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MAbs. 2013 May-Jun;5(3):479-90. doi: 10.4161/mabs.23995. Epub 2013 Apr 5.

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry.

Author information

1
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS); UDS-CNRS UMR 7140; Université de Strasbourg; Strasbourg, France.
2
Beckman Coulter Inc.; Brea, CA, USA.
3
Plateforme Protéomique; Université de Strasbourg; Institut de Biologie Moléculaire et Cellulaire; Strasbourg, France.
4
Centre d'immunologie Pierre Fabre; Saint-Julien-en-Genevois, France.

Abstract

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) - tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.

KEYWORDS:

CESI-MS/MS; N-linked glycosylation; mass spectrometry; microheterogeneity; microvariant; monoclonal antibody; peptide mapping; posttranslational modifications; sheathless capillary electrophoresis

PMID:
23563524
PMCID:
PMC4169039
DOI:
10.4161/mabs.23995
[Indexed for MEDLINE]
Free PMC Article

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