Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 412, Issue 23, Pages 5729-5741Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-020-02794-3
Keywords
Follicle-stimulating hormone; Glycoprotein; Human chorionic gonadotropin; Intact protein; Mass spectrometry; Nano-liquid chromatography
Funding
- Institut Pierre-Gilles de Gennes (Laboratoire d'excellence, Investissements d'avenir program) of PSL University [ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31]
- Conseil Regional d'ile-de-France
- DIM Analytics
- association Al Ihssan
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Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the alpha-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the alpha-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle (R), was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCG alpha glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCG alpha was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle (R). The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon (R), and 30 FSH alpha glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations.
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