Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques

Abstract

The European Medicines Agency received recently the first marketing authorization application for a biosimilar monoclonal antibody (mAb) and adopted the final guidelines on biosimilar mAbs and Fc-fusion proteins. The agency requires high similarity between biosimilar and reference products for approval. Specifically, the amino acid sequences must be identical. The glycosylation pattern of the antibody is also often considered to be a very important quality attribute due to its strong effect on quality, safety, immunogenicity, pharmacokinetics and potency. Here, we describe a case study of cetuximab, which has been marketed since 2004. Biosimilar versions of the product are now in the pipelines of numerous therapeutic antibody biosimilar developers. We applied a combination of intact, middle-down, middle-up and bottom-up electrospray ionization and matrix assisted laser desorption ionization mass spectrometry techniques to characterize the amino acid sequence and major post-translational modifications of the marketed cetuximab product, with special emphasis on glycosylation. Our results revealed a sequence error in the reported sequence of the light chain in databases and in publications, thus highlighting the potency of mass spectrometry to establish correct antibody sequences. We were also able to achieve a comprehensive identification of cetuximab’s glycoforms and glycosylation profile assessment on both Fab and Fc domains. Taken together, the reported approaches and data form a solid framework for the comparability of antibodies and their biosimilar candidates that could be further applied to routine structural assessments of these and other antibody-based products.

Keywords: :

• cetuximab
• sequence
• mass spectrometry
• glycosylation
• biosimilar

Submitted

05/08/2013

Revised

06/13/2013

Accepted

06/15/2013

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Supplemental Materials

All supplemental materials may be found here: www.landesbioscience.com/journals/mabs/article/24323.

Acknowledgments

We thank Stephanie Kaspar and Peter Brechlin for help with the bottom-up measurements and Ulrike Schweiger-Hufnagel (all Bruker-Daltonik, Bremen) for critically reading the manuscript. We further thank Jason Rouse’s team (Pfizer, Andover, MA) and Fredrik Olssen (Genovis, Lund) for many helpful discussions on subunit analysis of antibodies.