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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

Abstract

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

Keywords

Antibody drug Homologous antibody Mass spectrometry Selected reaction monitoring 

Abbreviations

CDR

Complementarity-determining region

CE

Collision energy

CXP

Collision cell exit potential

DP

Declustering potential

D-PBS

Dulbecco’s phosphate-buffered saline

DTT

Dithiothreitol

ESI

Electrospray ionization

EP

Entrance potential

IAA

Iodoacetamide

IS

Internal standard

LBA

Ligand binding assay

LC

Liquid chromatography

mAb

Monoclonal antibody

MS

Mass spectrometry

pSRM

Predicted SRM

QC

Quality control

SRM

Selected reaction monitoring

SIL

Stable isotope-labeled

VH

Heavy-chain variable region

VL

Light-chain variable region

Notes

Acknowledgements

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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