Mass Spectrometry and HPLC with Fluorescent Detection-Based Orthogonal Approaches to Characterize N-Linked Oligosaccharides of Recombinant Monoclonal Antibodies

  • Adam W. Lucka
  • Bruce R. Kilgore
  • Rekha Patel
  • Bruce A. AndrienJr
  • Shirish T. Dhume
Part of the Methods in Molecular Biology™ book series (MIMB, volume 446)


A number of HPLC and mass spectrometric techniques are used to characterize post-translational modification in recombinant monoclonal antibodies (MAbs) using the intact glycoprotein and free glycans. LC separation utilizing fluorescent detection technique allows tentative structural assignment of MAb oligosaccharides. Intact molecular weight analysis via electrospray allows for an accurate mass determination and observation of the native glycoform mass envelope. N-linked oligosaccharides are then analyzed by MALDI-ToF. Their structures are further confirmed by analyzing the fragmentation patterns formed by MS/MS. All these techniques provide useful information when performed in isolation. However, the combined information allows for definitive and robust characterization of the N-linked glycans from recombinant MAbs.

Key Words

Monoclonal antibodies MAbs recombinant IgG N-linked oligosac-charides glycans saccharides HPLC fluorescence anthranilic acid mass spectrometry ESI-ToF MALDI-ToF 


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Adam W. Lucka
    • 1
  • Bruce R. Kilgore
    • 1
  • Rekha Patel
    • 1
  • Bruce A. AndrienJr
    • 1
  • Shirish T. Dhume
    • 1
  1. 1.Alexion PharmaceuticalsCheshireCT

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