Abstract
Immunotherapies are a hot topic, with the potential to impact our understanding of the immune system and treat a diverse array of conditions. Therapeutic monoclonal antibodies (mAbs) are part of this revolution, and clinical chemists are aware of the success of the biologic drugs. Antibodies are not just immunoassay reagents anymore but are also present in clinical serum samples from more and more patients each day. The clinical laboratory will have many roles as mAb therapies expand, including the development of new assays to differentiate a mAb from an endogenous, disease-causing clone and monitoring therapeutic drugs for better patient outcomes and assessing for the loss of response to therapy.
Therapeutic mAbs use has expanded significantly in the last 5 years, and depending on their target or their concentration, they may impact routine clinical testing for patients. Optimizing therapy during the induction phase to keep the mAb concentrations above certain thresholds has proven to be associated with improved responses and better outcomes in chronic conditions such as inflammatory bowel disease. This chapter will describe a LC-MS/MS protocol for analysis of tryptic peptides unique to infliximab (clonotypic peptides) for quantitation of the mAb. The protocol can be adapted to other mAbs with similar outcomes and is a useful, relatively simple strategy for measurement of mAbs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ladwig PM, Barnidge DR, Willrich MAV (2017) Mass spectrometry approaches for identification and quantitation of therapeutic monoclonal antibodies in the clinical laboratory. Clin Vaccine Immunol 24(5). https://doi.org/10.1128/CVI.00545-16
Ordas I, Mould DR, Feagan BG, Sandborn WJ (2012) Anti-TNF monoclonal antibodies in inflammatory bowel disease: pharmacokinetics-based dosing paradigms. Clin Pharmacol Ther 91(4):635–646. https://doi.org/10.1038/clpt.2011.328
Lazar-Molnar E, Delgado JC (2016) Immunogenicity assessment of tumor necrosis factor antagonists in the clinical laboratory. Clin Chem 62(9):1186–1198. https://doi.org/10.1373/clinchem.2015.242875
Pavlov IY, Carper J, Lazar-Molnar E, Delgado JC (2016) Clinical laboratory application of a reporter-gene assay for measurement of functional activity and neutralizing antibody response to infliximab. Clin Chim Acta 453:147–153. https://doi.org/10.1016/j.cca.2015.12.015
Wang SL, Ohrmund L, Hauenstein S, Salbato J, Reddy R, Monk P, Lockton S, Ling N, Singh S (2012) Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum. J Immunol Methods 382(1–2):177–188. https://doi.org/10.1016/j.jim.2012.06.002
Willrich MA, Murray DL, Barnidge DR, Ladwig PM, Snyder MR (2015) Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC-MS/MS. Int Immunopharmacol 28(1):513–520. https://doi.org/10.1016/j.intimp.2015.07.007
Jawa V, Joubert MK, Zhang Q, Deshpande M, Hapuarachchi S, Hall MP, Flynn GC (2016) Evaluating immunogenicity risk due to host cell protein impurities in antibody-based biotherapeutics. AAPS J 18(6):1439–1452. https://doi.org/10.1208/s12248-016-9948-4
Wang D, Wynne C, Gu F, Becker C, Zhao J, Mueller HM, Li H, Shameem M, Liu YH (2015) Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry. Anal Chem 87(2):914–921. https://doi.org/10.1021/ac503158g
Zhang B, Jeong J, Burgess B, Jazayri M, Tang Y, Taylor Zhang Y (2016) Development of a rapid RP-UHPLC-MS method for analysis of modifications in therapeutic monoclonal antibodies. J Chromatogr B Analyt Technol Biomed Life Sci 1032:172–181. https://doi.org/10.1016/j.jchromb.2016.05.017
Kohlhagen MC, Barnidge DR, Mills JR, Stoner J, Gurtner KM, Liptac AM, Lofgren DI, Vanderboom PM, Dispenzieri A, Katzmann JA, Willrich MA, Snyder MR, Murray DL (2016) Screening method for M-proteins in serum using nanobody enrichment coupled to MALDI-TOF mass spectrometry. Clin Chem. https://doi.org/10.1373/clinchem.2015.253781
Ladwig PM, Barnidge DR, Willrich MA (2016) Quantification of the IgG2/4 kappa monoclonal therapeutic Eculizumab from serum using Isotype specific affinity purification and microflow LC-ESI-Q-TOF mass spectrometry. J Am Soc Mass Spectrom. https://doi.org/10.1007/s13361-016-1566-y
Mills JR, Barnidge DR, Murray DL (2015) Detecting monoclonal immunoglobulins in human serum using mass spectrometry. Methods 81:56–65. https://doi.org/10.1016/j.ymeth.2015.04.020
Mills JR, Kohlhagen MC, Dasari S, Vanderboom PM, Kyle RA, Katzmann JA, Willrich MA, Barnidge DR, Dispenzieri A, Murray DL (2016) Comprehensive assessment of M-proteins using nanobody enrichment coupled to MALDI-TOF mass spectrometry. Clin Chem 62(10):1334–1344. https://doi.org/10.1373/clinchem.2015.253740
Anderson L, Hunter CL (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 5(4):573–588. https://doi.org/10.1074/mcp.M500331-MCP200
Ladwig PM, Barnidge DR, Snyder MR, Katzmann JA, Murray DL (2014) Quantification of serum IgG subclasses by use of subclass-specific tryptic peptides and liquid chromatography—tandem mass spectrometry. Clin Chem 60(8):1080–1088. https://doi.org/10.1373/clinchem.2014.222208
CLSI (2004) Evaluation of precision performance of quantitative measurement methods: approved guideline, 2nd edn. CLSI document EP05-A2 edn. Clinical and Laboratory Standards Institute, Wayne, PA
Acknowledgments
The author would like to thank her colleagues Dr. Melissa R. Snyder and Dr. David L. Murray, who were mentors of this test development project, and Dr. David Barnidge and Paula Ladwig who were instrumental in the analytical development experiments and assay performance validation. The author reports an intellectual property/royalty income interest in an LC-MS/MS-based method for measurement of therapeutic monoclonal antibodies.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Willrich, M.A.V. (2019). Analysis of Tryptic Peptides from Therapeutic Monoclonal Antibodies Using LC-MS/MS. In: Langman, L., Snozek, C. (eds) LC-MS in Drug Analysis. Methods in Molecular Biology, vol 1872. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8823-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8823-5_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8822-8
Online ISBN: 978-1-4939-8823-5
eBook Packages: Springer Protocols