Analytical and Bioanalytical Chemistry

, Volume 407, Issue 1, pp 355–366 | Cite as

Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests

  • Gerd Vanhoenacker
  • Isabel Vandenheede
  • Frank David
  • Pat Sandra
  • Koen SandraEmail author
Research Paper
Part of the following topical collections:
  1. Multidimensional Chromatography


Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and 2D retention time opening interesting perspectives for use in QA/QC testing.


Comprehensive two-dimensional liquid chromatography (LC×LC) UV detection Q-TOF MS Monoclonal antibodies Biosimilars Peptide mapping 



The authors would like to acknowledge Agilent Technologies for contributing to this study.

Supplementary material

216_2014_8299_MOESM1_ESM.pdf (4.1 mb)
ESM 1 (PDF 4154 kb)


  1. 1.
    Walsh G (2010) Biopharmaceutical benchmarks. Nat Biotechnol 28:917–924CrossRefGoogle Scholar
  2. 2.
    Berkowitz SA, Engen JR, Mazzeo JR, Jones GB (2012) Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov 11:527–540CrossRefGoogle Scholar
  3. 3.
    Elvin JG, Couston RG, van der Walle CF (2013) Therapeutic antibodies: market considerations, disease targets and bioprocessing. Int J Pharm 440:83–98CrossRefGoogle Scholar
  4. 4.
    Sandra K, Vandenheede I, Sandra P (2014) Modern chromatographic and mass spectrometric techniques for protein biopharmaceutical characterization. J Chromatogr A 1335:81–103CrossRefGoogle Scholar
  5. 5.
    Sandra P, Vanhoenacker G (2007) Elevated temperature-extended column length conventional liquid chromatography to increase peak capacity for the analysis of tryptic digests. J Sep Sci 30:241–244CrossRefGoogle Scholar
  6. 6.
    Sandra K, Moshir M, D’hondt F, Verleysen K, Kas K, Sandra P (2008) Highly efficient peptide separations in proteomics. Part 1. Unidimensional high performance liquid chromatography. J Chromatogr B 877:1019–1039CrossRefGoogle Scholar
  7. 7.
    Davis JM, Giddings JC (1983) Statistical theory of component overlap in multicomponent chromatograms. Anal Chem 55:418–424CrossRefGoogle Scholar
  8. 8.
    Giddings JC (1995) Sample dimensionality: a predictor of order–disorder in component peak distribution in multidimensional separation. J Chromatogr A 703:3–15CrossRefGoogle Scholar
  9. 9.
    Francois I, Sandra K, Sandra P (2009) Comprehensive liquid chromatography: fundamental aspects and practical considerations—a review. Anal Chim Acta 64:14–31CrossRefGoogle Scholar
  10. 10.
    Guiochon G, Marchetti N, Mriziq K, Shalliker RA (2008) J Chromatogr A 1189:109–168CrossRefGoogle Scholar
  11. 11.
    François I, Cabooter D, Sandra K, Lynen F, Desmet G, Sandra P (2009) Tryptic digest analysis by comprehensive reversed phase × two reversed phase liquid chromatography (RP-LC × 2 RP-LC) at different pH’s. J Sep Sci 32:1137–1144CrossRefGoogle Scholar
  12. 12.
    Jandera P (2011) Multidimensional liquid chromatography: theoretical considerations. In: Mondello L (ed) Comprehensive chromatography in combination with mass spectrometry. WileyGoogle Scholar
  13. 13.
    François I, Sandra K, Sandra P (2011) History, evolution, and optimization aspects of comprehensive two-dimensional liquid chromatography. In: Mondello L (ed) Comprehensive chromatography in combination with mass spectrometry. WileyGoogle Scholar
  14. 14.
    Mondello L, Donato P, Cacciola F, Fanali C, Dugo P (2010) RP-LC × RP-LC analysis of a tryptic digest using a combination of totally porous and partially porous stationary phases. J Sep Sci 33:1454–1461CrossRefGoogle Scholar
  15. 15.
    Donato P, Cacciola F, Sommella E, Fanali C, Dugo L, Dachà M, Campiglia P, Novellino E, Dugo P, Mondello L (2011) Online comprehensive RPLC×RPLC with mass spectrometry detection for the analysis of proteome samples. Anal Chem 83:2485–2491CrossRefGoogle Scholar
  16. 16.
    D’Attoma A, Grivel C, Heinisch S (2012) On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: orthogonality and practical peak capacity considerations. J Chromatogr A 1262:148–159CrossRefGoogle Scholar
  17. 17.
    D’Attoma A, Heinisch S (2013) On-line comprehensive two dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part II: application to the separation of peptides. J Chromatogr A 1306:27–36CrossRefGoogle Scholar
  18. 18.
    Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783–792CrossRefGoogle Scholar
  19. 19.
    Sandra K, Moshir M, D’hondt F, Tuytten R, Verleysen K, Kas K, Francois I, Sandra P (2009) Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques. J Chromatogr B 877:1019–1039CrossRefGoogle Scholar
  20. 20.
    Gilar M, Olivova P, Daly AE, Gebler JC (2005) Orthogonality of separation in two-dimensional liquid chromatography. Anal Chem 77:6426–6434CrossRefGoogle Scholar
  21. 21.
    Jandera P (2012) Programmed elution in comprehensive two-dimensional liquid chromatography. J Chromatogr A 1255:112–129CrossRefGoogle Scholar
  22. 22.
    Garcia MC (2005) The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography-electrospray mass spectrometry. J Chromatogr B 825:111–123CrossRefGoogle Scholar
  23. 23.
    Vanhoenacker G, Sandra K, Vandenheede I, David F, Sandra P, Huber U, Naegele E (2013) Analysis of monoclonal antibody digests with the Agilent 1290 Infinity 2D-LC Solution. Agilent Technologies Application Note 5991-2880ENGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Gerd Vanhoenacker
    • 1
  • Isabel Vandenheede
    • 1
  • Frank David
    • 1
  • Pat Sandra
    • 1
  • Koen Sandra
    • 1
    Email author
  1. 1.Research Institute for Chromatography & MetablysKortrijkBelgium

Personalised recommendations