Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

  • Lidong He
  • Lissa C. Anderson
  • David R. Barnidge
  • David L. Murray
  • Christopher L. Hendrickson
  • Alan G. Marshall
Focus: 28th Sanibel Conference, Characterization of Protein Therapeutics by MS: Research Article

Abstract

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2–0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Graphical Abstract

Keywords

Fourier transform Ion cyclotron resonance FTMS Electrospray MS/MS Middle-down Collision-induced dissociation CID Electron transfer dissociation ETD Multiple myeloma Isotype Variable region 

Supplementary material

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References

  1. 1.
    Beck, A., Wurch, T., Bailly, C., Corvaia, N.: Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10, 345–352 (2010)CrossRefGoogle Scholar
  2. 2.
    Ecker, D.M., Jones, S.D., Levine, H.L.: The therapeutic monoclonal antibody market. mAbs 7, 9–14 (2015)CrossRefGoogle Scholar
  3. 3.
    Scott, A.M., Wolchok, J.D., Old, L.J.: Antibody therapy of cancer. Nat. Rev. Cancer. 12, 278–287 (2012)CrossRefGoogle Scholar
  4. 4.
    Chan, A.C., Carter, P.J.: Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol. 10, 301–316 (2010)CrossRefGoogle Scholar
  5. 5.
    Bouchard, H., Viskov, C., Garcia-Echeverria, C.: Antibody-drug conjugates—a new wave of cancer drugs. Bioorg. Med. Chem. Lett. 24, 5357–5363 (2014)CrossRefGoogle Scholar
  6. 6.
    Sondermann, P., Szymkowski, D.E.: Harnessing Fc receptor biology in the design of therapeutic antibodies. Curr. Opin. Immunol. 40, 78–87 (2016)CrossRefGoogle Scholar
  7. 7.
    Murray, D., Barnidge, D.: Characterization of immunoglobulin by mass spectrometry with applications for the clinical laboratory. Crit. Rev. Clin. Lab. Sci. 50, 91–102 (2013)CrossRefGoogle Scholar
  8. 8.
    Beck, A., Wagner-Rousset, E., Ayoub, D., Van Dorsselaer, A., Sanglier-Cianferani, S.: Characterization of therapeutic antibodies and related products. Anal. Chem. 85, 715–736 (2013)CrossRefGoogle Scholar
  9. 9.
    Dekker, L., Wu, S., Vanduijn, M., Tolic, N., Stingl, C., Zhao, R., Luider, T., Pasa-Tolic, L.: An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies. Proteomics 14, 1239–1248 (2014)CrossRefGoogle Scholar
  10. 10.
    Srzentic, K., Fornelli, L., Laskay, U.A., Monod, M., Beck, A., Ayoub, D., Tsybin, Y.O.: Advantages of extended bottom-up proteomics using Sap9 for analysis of monoclonal antibodies. Anal. Chem. 86, 9945–9953 (2014)CrossRefGoogle Scholar
  11. 11.
    Pang, Y., Wang, W.H., Reid, G.E., Hunt, D.F., Bruening, M.L.: Pepsin-containing membranes for controlled monoclonal antibody digestion prior to mass spectrometry analysis. Anal. Chem. 87, 10942–10949 (2015)CrossRefGoogle Scholar
  12. 12.
    Zhang, L., English, A.M., Bai, D.L., Ugrin, S.A., Shabanowitz, J., Ross, M.M., Hunt, D.F., Wang, W.H.: Analysis of monoclonal antibody sequence and post-translational modifications by time-controlled proteolysis and tandem mass spectrometry. Mol. Cell. Proteom. 15, 1479–1488 (2016)CrossRefGoogle Scholar
  13. 13.
    Gahoual, R., Busnel, J.M., Beck, A., Francois, Y.N., Leize-Wagner, E.: Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry. Anal. Chem. 86, 9074–9081 (2014)CrossRefGoogle Scholar
  14. 14.
    Mukherjee, R., Adhikary, L., Khedkar, A., Iyer, H.: Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by 'bottom-up' mass spectrometry with electron transfer dissociation. Rapid Commun. Mass Spectrom. 24, 879–884 (2010)CrossRefGoogle Scholar
  15. 15.
    Zhang, Z., Pan, H., Chen, X.: Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom. Rev. 28, 147–176 (2009)CrossRefGoogle Scholar
  16. 16.
    Mao, Y., Valeja, S.G., Rouse, J.C., Hendrickson, C.L., Marshall, A.G.: Top-down structural analysis of an intact monoclonal antibody by electron capture dissociation-Fourier transform ion cyclotron resonance-mass spectrometry. Anal. Chem. 85, 4239–4246 (2013)CrossRefGoogle Scholar
