An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level.

For the first time, the human Chorionic Gonadotropin (hCG) hormone at the intact level was characterized by reversed phase liquid chromatography (RPLC) coupled with high resolution mass spectrometry (HRMS). This heterodimeric protein is specific to human pregnancy, consists in an α and a β subunit, so-called hCGα and hCGβ, respectively, and has 8 glycosylation sites leading to a high number of isoforms. First, the LC method was optimized to separate the largest number of isoforms and also to facilitate the MS ionization process and data treatment. The initial mobile phase composition, slope of the gradient, and column temperature were appropriately selected to maximize the number of separated isoforms. Moreover, the MS detection parameters were adjusted to i) promote the efficient transfer of the heaviest ions, ii) avoid or limit the fragmentation of the ions and iii) improve the sensitivity. The repeatability of the final method in terms of retention times and peak areas was assessed. The method was next used to characterize two hCG-based drugs: Ovitrelle® (a recombinant hCG, r-hCG) and Pregnyl® (hCG isolated from urine of pregnant women, u-hCG). After the deconvolution step, the analytical method did not allow to observe the isoforms of the hCGβ. This may be due to its dramatic higher heterogeneity induced by its 6 glycosylation sites and a lack of ionization in the MS source. Nevertheless, the results revealed the presence of more than 30 hCGα isoforms, which differ by their number and their nature in the two drugs. Then, the molecular weights of the N-glycans already described in the literature for hCG were compiled in a database to identify the hCGα glycoforms by mass matching. This strategy was successfully applied for the identification of five glycoforms for both r-hCG and u-hCG. This work demonstrates for the first time the potential of RPLC-HRMS for the identification of the intact hCGα glycoforms.