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 Sandra
Research Paper
Part of the following topical collections:
  1. Multidimensional Chromatography

Abstract

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.

Keywords

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

Supplementary material

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

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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
  1. 1.Research Institute for Chromatography & MetablysKortrijkBelgium

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