Analytical and Bioanalytical Chemistry

, Volume 407, Issue 23, pp 7055–7066 | Cite as

Analytical characterization of IgG Fc subclass variants through high-resolution separation combined with multiple LC-MS identification

  • Xiaomei (Annie) He
  • Nathaniel Washburn
  • Enrique Arevalo
  • John H. RobbleeEmail author
Research Paper


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.

Graphical Abstract

Identification of sequence variants in IgG Fc mixtures


Serum IgGFc LC-MS Sequence variants Glycopeptide Quantitation 



We would like to thank Carlos Bosques for helpful comments and a thorough review of the manuscript.

Supplementary material

216_2015_8863_MOESM1_ESM.pdf (121 kb)
ESM 1 (PDF 120 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Xiaomei (Annie) He
    • 2
  • Nathaniel Washburn
    • 1
  • Enrique Arevalo
    • 1
  • John H. Robblee
    • 2
    Email author
  1. 1.Research DepartmentMomenta Pharmaceuticals Inc.CambridgeUSA
  2. 2.Biosimilars DepartmentMomenta Pharmaceuticals Inc.CambridgeUSA

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