TY - JOUR
T1 - Analytical Investigation of the Profile of Human Chorionic Gonadotropin in Highly Purified Human Menopausal Gonadotrophin Preparations
AU - Capolupo, Angela
AU - Petrocchi, Sofia
AU - Melchiorre, Maura
AU - Jonas, Kim
AU - D'Hooghe, Thomas
AU - Hanyaloglu, Aylin
AU - Sunkara, Sesh
AU - Palmese, Angelo
AU - Ozgumus, Beste
AU - Amoresano, Angela
AU - Angiuoni, Gabriella
AU - Montenegro, Susana
AU - Simone, Patrizia
AU - Lispi, Monica
N1 - Publisher Copyright:
© 2024 by the authors.
PY - 2024/8/29
Y1 - 2024/8/29
N2 - Highly purified human menopausal gonadotropin (HP-hMG [Menopur
®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur
® and two of Menogon
® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid
® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel
® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid
®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.
AB - Highly purified human menopausal gonadotropin (HP-hMG [Menopur
®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur
® and two of Menogon
® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid
® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel
® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid
®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.
KW - Female
KW - Humans
KW - Chorionic Gonadotropin/analysis
KW - Chromatography, Liquid/methods
KW - Follicle Stimulating Hormone/urine
KW - Glycopeptides/analysis
KW - Glycosylation
KW - Luteinizing Hormone/urine
KW - Menotropins/urine
KW - Oxidation-Reduction
KW - Polysaccharides/analysis
KW - Tandem Mass Spectrometry/methods
KW - Menopause
KW - Postmenopause
UR - http://www.scopus.com/inward/record.url?scp=85204105762&partnerID=8YFLogxK
U2 - 10.3390/ijms25179405
DO - 10.3390/ijms25179405
M3 - Article
C2 - 39273352
SN - 1661-6596
VL - 25
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 17
M1 - 9405
ER -