Analysis of Tryptic Peptides from Therapeutic Monoclonal Antibodies Using LC-MS/MS

  • Maria Alice V. Willrich
Part of the Methods in Molecular Biology book series (MIMB, volume 1872)


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.

Key words

LC-MS/MS Infliximab Therapeutic monoclonal antibodies Trypsin Tryptic peptides Method development 



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.


  1. 1.
    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).
  2. 2.
    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. CrossRefPubMedGoogle Scholar
  3. 3.
    Lazar-Molnar E, Delgado JC (2016) Immunogenicity assessment of tumor necrosis factor antagonists in the clinical laboratory. Clin Chem 62(9):1186–1198. CrossRefPubMedGoogle Scholar
  4. 4.
    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. CrossRefPubMedGoogle Scholar
  5. 5.
    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. CrossRefPubMedGoogle Scholar
  6. 6.
    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. CrossRefPubMedGoogle Scholar
  7. 7.
    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. CrossRefPubMedGoogle Scholar
  8. 8.
    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. CrossRefPubMedGoogle Scholar
  9. 9.
    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. CrossRefPubMedGoogle Scholar
  10. 10.
    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. CrossRefGoogle Scholar
  11. 11.
    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. CrossRefGoogle Scholar
  12. 12.
    Mills JR, Barnidge DR, Murray DL (2015) Detecting monoclonal immunoglobulins in human serum using mass spectrometry. Methods 81:56–65. CrossRefPubMedGoogle Scholar
  13. 13.
    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. CrossRefPubMedGoogle Scholar
  14. 14.
    Anderson L, Hunter CL (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics 5(4):573–588. CrossRefPubMedGoogle Scholar
  15. 15.
    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. CrossRefPubMedGoogle Scholar
  16. 16.
    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, PAGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and PathologyMayo ClinicRochesterUSA

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