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Determination of Antibody Glycosylation by Mass Spectrometry

  • Christiane JägerEmail author
  • Claudia Ferrara
  • Pablo Umaña
  • Anne Zeck
  • Jörg Thomas Regula
  • Hans Koll
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 901)

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.

Key words

N-linked glycosylation MALDI-TOF LC-MS ESI-MS IdeS IgG PNGase F Endoglycosidase 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Christiane Jäger
    • 1
    Email author
  • Claudia Ferrara
    • 1
  • Pablo Umaña
    • 1
  • Anne Zeck
    • 2
  • Jörg Thomas Regula
    • 3
  • Hans Koll
    • 3
  1. 1.pRED, Pharma Research and Early DevelopmentRoche Glycart AGSchlierenSwitzerland
  2. 2.NMI, Naturwissenschaftliches und Medizinisches InstitutUniversität TübingenReutlingenGermany
  3. 3.pRED, Pharma Research and Early DevelopmentRoche Diagnostics GmbHPenzbergGermany

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