  17. 17.
    Fornelli, L., Damoc, E., Thomas, P.M., Kelleher, N.L., Aizikov, K., Denisov, E., Makarov, A., Tsybin, Y.O.: Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS. Mol. Cell. Proteom. 11, 1758–1767 (2012)CrossRefGoogle Scholar
  18. 18.
    Wang, D., Wynne, C., Gu, F., Becker, C., Zhao, J., Mueller, H.M., Li, H., Shameem, M., Liu, Y.H.: Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry. Anal. Chem. 87, 914–921 (2015)CrossRefGoogle Scholar
  19. 19.
    Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A.A., Zhang, Z.: Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer. J. Am. Soc. Mass Spectrom. 20, 1415–1424 (2009)CrossRefGoogle Scholar
  20. 20.
    Zhang, Z., Shah, B.: Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry. Anal. Chem. 79, 5723–5729 (2007)CrossRefGoogle Scholar
  21. 21.
    Fornelli, L., Ayoub, D., Aizikov, K., Beck, A., Tsybin, Y.O.: Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry. Anal. Chem. 86, 3005–3012 (2014)CrossRefGoogle Scholar
  22. 22.
    Cotham, V.C., Brodbelt, J.S.: Characterization of therapeutic monoclonal antibodies at the subunit-level using middle-down 193 nm ultraviolet photodissociation. Anal. Chem. 88, 4004–4013 (2016)CrossRefGoogle Scholar
  23. 23.
    Sjogren, J., Olsson, F., Beck, A.: Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis. Analyst 141, 3114–3125 (2016)CrossRefGoogle Scholar
  24. 24.
    Hendrickson, C.L., Quinn, J.P., Kaiser, N.K., Smith, D.F., Blakney, G.T., Chen, T., Marshall, A.G., Weisbrod, C.R., Beu, S.C.: 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer: a national resource for ultrahigh resolution mass analysis. J. Am. Soc. Mass Spectrom. 26, 1626–1632 (2015)CrossRefGoogle Scholar
  25. 25.
    Shi, S.D.H., Drader, J.J., Freitas, M.A., Hendrickson, C.L., Marshall, A.G.: Comparison and interconversion of the two most common frequency-to-mass calibration functions for Fourier transform ion cyclotron resonance mass spectrometry. Int. J. Mass Spectrom. 195, 591–598 (2000)CrossRefGoogle Scholar
  26. 26.
    Ledford Jr., E.B., Rempel, D.L., Gross, M.L.: Space charge effects in Fourier transform mass spectrometry. Mass calibration. Anal. Chem. 56, 2744–2748 (1984)CrossRefGoogle Scholar
  27. 27.
    Fellers, R.T., Greer, J.B., Early, B.P., Yu, X., LeDuc, R.D., Kelleher, N.L., Thomas, P.M.: ProSight Lite: graphical software to analyze top-down mass spectrometry data. Proteomics 15, 1235–1238 (2015)CrossRefGoogle Scholar
  28. 28.
    Agarwal, A., Ghobrial, I.M.: Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma: a review of the current understanding of epidemiology, biology, risk stratification, and management of myeloma precursor disease. Clin. Cancer Res. 19, 985–994 (2013)CrossRefGoogle Scholar
  29. 29.
    Kyle, R.A., Buadi, F., Rajkumar, S.V.: Management of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Oncology (Williston Park) 25(578–586) (2011)Google Scholar
  30. 30.
    van de Donk, N.W., Mutis, T., Poddighe, P.J., Lokhorst, H.M., Zweegman, S.: Diagnosis, risk stratification and management of monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. Int. J. Lab. Hematol. 38(Suppl 1), 110–122 (2016)CrossRefGoogle Scholar
  31. 31.
    Durie, B.G., Salmon, S.E.: A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment, and survival. Cancer 36, 842–854 (1975)CrossRefGoogle Scholar
  32. 32.
    Jaskowski, T.D., Litwin, C.M., Hill, H.R.: Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis. Clin. Vaccine Immunol. 13, 277–280 (2006)CrossRefGoogle Scholar
  33. 33.
    Barnidge, D.R., Dasari, S., Botz, C.M., Murray, D.H., Snyder, M.R., Katzmann, J.A., Dispenzieri, A., Murray, D.L.: Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy. J. Proteome Res. 13, 1419–1427 (2014)CrossRefGoogle Scholar
