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Structural Characterization of Antibodies by Mass Spectrometry

  • Marie-Claire Janin-Bussat
  • Jean-Marc Strub
  • Elsa Wagner-Rousset
  • Olivier Colas
  • Christine Klinguer-Hamour
  • Nathalie Corvaia
  • Alain van Dorsselaer
  • Alain BeckEmail author

Among all analytical methods used to characterize monoclonal antibodies (mAbs), mass spectrometry plays an increasingly important role for both global and fine structural characterization of therapeutic candidates. In this chapter we discuss and provide detailed protocols for intact antibody and light and heavy chain primary structure characterisation by LC-ESI-TOF, glycosylation and production-system fingerprinting, structural isotyping and disulfide connection determination (humanised IgG1 vs IgG4) and finally non-stable “hot spots” mapping by Mass Spectrometry (de-amidation by MALDI-TOF and LC-IT MS/MS).

Keywords

Heavy Chain Pyroglutamic Acid Intact Antibody Nominal Molecular Weight Deamidation Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Atmanène C, Wagner-Rousset E, Malissard M, Chol B, Robert A, Corvaïa N, Van Dorsselaer A, Beck A, Sanglier-Cianferani S. (2009) Extending Mass Spectrometry contribution to therapeutic monoclonal antibody lead selection and development: characterization of antigen/antibody immune complexes using non-covalent ESI-MS. Analytical Chemistry. In Press Google Scholar
  2. Banks DD, Gadgil HS, Pipes GD, Bondarenko PV, Hobbs V, Scavezze JL, Kim J, Jiang XR, Mukku V, Dillon TM (2008) Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody improves homogeneity, stability, and biological activity. J Pharm Sci 97:775–790PubMedCrossRefGoogle Scholar
  3. Beck A, Bussat MC, Zorn N, Robillard V, Klinguer-Hamour C, Chenu S, Goetsch L, Corvaia N, Van Dorsselaer A, Haeuw JF (2005) Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells. J Chromatogr B Analyt Technol Biomed Life Sci 819:203–218PubMedCrossRefGoogle Scholar
  4. Beck A, Klinguer-Hamour C, Bussat MC, Champion T, Haeuw JF, Goetsch L, Wurch T, Sugawara M, Milon A, Van Dorsselaer A, Nguyen T, Corvaia N (2007) Peptides as tools and drugs for immunotherapies. J Pept Sci 13:588–602PubMedCrossRefGoogle Scholar
  5. Beck A, Wagner-Rousset E, Bussat MC, Lokteff M, Klinguer-Hamour C, Haeuw JF, Goetsch L, Wurch T, Van Dorsselaer A, Corvaia N (2008a) Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins. Curr Pharm Biotechnol 9:482–501PubMedCrossRefGoogle Scholar
  6. Beck A, Wagner-Rousset E, Goetsch L, Corvaïa N (2008b) Therapeutic antibodies: structure assessment by mass spectrometry from screening to clinical batches. Screen Trends Drug Discov 9:18–20Google Scholar
  7. Beck A, Wurch T, Corvaïa N (2008c) Editorial: therapeutic antibodies and derivatives: from the bench to the clinic. Curr Pharma Biotechnol 9:421–422CrossRefGoogle Scholar
  8. Chelius D, Rehder DS, Bondarenko PV (2005) Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal Chem 77:6004–6011PubMedCrossRefGoogle Scholar
  9. Chelius D, Huff Wimer ME, Bondarenko PV (2006a) Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody. J Am Soc Mass Spectrom 17:1590–1598PubMedCrossRefGoogle Scholar
  10. Chelius D, Jing K, Lueras A, Rehder DS, Dillon TM, Vizel A, Rajan RS, Li T, Treuheit MJ, Bondarenko PV (2006b) Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies. Anal Chem 78:2370–2376PubMedCrossRefGoogle Scholar
  11. Chu GC, Chelius D, Xiao G, Khor HK, Coulibaly S, Bondarenko PV (2007) Accumulation of succinimide in a recombinant monoclonal antibody in mildly acidic buffers under elevated temperatures. Pharm Res 24:1145–1156PubMedCrossRefGoogle Scholar
