Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The alpha-subunit structure, hCG alpha, has 2 N-glycosylation sites, while the beta subunit, hCG beta, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle (R), as standard. It allowed the detection of about 30 hCG alpha glycoforms, but didn't allow the detection of hCG beta glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 mu l, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCG alpha and hCG beta glycoforms, which allowed for the first time the detection of 33 hCG beta glycoforms at intact level. In addition, a higher number of hCG alpha glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% ( n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% ( n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations. (c) 2021 Elsevier B.V. All rights reserved.

Automated summary

The hCG protein, consisting of alpha and beta subunits with various glycoforms, was previously analyzed for glycoforms at the intact level. A modified method now allows for improved separation and detection of hCG alpha and beta glycoforms, with a higher number of glycoforms detected. The new method shows potential for rapid quality control of glycoprotein-based pharmaceutical preparations.