  34. 34.
    Botz, C.M., Barnidge, D.R., Murray, D.L., Katzmann, J.A.: Detecting monoclonal light chains in urine: microLC-ESI-Q-TOF mass spectrometry compared to immunofixation electrophoresis. Br. J. Haematol. 167, 437–438 (2014)CrossRefGoogle Scholar
  35. 35.
    Barnidge, D.R., Kohlhagen, M.C., Zheng, S., Willrich, M.A., Katzmann, J.A., Pittock, S.J., Murray, D.L.: Monitoring oligoclonal immunoglobulins in cerebral spinal fluid using microLC-ESI-Q-TOF mass spectrometry. J. Neuroimmunol. 288, 123–126 (2015)CrossRefGoogle Scholar
  36. 36.
    Barnidge, D.R., Krick, T.P., Griffin, T.J., Murray, D.L.: Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect monoclonal immunoglobulin light chains in serum and urine. Rapid Commun. Mass Spectrom. 29, 2057–2060 (2015)CrossRefGoogle Scholar
  37. 37.
    Mills, J.R., Barnidge, D.R., Murray, D.L.: Detecting monoclonal immunoglobulins in human serum using mass spectrometry. Methods 81, 56–65 (2015)CrossRefGoogle Scholar
  38. 38.
    Barnidge, D.R., Dispenzieri, A., Merlini, G., Katzmann, J.A., Murray, D.L.: Monitoring free light chains in serum using mass spectrometry. Clin. Chem. Lab. Med. 54, 1073–1083 (2016)CrossRefGoogle Scholar
  39. 39.
    Kohlhagen, M.C., Barnidge, D.R., Mills, J.R., Stoner, J., Gurtner, K.M., Liptac, A.M., Lofgren, D.I., Vanderboom, P.M., Diepenzieri, A., Katzmann J.A., Willrich, M.A., Snyder, M.R., Murray, D.L.: Screening method for M-proteins in serum using nanobody enrichment coupled to MALDI-TOF mass spectrometry. Clin. Chem. 62, 1345–1352 (2016)Google Scholar
  40. 40.
    Leung, N., Barnidge, D.R., Hutchison, C.A.: Laboratory testing in monoclonal gammopathy of renal significance (MGRS). Clin. Chem. Lab. Med. 54, 929–937 (2016)CrossRefGoogle Scholar
  41. 41.
    Mills, J.R., Cornec, D., Dasari, S., Ladwig, P.M., Hummel, A.M., Cheu, M.: Using mass spectrometry to quantify rituximab and perform individualized immunoglobulin phenotyping in ANCA-associated vasculitis. Anal. Chem. 88, 6317–6325 (2016)CrossRefGoogle Scholar
  42. 42.
    Mills, J.R., Kohlhagen, M.C., Dasari, S., Vanderboom, P.M., Kyle, R.A., Katzmann, J.A., Willrich, M.A., Barnidge, D.R., Dispenzieri, A., Murray, D.L.: Comprehensive assessment of M-proteins using nanobody enrichment coupled to MALDI-TOF mass spectrometry. Clin. Chem. 62, 1334–1344 (2016)Google Scholar
  43. 43.
    Barnidge, D.R., Dasari, S., Ramirez-Alvarado, M., Fontan, A., Willrich, M.A., Tschumper, R.C., Jelinek, D.F., Snyder, M.R., Dispenzieri, A., Katzmann, J.A., Murray, D.L.: Phenotyping polycloncal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometer. J. Protome Res. 13, 5198–5205 (2014)CrossRefGoogle Scholar
  44. 44.
    Senko, M.W., Beu, S.C., McLaffertycor, F.W.: Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions. J. Am. Soc. Mass Spectrom. 6, 229–233 (1995)CrossRefGoogle Scholar
  45. 45.
    Horn, D.M., Zubarev, R.A., McLafferty, F.W.: Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. J. Am. Soc. Mass Spectrom. 11, 320–332 (2000)CrossRefGoogle Scholar
  46. 46.
    An, Y., Zhang, Y., Mueller, H.M., Shameem, M., Chen, X.: A new tool for monoclonal antibody analysis: application of IdeS protelysis in IgG domain-specific characterization. mAbs 6, 879–893 (2014)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Lidong He
    • 1
  • Lissa C. Anderson
    • 2
  • David R. Barnidge
    • 3
  • David L. Murray
    • 3
  • Christopher L. Hendrickson
    • 1
    • 2
  • Alan G. Marshall
    • 1
    • 2
  1. 1.Department of Chemistry and BiochemistryFlorida State UniversityTallahasseeUSA
  2. 2.National High Magnetic Field LaboratoryFlorida State UniversityTallahasseeUSA
  3. 3.Department of Laboratory Medicine and PathologyMayo ClinicRochesterUSA

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