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

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: :

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

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Rabah Gahoual would like to thank the MRT for funding his Ph.D work. LSMIS would like to thank Beckman Coulter Inc. for lending a CESI prototype, Bruker Daltonics lending the microTOF-Q II and M. Anselme from Beckman Coulter Inc. for his help. The authors would also thanks Dr. E. Wagner-Rousset, Dr. D. Ayoub, MC. Janin-Bussat and O. Colas (Centre d’Immunologie Pierre Fabre, Saint-Julien en Genevois, France) for discussions around sample preparation and antibody LC-MS characterization.