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Therapeutic Monoclonal Antibody Intact Mass Analysis by Capillary Electrophoresis–Mass Spectrometry

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Capillary Electrophoresis-Mass Spectrometry

Abstract

The characterization of monoclonal antibody (mAb) therapeutics via mass spectroscopy is of important value in determining sequence integrity and identifying post-translational modifications. The monoclonal antibodies are commonly either reduced to generate heavy chain and light chain, or enzymatically cleaved to produce characteristic domains for subunit intact mass analysis. Toward this end, liquid chromatography coupled with mass spectrometry (LC-MS) is usually applied for the separation of these antibody subunits followed by on-line mass analysis. Capillary electrophoresis (CE) is an emerging separation technique that provides excellent protein separation efficiency at ambient temperature. The recent advancement on the coupling of capillary electrophoresis with mass spectrometer has essentially eliminated the technical obstacle for the broad application of CE-MS in the intact mass analysis of monoclonal antibody therapeutics. In this chapter, we will discuss several commercially available CE-MS interfaces and their applications, followed by demonstration of the CE-MS intact mass analysis procedure that has been developed for therapeutic protein characterization.

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References

  1. Rehder DS, Dillon TM, Pipes GD, Bondarenko PV (2006) Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics. J Chromatogr A 1102:164–175

    Article  CAS  PubMed  Google Scholar 

  2. Beck A, Wagner-Rousset E, Ayoub D, Van Dorsselaer A, Sanglier-Cianférani S (2013) Characterization of therapeutic antibodies and related products. Anal Chem 85:715–736

    Article  CAS  PubMed  Google Scholar 

  3. Fekete S, Guillarme D, Sandra P, Sandra K (2016) Chromatographic, electrophoretic, and mass spectrometric methods for the analytical characterization of protein biopharmaceuticals. Anal Chem 88:480–507

    Article  CAS  PubMed  Google Scholar 

  4. Han M, Rock BM, Pearson JT, Rock DA (2016) Intact mass analysis of monoclonal antibodies by capillary electrophoresis–mass spectrometry. J Chromatogr B 1011:24–32

    Article  CAS  Google Scholar 

  5. Zhao Y, Sun L, Knierman MD, Dovichi NJ (2016) Fast separation and analysis of reduced monoclonal antibodies with capillary zone electrophoresis coupled to mass spectrometry. Talanta 148:529–533

    Article  CAS  PubMed  Google Scholar 

  6. Said N et al (2016) Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis—tandem mass spectrometry as nanoESI infusion platform and separation method. Anal Chim Acta 918:50–59

    Article  CAS  PubMed  Google Scholar 

  7. Babu SCV, Gudihal R (2014) Characterization of monoclonal antibodies using capillary ionization-mass spectrometry. Agil. Technol. Publ. number 5991–5212EN

    Google Scholar 

  8. Knierman MD (2015) Protein analysis by CE/MS using a 6550 Q-TOF. http://cnpg.comparenetworks.com/174788-Protein-Analysis-by-CE-MS-using-a-6550-Q-TOF/ 174788

  9. Smith SL (1996) Ten years of Orthoclone OKT3 (muromonab-CD3): a review. J Transpl Coord 6:109–119

    Article  CAS  PubMed  Google Scholar 

  10. Reichert JM (2016) Antibodies to watch in 2016. MAbs 8:197–204

    Article  CAS  PubMed  Google Scholar 

  11. Beck A (2011) Biosimilar, biobetter and next generation therapeutic antibodies. MAbs 3:107–110

    Google Scholar 

  12. Goetze AM, Schenauer MR, Flynn GC (2010) Assessing monoclonal antibody product quality attribute criticality through clinical studies. MAbs 2:500–507

    Article  PubMed  PubMed Central  Google Scholar 

  13. Bondarenko PV, Second TP, Zabrouskov V, Makarov AA, Zhang Z (2009) 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

    Article  CAS  PubMed  Google Scholar 

  14. Vlasak J, Ionescu R (2011) Fragmentation of monoclonal antibodies. MAbs 3:253–263

    Article  PubMed  PubMed Central  Google Scholar 

  15. Zhao SS, Chen DDY (2014) Applications of capillary electrophoresis in characterizing recombinant protein therapeutics. Electrophoresis 35:96–108

    Article  CAS  PubMed  Google Scholar 

  16. Salas-Solano O et al (2006) Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies. Anal Chem 78:6583–6594

    Article  CAS  PubMed  Google Scholar 

  17. Michels DA, Brady LJ, Guo A, Balland A (2007) Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection. Anal Chem 79:5963–5971

    Article  CAS  PubMed  Google Scholar 

  18. Michels DA, Parker M, Salas-Solano O (2012) Quantitative impurity analysis of monoclonal antibody size heterogeneity by CE-LIF: example of development and validation through a quality-by-design framework. Electrophoresis 33:815–826

