Abstract
We present here an analytical protocol for the sensitive, specific, and accurate absolute quantification of cetuximab, a human:murine chimeric monoclonal antibody, using mass spectrometry. Extraction from human serum is performed with micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of cetuximab. This specific immunocapture step allows sample purification and, in parallel, assessment of the antibody’s biological potency. The eluted mAb is digested with trypsin and specific peptides from light and heavy chains are monitored by liquid chromatography coupled with tandem mass spectrometry operated in the selected reaction monitoring (SRM) mode. The limit of quantification of the assay was 20 ng/mL in serum.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dubois M, Fenaille F, Clement G, Lechmann M, Tabet JC, Ezan E et al (2008) Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum. Anal Chem 80:1737–1745
Hagman C, Ricke D, Ewert S, Bek S, Falchetto R, Bitsch F (2008) Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry. Anal Chem 80:1290–1296
Heudi O, Barteau S, Zimmer D, Schmidt J, Bill K, Lehmann N et al (2008) 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 80:4200–4207
Yang Z, Hayes M, Fang X, Daley MP, Ettenberg S, Tse FL (2007) LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup. Anal Chem 79:9294–9301
Becher F, Duriez E, Volland H, Tabet JC, Ezan E (2007) Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry. Anal Chem 79:659–665
Van den Broek I, Sparidans RW, Huitema ADR, Schellens JHM, Beijnen JH (2006) Development and validation of a quantitative assay for the measurement of two HIV-fusion inhibitors, enfuvirtide and tifuvirtide, and one metabolite of enfuvirtide (M-20) in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 837:49–58
Brun V, Dupuis A, Adrait A, Marcellin M, Thomas D, Court M et al (2007) Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics 6:2139–2149
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Becher, F., Dubois, M., Fenaille, F., Ezan, E. (2013). Mass Spectrometry Protocol for the Absolute Quantification of a Monoclonal Antibody in Serum with Immunopurification. In: Beck, A. (eds) Glycosylation Engineering of Biopharmaceuticals. Methods in Molecular Biology, vol 988. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-327-5_22
Download citation
DOI: https://doi.org/10.1007/978-1-62703-327-5_22
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-326-8
Online ISBN: 978-1-62703-327-5
eBook Packages: Springer Protocols