Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

  • Lidong He
  • Lissa C. Anderson
  • David R. Barnidge
  • David L. Murray
  • Christopher L. Hendrickson
  • Alan G. Marshall
Focus: 28th Sanibel Conference, Characterization of Protein Therapeutics by MS: Research Article

DOI: 10.1007/s13361-017-1602-6

Cite this article as:
He, L., Anderson, L.C., Barnidge, D.R. et al. J. Am. Soc. Mass Spectrom. (2017) 28: 827. doi:10.1007/s13361-017-1602-6


With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2–0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Graphical Abstract


Fourier transform Ion cyclotron resonance FTMS Electrospray MS/MS Middle-down Collision-induced dissociation CID Electron transfer dissociation ETD Multiple myeloma Isotype Variable region 

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Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Lidong He
    • 1
  • Lissa C. Anderson
    • 2
  • David R. Barnidge
    • 3
  • David L. Murray
    • 3
  • Christopher L. Hendrickson
    • 1
    • 2
  • Alan G. Marshall
    • 1
    • 2
  1. 1.Department of Chemistry and BiochemistryFlorida State UniversityTallahasseeUSA
  2. 2.National High Magnetic Field LaboratoryFlorida State UniversityTallahasseeUSA
  3. 3.Department of Laboratory Medicine and PathologyMayo ClinicRochesterUSA

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