  12. Cohen SL, Price C, Vlasak J (2007) Beta-elimination and peptide bond hydrolysis: two distinct mechanisms of human IgG1 hinge fragmentation upon storage. J Am Chem Soc 129:6976–6977PubMedCrossRefGoogle Scholar
  13. Cordoba AJ, Shyong BJ, Breen D, Harris RJ (2005) Non-enzymatic hinge region fragmentation of antibodies in solution. J Chromatogr B Analyt Technol Biomed Life Sci 818:115–121PubMedCrossRefGoogle Scholar
  14. Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:1591–1597PubMedCrossRefGoogle Scholar
  15. Dick LW Jr, Kim C, Qiu D, Cheng KC (2007) Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides. Biotechnol Bioeng 97:544–553PubMedCrossRefGoogle Scholar
  16. Dillon TM, Bondarenko PV, Speed RM (2004) Development of an analytical reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry method for characterization of recombinant antibodies. J Chromatogr A 1053:299–305PubMedGoogle Scholar
  17. Dillon TM, Bondarenko PV, Rehder DS, Pipes GD, Kleemann GR, Ricci MS (2006) Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. J Chromatogr A 1120:112–120PubMedCrossRefGoogle Scholar
  18. Gadgil HS, Bondarenko PV, Pipes GD, Dillon TM, Banks D, Abel J, Kleemann GR, Treuheit MJ (2006a) Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis. Anal Biochem 355:165–174PubMedCrossRefGoogle Scholar
  19. Gadgil HS, Pipes GD, Dillon TM, Treuheit MJ, Bondarenko PV (2006b) Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies. J Am Soc Mass Spectrom 17:867–872PubMedCrossRefGoogle Scholar
  20. Gadgil HS, Bondarenko PV, Treuheit MJ, Ren D (2007) Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method. Anal Chem 79:5991–5999PubMedCrossRefGoogle Scholar
  21. Huang LH, Biolsi S, Bales KR, Kuchibhotla U (2006) Impact of variable domain glycosylation on antibody clearance: An LC/MS characterization. Anal Biochem 349:197–207PubMedCrossRefGoogle Scholar
  22. Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC (2007) Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem 360:75–83PubMedCrossRefGoogle Scholar
  23. Le JC, Bondarenko PV (2005) Trap for MAbs: characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry. J Am Soc Mass Spectrom 16:307–311PubMedCrossRefGoogle Scholar
  24. Liu H, Gaza-Bulseco G, Sun J (2006) Characterization of the stability of a fully human monoclonal IgG after prolonged incubation at elevated temperature. J Chromatogr B Analyt Technol Biomed Life Sci 837:35–43PubMedCrossRefGoogle Scholar
  25. Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J (2008a) Heterogeneity of monoclonal antibodies. J Pharm Sci 97:2426–2447PubMedCrossRefGoogle Scholar
  26. Liu H, Gaza-Bulseco G, Xiang T, Chumsae C (2008b) Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody. Mol Immunol 45:701–708PubMedCrossRefGoogle Scholar
  27. Lyubarskaya Y, Houde D, Woodard J, Murphy D, Mhatre R (2006) Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity. Anal Biochem 348:24–39PubMedCrossRefGoogle Scholar
  28. Olivova P, Chen W, Chakraborty AB, Gebler JC (2008) Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 22:29–40PubMedCrossRefGoogle Scholar
  29. Qian J, Liu T, Yang L, Daus A, Crowley R, Zhou Q (2007) Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion. Anal Biochem 364:8–18PubMedCrossRefGoogle Scholar
  30. Raju TS, Scallon BJ (2006) Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain. Biochem Biophys Res Commun 341:797–803PubMedCrossRefGoogle Scholar
  31. 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 1102:164–175PubMedCrossRefGoogle Scholar
