■ Development of analytical methods for the characterization and quantitation of glycoproteins in biological samples: application to the human chorionic gonadotropin (hCG)

Description of the PhD project

Glycosylation is one of the most common post-translational modifications of proteins that affects their biological activity. Its characterization is of great interest for diagnostic and therapy. However, number and types of glycosylation sites, degrees of site occupancy and the different structures of glycans can lead to a large number of glycoforms for a given protein. To characterize them, it is necessary to develop efficient analytical methods, which constitutes a great challenge. Most of them are based on bottom-up that consists in the analysis of the protein at the glycopeptide or glycan level after its enzymatic digestion. This digestion is conventionally carried out during 16 h in solution with enzyme/substrate (E/S) ratio of about 1:50 to limit the protease autoprotolysis. The development of Immobilized Enzyme Reactors (IMERs) overcomes this limitation by immobilizing proteases (E/S ratios up to 4,000), leading to a digestion time of less than 1 h. These IMERs are also reusable and easily automated. One of the objectives of this project is to synthesize and characterize miniaturized IMERs.
The bottom-up approach allows obtaining the localization and composition of glycans on the protein, but it also leads to a loss of information on the nature of the glycans present on a given protein. This is why there is an ongoing effort to analyze glycoproteins at the intact level. Different electrophoretic and chromatographic separation methods can be considered. This second objective deals with the development of new analytical methods for the analysis of glycoproteins at the intact level.
At last, to characterize a glycoprotein in biological samples, its selective extraction has to be done thanks to the immobilization of specific antibodies on a solid support to form a so-called immunosorbent (IS). This is the third objective: synthesis of ISs, optimization of the extraction protocol and their miniaturization. The final objective will be to couple on-line the different extraction, separation, and detection steps to get fast, more reliable, and low sample consumption analysis.
This project will deal with a model protein: the human chorionic gonadotropin (hCG). It is a hormone essential for preservation of pregnancy and placenta development and composed of 2 non-covalently linked glycosylated subunits, hCGα and hCGβ. The α-subunit is common to other gonadotropin hormones, while the β-subunit is specific of hCG. hCGα has 2 N-glycosylation sites and hCGβ 2 N- and 4 O-glycosylation sites. With 8 potential glycans, hCG is a highly glycosylated protein. It was observed that different placental sources produce different hCG isoforms and that the relative proportion of different glycoforms, the biological activity, and the half-life of hCG vary during pregnancy. This is why one can expect that some hCG glycoforms could be maternal biomarkers to identify "at-risk" pregnancies. This is why it is so relevant to characterize them.


Analytical sciences, Separation methods, Liquid chromatography, Capillary electrophoresis, Mass spectrometry, Protein analysis, Glycoproteins, human chorionic gonadotropin, immunoextraction, sample handling

Research unit

UMR8231 Chemistry, Biology & Innovation

Description of the research Unit/subunit

Research objectives of analytical chemists are strongly dependent on asks from the society. In many areas (health, food, environment), the development of rapid methods, with low costs, easy to use, environmentally friendly that can be handled low amount of sample (drop of blood or less) is required. There is also a need for solving very complex samples (proteomics, natural or petroleum products) and for trace analysis in complex samples.
In order to answer to these demands, the Analytical and Bioanalytical Sciences and Miniaturization Laboratory headed by Prof. V. Pichon has focused its research on the development of multidimensional chromatographic modes for solving complex samples and of highly selective extraction sorbents for trace analysis. Miniaturization has been also considered for the rapid analysis of low sample volumes.
The team at ESCPI Paris - PSL University belongs to the UMR CBI 8231 and is affiliated to CNRS (https://www.lsabm.espci.fr/spip.php?rubrique8). It combines strong competences in chromatography, sample treatment, trace/ultra-trace analyses, hyphenation with mass spectrometry, capillary electrophoresis, miniaturization, chemometric, and method validation.

