Development of new analytical methods for the analysis at the intact level of glycoforms of hCG and other gonadotropins by nano liquid chromatography hyphenated to high resolution mass spectrometry ; Développement de nouvelles méthodes analytiques pour l'analyse au niveau intact de glycoformes d'hCG et d'autres gonadotrophines par nano chromatographie liquide couplée à la spectrométrie de masse haute résolution

Glycosylation is the attachment of glycans to a protein, so-called a glycoprotein. It is one of the most common post-translational modifications and it impacts protein activity and stability. Human chorionic gonadotropin (hCG) is a highly glycosylated protein, composed of two subunits, hCGα and hCGβ, having in total four N- and four O-glycosylation sites, leading to a very high number of glycoforms. However, the glycosylation state of this hormone essential for pregnancy was associated with some pregnancy pathologies. For this reason, it is necessary to be able to analyze the hCG glycoforms present in biological samples. In this work, new analytical methods for the analysis at the intact level of hCG glycoforms by nano liquid chromatography (nanoLC) hyphenated with mass spectrometry (MS) were developed. The miniaturization of the separation allowed an increase in sensitivity and its coupling with a high mass accuracy and high mass resolution analyzer, an Orbitrap, allowed the detection and semi-quantification of several α-subunit glycoforms in hCG-based drugs, but also of another gonadotropin sharing the same α-subunit, the follicle-stimulating hormone (FSH). The optimization of both a preconcentration step in the nanoLC-MS set-up and of the mobile phase gradient and nature allowed the detection of hCGβ glycoforms for the first time and also of a higher number of hCGα glycoforms. Next this analytical method using either formic acid or trifluoroacetic acid as mobile phase additive was evaluated for the detection of the α- and β-subunits of different recombinant or urinary gonadotropin-based drugs. Finally, with the aim to determine hCG glycoforms in serum of pregnant women, i.e complex biological samples, rather than in concentrated and fairly pure gonadotropin-based drugs, it was necessary to develop a selective extraction step of hCG glycoforms before their analysis in nanoLC-MS. This is why, immunosorbents were synthesized by immobilizing two different anti-hCG antibodies on a solid sorbent. The primary ...