    Article  CAS  PubMed  Google Scholar 

  19. Glover ZW et al (2016) Compatibility and stability of pertuzumab and trastuzumab admixtures in i.v. infusion bags for coadministration. J Pharm Sci 102:794–812

    Article  CAS  Google Scholar 

  20. Yin S, Pastuskovas CV, Khawli LA, Stults JT (2013) Characterization of therapeutic monoclonal antibodies reveals differences between in vitro and in vivo time-course studies. Pharm Res 30:167–178 (2013)

    Google Scholar 

  21. Székely A et al (2014) Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries. Electrophoresis 35:2155–2162

    PubMed  Google Scholar 

  22. Lin J, Tan Q, Wang S (2011) A high-resolution capillary isoelectric focusing method for the determination of therapeutic recombinant monoclonal antibody. J Sep Sci 34:1696–1702

    Article  CAS  PubMed  Google Scholar 

  23. Salas-Solano O et al (2012) Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study. J Sep Sci 35:3124–3129

    Article  CAS  PubMed  Google Scholar 

  24. Righetti PG, Sebastiano R, Citterio A (2013) Capillary electrophoresis and isoelectric focusing in peptide and protein analysis. Proteomics 13:325–340

    Article  CAS  PubMed  Google Scholar 

  25. Cao J, Sun W, Gong F, Liu W (2014) Charge profiling and stability testing of biosimilar by capillary isoelectric focusing. Electrophoresis 35:1461–1468

    Article  CAS  PubMed  Google Scholar 

  26. He Y et al (2010) Analysis of identity, charge variants, and disulfide isomers of monoclonal antibodies with capillary zone electrophoresis in an uncoated capillary column. Anal Chem 82:3222–3230

    Article  CAS  PubMed  Google Scholar 

  27. He Y, Isele C, Hou W, Ruesch M (2011) Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary. J Sep Sci 34:548–555

    Article  CAS  PubMed  Google Scholar 

  28. Moritz B et al (2015) Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies. J Chromatogr B 983:101–110

    Article  CAS  Google Scholar 

  29. Gennaro LA, Salas-Solano O (2008) On-line CE–LIF–MS technology for the direct characterization of N-linked glycans from therapeutic antibodies. Anal Chem 80:3838–3845

    Article  CAS  PubMed  Google Scholar 

  30. Smith RD, Udseth HR (1988) Capillary zone electrophoresis-MS. Nature 331:639–640

    Article  CAS  PubMed  Google Scholar 

  31. Servais A-C, Crommen J, Fillet M (2006) Capillary electrophoresis–mass spectrometry, an attractive tool for drug bioanalysis and biomarker discovery. Electrophoresis 27:2616–2629

    Article  CAS  PubMed  Google Scholar 

  32. Monton MRN, Soga T (2007) Metabolome analysis by capillary electrophoresis–mass spectrometry. J Chromatogr A 1168:237–246

    Article  CAS  PubMed  Google Scholar 

  33. Mokaddem M, Gareil P, Belgaied J-E, Varenne A (2008) A new insight into suction and dilution effects in capillary electrophoresis coupled to mass spectrometry via an electrospray ionization interface. Part I-Suction effect. Electrophoresis 29:1957–1964

    Article  CAS  PubMed  Google Scholar 

  34. Zhao SS, Zhong X, Chen DDY (2012) Atmospheric pressure ion lens extends the stable operational region of an electrospray ion source for capillary electrophoresis–mass spectrometry. Electrophoresis 33:1322–1330

    Article  CAS  PubMed  Google Scholar 

  35. Zhao SS, Zhong X, Tie C, Chen DDY (2012) Capillary electrophoresis–mass spectrometry for analysis of complex samples. Proteomics 12:2991–3012

    Article  CAS  PubMed  Google Scholar 

  36. Lindenburg PW, Haselberg R, Rozing G, Ramautar R (2015) Developments in interfacing designs for CE-MS: towards enabling tools for proteomics and metabolomics. Chromatographia 78:367–377

    Article  CAS  Google Scholar 

  37. Moini M (2007) Simplifying CE-MS operation. 2. Interfacing low-flow separation techniques to mass spectrometry using a porous tip. Anal Chem 79:4241–4246

    Article  CAS  PubMed  Google Scholar 

  38. Gahoual R, Busnel J-M, Wolff P, François YN, Leize-Wagner E (2014) Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes. Anal Bioanal Chem 406:1029–1038

    Article  CAS  PubMed  Google Scholar 

  39. Haselberg R, de Jong GJ, Somsen GW (2013) Low-flow sheathless capillary electrophoresis–mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins. Anal Chem 85:2289–2296

    Article  CAS  PubMed  Google Scholar 

  40. Gahoual R et al (2014) Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry. MAbs 6:1464–1473

    Article  PubMed  PubMed Central  Google Scholar 

  41. Wojcik R, Dada OO, Sadilek M, Dovichi NJ (2010) Simplified capillary electrophoresis nanospray sheath-flow interface for high efficiency and sensitive peptide analysis. Rapid Commun Mass Spectrom 24:2554–2560

