Quantification of glycosylations in therapeutic antibodies based on FTIR spectroscopy

FTIR spectroscopy is a long-established analytical tool and has already been widely used to analyse protein structure. These analyses are mainly based on the region of the FTIR spectrum between 1700 and 1500 cm-1. It is also established that glycosylations are responsible for absorption in other areas of the FTIR spectrum of proteins. The most interesting area for studying saccharides is found between 1200 and 1000 cm-1, due to the vibrations of C-O bonds.


The purpose of this study is to evaluate if FTIR spectroscopy could provide qualitative and quantitative information concerning protein glycosylation. A FTIR-based approach would offer 2 key advantages:

  • An analysis that can be performed on the whole protein (without cleavage, separation steps or labelling);
  • An extremely short processing time (a quick measurement and the possibility to automate the data analysis).

 

A database of FTIR spectra of monosaccharides, glycans and glycoproteins has thus been established. In terms of qualitative analysis, we have shown that each monosaccharide and glycan has a distinct FTIR fingerprint. Furthermore, we have evidenced that the FTIR spectra of glycoproteins in the area associated with saccharides is significantly different according to the glycosylation profile. This was also demonstrated by comparing 15 therapeutic antibodies which have very similar glycosylation profiles. It thus underlines the sensitivity and the benefits of the approach i.e. to provide comparability study for biosimilar development.
In terms of quantitative analysis, we have calibrated a model to predict the global glycosylation level. Furthermore, we will attempt to build predictive model for the major glycan and monosaccharide content in antibodies.

Authors

Allison Derenne
Erik Goormaghtigh

Organisations

Université libre de Bruxelles, Faculty of Sciences, Laboratory of structure and function of biological membranes

Presenting author

Allison Derenne, Researcher, Université libre de Bruxelles
alderenn@ulb.ac.be
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