Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry

Mass Spectrom Rev. 2017 Nov;36(6):734-754. doi: 10.1002/mas.21500. Epub 2016 Apr 20.


In the last decade, the advancement of liquid chromatography mass spectrometry (LC/MS) techniques has enabled their broad application in protein characterization, both quantitatively and qualitatively. Owing to certain important merits of LC/MS techniques (e.g., high selectivity, flexibility, and rapid method development), LC/MS assays are often deemed as preferable alternatives to conventional methods (e.g., ligand-binding assays) for the analysis of protein biotherapeutics. At the discovery and development stages, LC/MS is generally employed for two purposes absolute quantification of protein biotherapeutics in biological samples and qualitative characterization of proteins. For absolute quantification of a target protein in bio-matrices, recent work has led to improvements in the efficiency of LC/MS method development, sample treatment, enrichment and digestion, and high-performance low-flow-LC separation. These advances have enhanced analytical sensitivity, specificity, and robustness. As to qualitative analysis, a range of techniques have been developed to characterize intramolecular disulfide bonds, glycosylation, charge variants, primary sequence heterogeneity, and the drug-to-antibody ratio of antibody drug conjugate (ADC), which has enabled a refined ability to assess product quality. In this review, we will focus on the discussion of technical challenges and strategies of LC/MS-based quantification and characterization of biotherapeutics, with the emphasis on the analysis of antibody-based biotherapeutics such as monoclonal antibodies (mAbs) and ADCs. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:734-754, 2017.

Keywords: antibody-drug conjugate; biotherapeutics; liquid chromatography; mass spectrometry; quantification.

Publication types

  • Review

MeSH terms

  • Animals
  • Antibodies, Monoclonal / analysis*
  • Antibodies, Monoclonal / therapeutic use
  • Biological Products / analysis
  • Chromatography, Liquid / methods*
  • Disulfides / analysis
  • Disulfides / chemistry
  • Drug Discovery / methods
  • Glycosylation
  • Humans
  • Immunoconjugates / analysis
  • Mass Spectrometry / methods*
  • Peptide Mapping / methods
  • Proteins / analysis*
  • Proteins / metabolism
  • Proteins / pharmacokinetics
  • Recombinant Proteins / analysis
  • Sensitivity and Specificity
  • Tissue Distribution


  • Antibodies, Monoclonal
  • Biological Products
  • Disulfides
  • Immunoconjugates
  • Proteins
  • Recombinant Proteins