Quantitative LC/ESI-SRM/MS of antibody biopharmaceuticals: use of a homologous antibody as an internal standard and three-step method development
- 445 Downloads
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.
KeywordsAntibody drug Homologous antibody Mass spectrometry Selected reaction monitoring
Collision cell exit potential
Dulbecco’s phosphate-buffered saline
Ligand binding assay
Selected reaction monitoring
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.
- 5.Philippidis A. The top 25 best-selling drugs of 2014, Genetic Engineering & Biotechnology News, Feb 23, 2015. (http://www.genengnews.com/keywordsandtools/print/3/37387/).
- 14.Furlong MT, Ouyang Z, Wu S, Tamura J, Olah T, Tymiak A, et al. A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies. Biomed Chromatogr. 2012;26:1024–32. doi: 10.1002/bmc.2759.Google Scholar
- 17.Zhang Q, Spellman DS, Song Y, Choi B, Hatcher NG, Tomazela D, et al. Generic automated method for liquid chromatography-multiple reaction monitoring mass spectrometry based monoclonal antibody quantitation for preclinical pharmacokinetic studies. Anal Chem. 2014;86:8776–84. doi: 10.1021/ac5019827.CrossRefGoogle Scholar
- 21.Kamiie J, Ohtsuki S, Iwase R, Ohmine K, Katsukura Y, Yanai K, et al. Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res. 2008;25:1469–83. doi: 10.1007/s11095-008-9532-4.CrossRefGoogle Scholar
- 23.Heudi O, Barteau S, Zimmer D, Schmidt J, Bill K, Lehmann N, et al. Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry. Anal Chem. 2008;80:4200–7. doi: 10.1021/ac800205s.CrossRefGoogle Scholar
- 26.Liu G, Ji QC, Dodge R, Sun H, Shuster D, Zhao Q, et al. Liquid chromatography coupled with tandem mass spectrometry for the bioanalysis of proteins in drug development: practical considerations in assay development and validation. J Chromatogr A. 2013;1284:155–62. doi: 10.1016/j.chroma.2013.02.016.CrossRefGoogle Scholar
- 28.Lame M, Yang H, Naughton S, Chambers E. An intact murine monoclonal antibody for use as a generic internal standard and workflow check standard in protein bioanalysis studies. Waters Co., Milford, MA, USA, Waters application note 720005543 ( http://www.waters.com/webassets/cms/library/docs/720005543en.pdf).
- 34.McGinley M, Jarrett D, Layne J, Chitty M, Farkas T. Optimising core-shell UHPLC columns for improving protein and peptide separations. Chromatography Today 4 2011;33–36. (http://www.chromatographytoday.com/articles/hplc-uhplc/31/michael_mcginley_deborah_jarrett_jeff_layne_mike_chitty_and_tivadar_farkas/optimising_core-shell_uhplc_columns_for_improving_protein_and_peptide_separations/1099/).
- 35.Duan X, Abuqayyas L, Dai L, Balthasar JP, Qu J. High-throughput method development for sensitive, accurate, and reproducible quantification of therapeutic monoclonal antibodies in tissues using orthogonal array optimization and nano liquid chromatography/selected reaction monitoring mass spectrometry. Anal Chem. 2012;84:4373–82. doi: 10.1021/ac2034166.CrossRefGoogle Scholar
- 36.Duan X, Dai L, Chen SC, Balthasar JP, Qu J. Nano-scale liquid chromatography/mass spectrometry and on-the-fly orthogonal array optimization for quantification of therapeutic monoclonal antibodies and the application in preclinical analysis. J Chromatogr A. 2012;1251:63–73. doi: 10.1016/j.chroma.2012.06.007.CrossRefGoogle Scholar