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

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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.

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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

V H :

Heavy-chain variable region

V L :

Light-chain variable region

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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.

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Authors and Affiliations

  1. Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, Miyagi, 980-8578, Japan

    Fumio Osaki & Tomoyuki Oe

  2. Analysis & Pharmacokinetics Research Labs, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki, 305-8585, Japan

    Fumio Osaki & Kenji Tabata

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  1. Fumio Osaki
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Correspondence to Tomoyuki Oe.

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Osaki, F., Tabata, K. & Oe, T. Quantitative LC/ESI-SRM/MS of antibody biopharmaceuticals: use of a homologous antibody as an internal standard and three-step method development. Anal Bioanal Chem 409, 5523–5532 (2017). https://doi.org/10.1007/s00216-017-0488-2

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