF, Casadevall A (2008) Isothermal titration calorimetry reveals differential binding thermodynamics of variable region-identical antibodies differing in constant region for a univalent ligand. J Biol Chem 283(46):31366–31370. doi: 10.1074/jbc.M806473200 CrossRefGoogle Scholar
- 13.Ivancic MM, Gadgil HS, Halsall HB, Treuheit MJ (2010) LC/MS analysis of complex multiglycosylated human alpha(1)-acid glycoprotein as a model for developing identification and quantitation methods for intact glycopeptide analysis. Anal Biochem 400(1):25–32. doi: 10.1016/j.ab.2010.01.026 CrossRefGoogle Scholar
- 15.Qian J, Liu T, Yang L, Daus A, Crowley R, Zhou Q (2007) Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion. Anal Biochem 364(1):8–18. doi: 10.1016/j.ab.2007.01.023 CrossRefGoogle Scholar
- 18.Selman MH, Derks RJ, Bondt A, Palmblad M, Schoenmaker B, Koeleman CA, van de Geijn FE, Dolhain RJ, Deelder AM, Wuhrer M (2012) Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface. J Proteome 75(4):1318–1329. doi: 10.1016/j.jprot.2011.11.003 CrossRefGoogle Scholar
- 27.Dechavanne C, Guillonneau F, Chiappetta G, Sago L, Levy P, Salnot V, Guitard E, Ehrenmann F, Broussard C, Chafey P, Le Port A, Vinh J, Mayeux P, Dugoujon JM, Lefranc MP, Migot-Nabias F (2012) Mass spectrometry detection of G3m and IGHG3 alleles and follow-up of differential mother and neonate IgG3. PLoS One 7(9):e46097. doi: 10.1371/journal.pone.0046097 CrossRefGoogle Scholar
- 28.Lu Q, Padler-Karavani V, Yu H, Chen X, Wu SL, Varki A, Hancock WS (2012) LC-MS analysis of polyclonal human anti-Neu5Gc xeno-autoantibodies immunoglobulin G subclass and partial sequence using multistep intravenous immunoglobulin affinity purification and multienzymatic digestion. Anal Chem 84(6):2761–2768. doi: 10.1021/ac2030893 CrossRefGoogle Scholar