Analytical and Bioanalytical Chemistry

, Volume 409, Issue 23, pp 5523–5532 | Cite as

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 OeEmail author
Research Paper


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 (V H and V L) 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.

Graphical abstract


Antibody drug Homologous antibody Mass spectrometry Selected reaction monitoring 



Complementarity-determining region


Collision energy


Collision cell exit potential


Declustering potential


Dulbecco’s phosphate-buffered saline




Electrospray ionization


Entrance potential




Internal standard


Ligand binding assay


Liquid chromatography


Monoclonal antibody


Mass spectrometry


Predicted SRM


Quality control


Selected reaction monitoring


Stable isotope-labeled


Heavy-chain variable region


Light-chain variable region



The authors thank Aiji Miyashita, Masako Furutani, Fujiko Takamura, and Dr. Tetsu Saito of Astellas Pharma Inc. for their valuable scientific discussions and Masamichi Yuda and Masashi Kawasaki of Astellas Pharma Inc. for providing antibodies.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

216_2017_488_MOESM1_ESM.pdf (4.9 mb)
ESM 1 (PDF 5012 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.Analysis & Pharmacokinetics Research LabsAstellas Pharma Inc.TsukubaJapan

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