Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry

Kristina Srzentić, Luca Fornelli, Yury O. Tsybin, Joseph A. Loo, Henrique Seckler, Jeffrey N. Agar, Lissa C. Anderson, Dina L. Bai, Alain Beck, Jennifer S. Brodbelt, Yuri E.M. van der Burgt, Julia Chamot-Rooke, Sneha Chatterjee, Yunqiu Chen, David J. Clarke, Paul O. Danis, Jolene K. Diedrich, Robert A. D'Ippolito, Mathieu Dupré, Natalia GasilovaYing Ge, Young Ah Goo, David R. Goodlett, Sylvester Greer, Kim F. Haselmann, Lidong He, Christopher L. Hendrickson, Joshua D. Hinkle, Matthew V. Holt, Sam Hughes, Donald F. Hunt, Neil L. Kelleher, Anton N. Kozhinov, Ziqing Lin, Christian Malosse, Alan G. Marshall, Laure Menin, Robert J. Millikin, Konstantin O. Nagornov, Simone Nicolardi, Ljiljana Paša-Tolić, Stuart Pengelley, Neil R. Quebbemann, Anja Resemann, Wendy Sandoval, Richa Sarin, Nicholas D. Schmitt, Jeffrey Shabanowitz, Jared B. Shaw, Michael R. Shortreed, Lloyd M. Smith, Frank Sobott, Detlev Suckau, Timothy Toby, Chad R. Weisbrod, Norelle C. Wildburger, John R. Yates, Sung Hwan Yoon, Nicolas L. Young, Mowei Zhou

Research output: Contribution to journalArticlepeer-review

69 Scopus citations


The Consortium for Top-Down Proteomics ( launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Original languageEnglish (US)
Pages (from-to)1783-1802
Number of pages20
JournalJournal of the American Society for Mass Spectrometry
Issue number9
StatePublished - Sep 2 2020


  • FTMS
  • Fourier transform mass spectrometry
  • MS/MS
  • Therapeutic protein
  • glycoform
  • intact mass measurement
  • tandem mass spectrometry

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy


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