    Article  CAS  PubMed  Google Scholar 

  42. Wojcik R, Vannatta M, Dovichi NJ (2010) Automated enzyme-based diagonal capillary electrophoresis: application to phosphopeptide characterization. Anal Chem 82:1564–1567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li Y, Wojcik R, Dovichi NJ (2011) A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection. J Chromatogr A 1218:2007–2011

    Article  CAS  PubMed  Google Scholar 

  44. Li Y et al (2012) CZE-ESI-MS/MS as an alternative proteomics platform to UPLC-ESI-MS/MS for samples of intermediate complexity. Anal Chem 84:1617–1622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sun L et al (2012) CZE-ESI-MS/MS system for analysis of subnanogram amounts of tryptic digests of a cellular homogenate. Proteomics 12:3013–3019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wojcik R, Li Y, MacCoss M, Dovichi NJ (2012) Capillary electrophoresis with Orbitrap-Velos mass spectrometry detection. Talanta 88:324–329

    Article  CAS  PubMed  Google Scholar 

  47. Zhu G et al (2012) A rapid cIEF–ESI–MS/MS method for host cell protein analysis of a recombinant human monoclonal antibody. Talanta 98:253–256

    Article  CAS  PubMed  Google Scholar 

  48. Hommerson P, Khan AM, de Jong GJ, Somsen GW (2011) Ionization techniques in capillary electrophoresis–mass spectrometry: principles, design, and application. Mass Spectrom Rev 30:1096–1120

    Article  CAS  PubMed  Google Scholar 

  49. Johnson T et al (2001) A CE–MALDI interface based on the use of prestructured sample supports. Anal Chem 73:1670–1675

    Article  CAS  PubMed  Google Scholar 

  50. Zuberovic A, Ullsten S, Hellman U, Markides KE, Bergquist J (2004) Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries. Rapid Commun Mass Spectrom 18:2946–2952

    Article  CAS  PubMed  Google Scholar 

  51. Biacchi M et al (2014) Analysis of monoclonal antibody by a novel CE-UV/MALDI-MS interface. Electrophoresis 35:2986–2995

    Article  CAS  PubMed  Google Scholar 

  52. Biacchi M et al (2015) glycoform separation and characterization of cetuximab variants by middle-up off-line capillary zone electrophoresis-UV/electrospray ionization-MS. Anal Chem 87:6240–6250

    Article  CAS  PubMed  Google Scholar 

  53. Batz NG, Mellors JS, Alarie JP, Ramsey JM (2014) Chemical vapor deposition of aminopropyl silanes in microfluidic channels for highly efficient microchip capillary electrophoresis-electrospray ionization-mass spectrometry. Anal Chem 86:3493–3500

    Article  CAS  PubMed  Google Scholar 

  54. Mellors JS, Gorbounov V, Ramsey RS, Ramsey JM (2008) Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry. Anal Chem 80:6881–6887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Redman EA, Batz NG, Mellors JS, Ramsey JM (2015) Integrated microfluidic capillary electrophoresis-electrospray ionization devices with online MS detection for the separation and characterization of intact monoclonal antibody variants. Anal Chem 87:2264–2272

    Article  CAS  PubMed  Google Scholar 

  56. Redman EA, Mellors JS, Starkey JA, Ramsey JM (2016) Characterization of intact antibody drug conjugate variants using microfluidic capillary electrophoresis–mass spectrometry. Anal Chem 88:2220–2226

    Article  CAS  PubMed  Google Scholar 

  57. Redman EA, Ramos-Payan M, Mellors JS, Ramsey JM (2016) Analysis of hemoglobin glycation using microfluidic CE-MS: a rapid, mass spectrometry compatible method for assessing diabetes management. Anal Chem 88:5324–5330

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Pharmacokinetics and Drug Metabolism, Amgen Inc, 1120 Veterans Boulevard, South San Francisco, California, USA

    Mei Han, Brooke M. Rock, Josh T. Pearson, Yunan Wang & Dan A. Rock

Authors
  1. Mei Han
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  2. Brooke M. Rock
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  3. Josh T. Pearson
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  4. Yunan Wang
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  5. Dan A. Rock
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Corresponding author

Correspondence to Mei Han .

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Editors and Affiliations

  1. CMP Scientific, Corp., Brooklyn, New York, USA

    James Q. Xia

  2. Department of Chemical and Systems Biology, Stanford School of Medicine, Stanford University, Stanford, California, USA

    Lichao Zhang

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Han, M., Rock, B.M., Pearson, J.T., Wang, Y., Rock, D.A. (2016). Therapeutic Monoclonal Antibody Intact Mass Analysis by Capillary Electrophoresis–Mass Spectrometry. In: Xia, J., Zhang, L. (eds) Capillary Electrophoresis-Mass Spectrometry. Springer, Cham. https://doi.org/10.1007/978-3-319-46240-0_3

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