Quantitative LC/ESI-SRM/MS of antibody biopharmaceuticals: use of a homologous antibody as an internal standard and three-step method development

Fumio Osaki, Kenji Tabata, Tomoyuki Oe

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)


Monoclonal antibody-based therapeutic agents (antibody drugs) have attracted considerable attention as a new type of drug. Concomitantly, the use of quantitative approaches for characterizing antibody drugs, such as liquid chromatography (LC)-mass spectrometry (MS), has increased. Generally, selective quantification of antibody drugs is done using unique peptides from variable regions (VH and VL) as surrogate peptides. Further, numerous internal standards (ISs) such as stable isotope-labeled (SIL)-intact proteins and SIL-surrogate peptides are used. However, developing LC-MS methodology for characterizing antibody drugs is time-consuming and costly. Therefore, LC-MS is difficult to apply for this purpose, particularly during the drug discovery stage when numerous candidates must be evaluated. Here, we demonstrate an efficient approach to developing a quantitative LC/electrospray ionization (ESI)-selected reaction monitoring (SRM)/MS method for characterizing antibody drugs. The approach consists of the following features: (i) standard peptides or SIL-IS are not required; (ii) a peptide from the homologous monoclonal antibody serves as an IS; (iii) method development is monitored using a spiked plasma sample and one quantitative MS analysis; and (iv) three predicted SRM assays are performed to optimize quantitative SRM conditions such as transition, collision energy, and declustering potential values. Using this strategy, we developed quantitative SRM methods for infliximab, alemtuzumab, and bevacizumab with sufficient precision (<20%)/accuracy (<±20%) for use in the drug discovery stage. We have also demonstrated that choosing a higher homologous peptide pair (from analyte mAb/IS mAb) is necessary to obtain the sufficient precision and accuracy. [Figure not available: see fulltext.].

Original languageEnglish
Pages (from-to)5523-5532
Number of pages10
JournalAnalytical and Bioanalytical Chemistry
Issue number23
Publication statusPublished - 2017 Sept 1


  • Antibody drug
  • Homologous antibody
  • Mass spectrometry
  • Selected reaction monitoring


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