Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

Short Communication

Abstract

For the first time, the characterization of intact 150-kDa monoclonal antibodies (MAbs) using a commercially available three-dimensional ion-trap mass spectrometer (IT-MS) is reported. The IT-MS analysis was performed on-line with reversed-phase high performance liquid chromatography (RP-HPLC) on a POROS column using a nontraditional solvent system of acetonitrile, isopropanol, ethanol, and water in formic acid. The operating parameters of the IT-MS were optimized by extending the mass range to m/z 4000 and elevating the tube lens offset voltage value to around −100 V. Mass accuracy better than 300 ppm (±40 Da) has been routinely achieved for these macromolecules. Multiple peaks 162 Da apart due to the hexose variants of the monoclonal IgG antibodies were partially resolved in mass spectra. Several commercial and chimeric antibodies have been investigated in this study.

References

  1. 1.
    Clark, M. R. Antibody Humanization: A Case of the “Emperor’s new clothes”. Immunol. Today. 2000, 21(8), 397–420.CrossRefGoogle Scholar
  2. 2.
    Lewis, D. A.; Guzzetta, A. W.; Handcock, W. S. Characterization of Humanized Anti-Tac, an Antibody Directed Against the Interleukin 2 Receptor, Using Electrospray Ionization Mass Spectrometry by Direct Infusion LC/MS and MS/MS. Anal. Chem. 1994, 66, 585–595.CrossRefGoogle Scholar
  3. 3.
    Fenn, J. B.; Mann, M.; Wang, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64–71.CrossRefGoogle Scholar
  4. 4.
    Wilm, M. S.; Mann, M. Analytical Properties of the Nanoelectrospray Ion Source. Anal. Chem. 1996, 68, 1–8.CrossRefGoogle Scholar
  5. 5.
    Le, J. C.; Hui, J.; Haniu, M.; Katta, V.; Rhode, M. F. J. Development and Evaluation of On-Line Nanoliter Flow Analysis of Proteins Digests by Pneumatic-Splitter Electrospray Liquid Chromatography Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 703–712.CrossRefGoogle Scholar
  6. 6.
    Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLafferty, F. Attomole-Sensitive Electrospray Source for Large-Molecule Mass Spectrometry. Anal. Chem. 1995, 67, 3802–3805.CrossRefGoogle Scholar
  7. 7.
    Alexander, J. A.; Hughes, E. D. Monitoring of IgG Antibody Thermal Stability by Micellar Electrokinetic Capillary Chromatography and Matrix-Assisted Laser Desorption/Ionnization Mass Spectrometry. Anal. Chem. 1995, 67, 3626–3632.CrossRefGoogle Scholar
  8. 8.
    Downard, K. M. Contributions of Mass Spectrometry to Structural Immunology. J. Mass Spectrom. 2000, 35, 493–503.CrossRefGoogle Scholar
  9. 9.
    Verentchikov, A. N.; Ens, W.; Standing, K. G. Reflecting Time-of-Flight Mass Spectrometer with an Electrospray Ion Source and Orthogonal Extraction. Anal. Chem. 1995, 66, 126–133.CrossRefGoogle Scholar
  10. 10.
    Tito, M. A.; Tars, K.; Valegard, K.; Hajdu, J.; Robinson, C. V. Probing Molecular Interaction in Intact Antibody: Antigen Complexes, an Electrospray Time-of Flight Mass Spectrometry Approach. Biophys. J. 2001, 81, 3503–3509.CrossRefGoogle Scholar
  11. 11.
    Robert, G. D.; Johnson, W. P.; Burman, S.; Anumula, K. R.; Carr, S. A. An integrated Strategy for Structure Characterization of the Protein and Carbohydrate Components of Monoclonal Antibodies: Application to Anti-Respiratory Syncytial Virus MAB. Anal. Chem. 1995, 67, 3613–3625.CrossRefGoogle Scholar
  12. 12.
    Bennett, K. L.; Smith, S. V.; Lambrecht, R. M.; Truscott, R. J. W.; Sheil, M. M. Rapid Characterization of Chemically-Modified Proteins by Electrospray Mass Spectrometry. Bioconj. Chem. 1996, 7, 16–22.CrossRefGoogle Scholar
  13. 13.
    Masuda, K.; Yamaguchi, Y.; Kato, K.; Takahashi, N.; Shimada, I.; Arata, Y. Pairing of Oligosaccharides in the Fc Region of Immunoglobulin G. FEBS Lett. 2000, 473, 349–357.CrossRefGoogle Scholar
  14. 14.
    Le, J. C.; Bondarenko, P. Trap for MAbs: Novel Approach to Characterize Intact Antibodies and Degradation Products Using RP-HPLC and Ion-Trap Mass Spectrometry. Proceedings of the 52nd ASMS Conference; Nashville, TN, May 2004.Google Scholar
  15. 15.