Name of the supervisor
Nathalie Delaunay (Nathalie.Delaunay@espci.fr)

Name of the co-supervisor
Valérie Pichon (Valerie.Pichon@espci.fr)

3i Aspects of the proposal

This project is connected to the KET biotechnology. Indeed, it deals with proteomics and targets the development of new analytical methods for the characterization and analysis of glycoproteins in biological samples or drug formulations that are obviously simpler samples. The glycoproteins can be either biomarkers or new biotherapeutic drugs, such as the monoclonal antibodies. This is why it can contribute to new growth and competitiveness in biotech or pharmaceutical industry, and also to improvement of Human health through the development of new diagnostic tests. Depending on the nature of the studied glycoprotein, different industrial partners could be considered. Concerning the first targeted glycoprotein, hCG, some contacts with pharmaceutical companies have been already made. For each new analytical tool or developed method, patent will be considered, and consequently its use thanks to the creation of a start-up company. The strong expertise in the creation of start-ups of the head of UMR 8231 CBI, Professor J. Bibette, will be very useful at this stage.

This project crosses the frontier between Chemistry and Biology. Indeed, it is based on the collaboration between 2 Laboratories:

  • Pathophysiology & Pharmacotoxicology of the Human Placenta, UMR-S1139, Inserm/Paris Descartes, headed by Dr Thierry Fournier
  • Analytical and Bioanalytical Sciences and Miniaturization Laboratory, UMR CBI 8231, ESCPI Paris - PSL University/CNRS, headed by Professor Valérie Pichon.

The first team has a unique scientific and technological expertise in Biology to study placenta, especially human trophoblast differentiation based on the pertinence of their in vitro models and their knowledge of the uteroplacental interface, which will facilitate this transversal project focusing on the characterization and actions on placental targets of soluble factors of trophoblast origin such as hCG glycoforms.

The second team combines strong competences in Analytical Chemistry, especially in chromatography, sample treatment, trace/ultra-trace analyses, hyphenation with mass spectrometry, capillary electrophoresis, miniaturization, chemometric, and method validation. This team is particularly recognized at the international level for their activity that concerns the development of solid-phase extraction methods and of selective sorbents such as immunosorbents.
This project combines their respective expertise to characterize the glycan structures of the key pregnancy hormone (hCG) to propose a hCG glycoforms signature of physiological and pathological pregnancies. Indeed, the consortium consists in:

1-Cell biologists and pathophysiologists specialized in human placentation, trophoblast differentiation and placental endocrinology
2- Experts in structural analysis of complex glycosidic molecules and in the study of the glycosylation of glycoproteins.

This project dealing with the study of hCG glycoforms started some years ago and two international collaborations took place with the University of Geneva and the Vrije Universiteit of Brussel. Two publications that we co-authored are the proof of this international mobility of our PhD students:

An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact levels, J. Camperi, A. Combes, J. Guibourdenche, D. Guillarme, V. Pichon, T. Fournier, N. Delaunay, J. Pharm. Biomed. Anal. 161 (2018) 35-44.
First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level, J. Camperi, B. De Cock, V. Pichon, A. Combes, J. Guibourdenche, T. Fournier, Y. Vander Heyden, D. Mangelings, N. Delaunay, Talanta 193 (2019) 77-86.
Indeed, Julien Camperi, a PhD student at ESPCI Paris – PSL University did some experiments with Davy Guillarme in the University of Geneva and Bart de Cock, a PhD student at the Vrije Universiteit Brussel did some experiments in ESPCI Paris – PSL University. This kind of international collaboration will be still used for the rest of the project.

Expected Profile of the candidate

The candidate should have a high-level background in Chemistry or Biochemistry and, more especially in Analytical Chemistry. Knowledge and training in the Separation Science field would be appreciated, especially in chromatographic and electrokinetic methods, sample treatment, and chemometrics.
A high interest for academic research and carrying out laboratory experiments, ability to write articles in English and make pedagogic and dynamic oral presentations of its work are required.
Autonomy, teamwork skills, open-mindedness, seriousness, motivation, and dynamism are also expected.

Important dates

Call for applications : from February 1st to March 31st 2019
Eligibility check results : Mid April
3i Committee evaluation results : Mid May
Interviews from the shortlisted candidates with the Selection Committee : Late June-Early July
Final results : Mid July