First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level

Julien Camperi; Bart De Cock; Valérie Pichon; Audrey Combes; Jean Guibourdenche; Thierry Fournier; Yvan Vander Heyden; Debby Mangelings; Nathalie Delaunay

doi:10.1016/j.talanta.2018.09.095

First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level

Talanta. 2019 Feb 1:193:77-86. doi: 10.1016/j.talanta.2018.09.095. Epub 2018 Sep 26.

Authors

Julien Camperi¹, Bart De Cock², Valérie Pichon³, Audrey Combes¹, Jean Guibourdenche⁴, Thierry Fournier⁵, Yvan Vander Heyden⁶, Debby Mangelings⁶, Nathalie Delaunay⁷

Affiliations

¹ Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI 8231 CNRS - ESPCI Paris, PSL University, Paris, France.
² Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI 8231 CNRS - ESPCI Paris, PSL University, Paris, France; Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
³ Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI 8231 CNRS - ESPCI Paris, PSL University, Paris, France; Sorbonne Université, Paris, France.
⁴ Laboratory of Hormonology, CHU Cochin AP-HP, Paris, France.
⁵ Laboratory of Physiopathology and PharmacoToxicology of the Human Placenta, UMR-S 1139 Inserm - University Paris Descartes, Sorbonne Paris Cité, Paris, France.
⁶ Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
⁷ Laboratory of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI 8231 CNRS - ESPCI Paris, PSL University, Paris, France. Electronic address: nathalie.delaunay@espci.fr.

PMID: 30368301
DOI: 10.1016/j.talanta.2018.09.095

Abstract

In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a hetero-dimeric glycoprotein, specific to the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize. First, Capillary Gel Electrophoresis (CGE) was used to separate the isoforms of two hCG-based drugs: Ovitrelle® (recombinant r-hCG) and Pregnyl (hCG isolated from the urine of pregnant women, u-hCG). As expected, CGE led to a better resolution than SDS-PAGE and confirmed the large heterogeneity of hCG. Different CGE profiles were obtained for the two hCG-based drugs, varying in number of peaks, migration times, and peak intensities, thus demonstrating that the drugs contain isoforms, different in nature and proportion. This result was confirmed by Capillary IsoElectrophoretic Focusing (CIEF). The pI ranges of the hCG isoforms were found between 3.4 and 4.7, and 4.5 and 5.2 for r-hCG and u-hCG, respectively. This information was further used to develop the separation of the hCG isoforms by Capillary Zone Electrophoresis (CZE). The pH, the nature, and the concentration of the background electrolyte as well as the nature and the content of its organic modifier were optimized. The use of a coated capillary to avoid protein adsorption was also evaluated. The final CZE-UV method allowed distinguishing at least 6 peaks, corresponding to different hCG isoforms. To significantly improve the level of information obtained, the CZE instrument was then coupled by means of an electrospray ionization source to a triple quadrupole (TQ) mass spectrometer. Two detection strategies were used, one focusing on the lower m/z values (100-1000) in order to identify some sugar residues as diagnostic ions to confirm the presence of glycan chains, and the second focusing on the higher m/z values (1000-2000), corresponding to the multiple charged intact protein isoforms. For both approaches, the fragmentor and capillary voltage values were optimized. The composition and the flow-rate of the sheath liquid were then optimized for the strategy focusing on the higher m/z values in order to both increase the charge state of the ionized intact isoforms and the signal-to-noise ratio. The final method was used to compare the two hCG-based drugs, demonstrating the potential of the developed CZE-MS method for isoforms fingerprinting.

Keywords: Capillary electrophoresis; Glycosylation; Human Chorionic Gonadotropin; Intact protein; Mass spectrometry.