The AAPS Journal

, Volume 12, Issue 4, pp 576–585

Ligand-Binding Mass Spectrometry to Study Biotransformation of Fusion Protein Drugs and Guide Immunoassay Development: Strategic Approach and Application to Peptibodies Targeting the Thrombopoietin Receptor

Authors

  • Michael P. Hall
    • Department of PKDMAmgen Inc.
  • Colin Gegg
    • Department of Protein ScienceAmgen Inc.
  • Kenneth Walker
    • Department of Protein ScienceAmgen Inc.
  • Christopher Spahr
    • Department of Protein ScienceAmgen Inc.
  • Robert Ortiz
    • Department of PKDMAmgen Inc.
  • Vimal Patel
    • Department of PKDMAmgen Inc.
  • Steven Yu
    • Department of PKDMAmgen Inc.
  • Liana Zhang
    • Department of PKDMAmgen Inc.
  • Hsieng Lu
    • Department of Protein ScienceAmgen Inc.
  • Binodh DeSilva
    • Department of PKDMAmgen Inc.
    • Department of PKDMAmgen Inc.
Research Article

DOI: 10.1208/s12248-010-9218-9

Cite this article as:
Hall, M.P., Gegg, C., Walker, K. et al. AAPS J (2010) 12: 576. doi:10.1208/s12248-010-9218-9

Abstract

The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc (“peptibodies”) using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.

Key words

fusion protein biotransformationin vivo stability of biopharmaceuticalsimmunoaffinity-mass spectrometryligand-binding assaypeptibodies

Copyright information

© American Association of Pharmaceutical Scientists 2010