    Dillon, T. M.; Speed Ricci, M.; Rehder, D.; Pipes, G.; Zhang, Y.; Stackhouse, N.; Huinker, A.; Treuheit, M. J.; Bondarenko, P. V. Rapid LC/MS Method for Monitoring and Characterizing Intact Therapeutic Monoclonal Antibodies. Proceedings of the 52nd ASMS Conference; Nashville, TN, May, 2004.Google Scholar
  16. 16.
    Dillon, T. M.; Bondarenko, P. V.; Speed Ricci, M. Development of an Analytical Reversed-Phase High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry Method for Characterization of Recombinant Antibodies. J. Chromatogr. A 2004, 1053, 299–305.Google Scholar
  17. 17.
    Eris, T.; Liu, J. Reversed Phase HPLC/MS Method for Analysis of Post-Translational Modification of Monoclonal Antibody in Cell Culture Media. Proceedings of the 50th ASMS Conference; Orlando, FL, June, 2002.Google Scholar
  18. 18.
    Battersby, J. E.; Snedecor, B.; Chen, C.; Champion, K. M.; Riddle, L.; Vanderlaan, M. Affinity Reversed Phase Liquid Chromatography Assay to Quantitate Recombinant Antibodies and Antibody Fragments in Fermentation Broth. J. Chromatogr. A 2000, 972, 61–76.Google Scholar
  19. 19.
    Apffel, A.; Fischer, S.; Goldberg, G.; Goodley, P. C.; Kuhlmann, F. E. Enhanced Sensitivity for Peptide Mapping with Electrospray Liquid Chromatography-Mass Spectrometry in the Presence of Signal Suppression Due to Trifluoroacetic Acid-Containing Mobile Phases. J. Chromatogr. A 1995, 712, 177–190.CrossRefGoogle Scholar
  20. 20.
    Medzihradszky, K. F.; Maltby, D. A.; Hall, S. C.; Settineri, C. A.; Burlingame, A. L. Characterization of Protein O-Glycosylation by Reversed-Phase Microbore Liquid Chromatography/Electrospray Mass Spectrometry, Complementary Mobile Phases, and Sequential Exoglycosidase Digestion. J. Am. Soc. Mass Spectrom. 1994, 5, 350–358.CrossRefGoogle Scholar
  21. 21.
    Paul, W.; Steinwedel, H. German Pattent 944900; 1956. U.S. Patent 2939 952; 1960.Google Scholar
  22. 22.
    Paul, W. Electromagnetic Traps for Charged and Neutral Particles (Nobel lecture). Angew. Chem. 1990, 29, 739–748.CrossRefGoogle Scholar
  23. 23.
    March, R. E. An Introduction to Quadrupole Ion Trap Mass Spectrometry. J. Mass Spectrom. 1997, 32, 351–369.CrossRefGoogle Scholar
  24. 24.
    Stafford, G. C. Jr.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Recent Improvements in Analytical Application of Advanced Ion-Trap Technology. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85–98.CrossRefGoogle Scholar
  25. 25.
    Mcluckey, S. A.; Van Berkel, G. J.; Goeringer, D. E.; Glish, G. L. Ion Trap MS of Externally Generated Ions. Anal. Chem. 1994, 66, 689–696.CrossRefGoogle Scholar
  26. 26.
    Fountain, S. T.; Lee, H.; Lubman, D. M. Mass-Selective TOF-MS with Ion-Trap Storage. Rapid Commun. Mass Spectrom. 1994, 68, 487–494.CrossRefGoogle Scholar
  27. 27.
    Cai, Y.; Peng, W. P.; Chang, H. C. Ion Trap Mass Spectrometry of Fluorescently Labeled Nanoparticles. Anal. Chem. 2003, 75, 1805–1811.CrossRefGoogle Scholar
  28. 28.
    Guan, S.; Marshall, A. G. Equilibrium Space Charge Distribution in a Quadrupole Ion Trap. J. Am. Soc. Mass Spectrom. 1994, 5, 64–71.CrossRefGoogle Scholar
  29. 29.
    Harris, R. J.; Kabakoff, B.; Macchi, F. D.; Shen, F. J.; Kwong, M. Y.; Andya, J. D.; Shire, S. J.; Bjork, N.; Totpal, K.; Chen, A. B. Identification of Multiple Sources of Charge Heterogeneity in a Recombinant Antibody. J. Chromatogr. B Biomed. Sci. Appl. 2001, 752, 233–245.CrossRefGoogle Scholar
  30. 30.
    Jones, M. D.; Hunt, J.; Liu, J. L.; Patterson, S. D.; Kohno, T.; Lu, H. S. Determination of Tumor Necrosis Factor Binding Protein Disulfide Structure: Deviation of the Fourth Domain Structure from the TNFR/NGFR Family Cysteine-Rich Region Signature. Biochemistry 1997, 36, 14914–14923.CrossRefGoogle Scholar
  31. 31.
    Mylchreest, I. C.; Hail, M. E.; Herron, J. R. Method and Apparatus for Focusing Ions in Viscous Flow Jet Expansion Region of an Electrospray Apparatus. Thermo Finnigan U.S. Patent 5157260; 1992.Google Scholar
  32. 32.
    Rockwood, A. L.; Busman, M.; Smith, R. D. Coulombic Effects in the Dissociation of Large Highly Charged Ions. Int. J. Mass Spectrom. Ion Processes 1991, 111, 103–129.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2004

Authors and Affiliations

  1. 1.Department of Pharmaceutics and Drug DeliveryAmgen Inc.Thousand OaksUSA

Personalised recommendations