  32. Siemiatkoski J, Lyubarskaya Y, Houde D, Tep S, Mhatre R (2006) A comparison of three techniques for quantitative carbohydrate analysis used in characterization of therapeutic antibodies. Carbohydr Res 341:410–419PubMedCrossRefGoogle Scholar
  33. Srebalus Barnes CA, Lim A (2007) Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals. Mass Spectrom Rev 26:370–388PubMedCrossRefGoogle Scholar
  34. Terashima I, Koga A, Nagai H (2007) Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H(2)(18)O. Anal Biochem 368:49–60PubMedCrossRefGoogle Scholar
  35. Tous GI, Wei ZP, Feng JH, Bilbulian S, Bowen S, Smith J, Strouse R, McGeehan P, Casas-Finet J, Schenerman MA (2005) Characterization of a novel modification to monoclonal antibodies: Thioether cross-link of heavy and light chains. Anal Chem 77:2675–2682PubMedCrossRefGoogle Scholar
  36. Vlasak J, Ionescu R (2008) Heterogeneity of Monoclonal Antibodies relvealed by charge-sensitive methods. Curr Pharm Biotechnol 9:468–481PubMedCrossRefGoogle Scholar
  37. Wagner-Rousset E, Bednarczyk A, Bussat MC, Colas O, Corvaia N, Schaeffer C, Van Dorsselaer A, Beck A (2008) The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies. J Chromatogr B Analyt Technol Biomed Life Sci 872:23–37PubMedCrossRefGoogle Scholar
  38. Wang L, Amphlett G, Lambert JM, Blattler W, Zhang W (2005) Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry. Pharm Res 22:1338–1349PubMedCrossRefGoogle Scholar
  39. Wei Z, Feng J, Lin HY, Mullapudi S, Bishop E, Tous GI, Casas-Finet J, Hakki F, Strouse R, Schenerman MA (2007) Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus. Anal Chem 79:2797–2805PubMedCrossRefGoogle Scholar
  40. Xiao G, Bondarenko PV, Jacob J, Chu GC, Chelius D (2007) 18O labeling method for identification and quantification of succinimide in proteins. Anal Chem 79:2714–2721PubMedCrossRefGoogle Scholar
  41. Yan B, Valliere-Douglass J, Brady L, Steen S, Han M, Pace D, Elliott S, Yates Z, Han Y, Balland A, Wang W, Pettit D (2007) Analysis of posttranslational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry. J Chromatogr A 1164:153–161PubMedCrossRefGoogle Scholar
  42. Yang J, Wang S, Liu J, Raghani A (2007) Determination of tryptophan oxidation of monoclonal antibody by reversed phase high performance liquid chromatography. J Chromatogr A 1156:174–182PubMedCrossRefGoogle Scholar
  43. Ying H, Liu H (2007) Identification of an alternative signal peptide cleavage site of mouse monoclonal antibodies by mass spectrometry. Immunol Lett 111:66–68PubMedCrossRefGoogle Scholar
  44. Yu L, Vizel A, Huff MB, Young M, Remmele RL Jr, He B (2006) Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development. J Pharm Biomed Anal 42:455–463PubMedCrossRefGoogle Scholar
  45. Zhang Z, Shah B (2007) Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry. Anal Chem 79:5723–5729PubMedCrossRefGoogle Scholar
  46. Zhang W, Marzilli LA, Rouse JC, Czupryn MJ (2002) Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody. Anal Biochem 311:1–9PubMedCrossRefGoogle Scholar
  47. Zhang Z, Pan H, Chen X (2009) Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom Rev 28:147–176PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Marie-Claire Janin-Bussat
    • 1
  • Jean-Marc Strub
    • 2
  • Elsa Wagner-Rousset
    • 1
  • Olivier Colas
    • 1
  • Christine Klinguer-Hamour
    • 1
  • Nathalie Corvaia
    • 1
  • Alain van Dorsselaer
    • 2
  • Alain Beck
    • 1
    Email author
  1. 1.Centre d’Immunologie Pierre Fabre (CIPF)Saint-Julien-en-GenevoisFrance
  2. 2.Laboratoire de Spectrométrie de Masse Biologique (LSMBO)Université de StrasbourgStrasbourgFrance

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