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Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars

Abstract : Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f ®)-with biosimilar preparations (Bemfola ® and Ovaleap ®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f ® or biosimilar (1 × 10 −3-1 × 10 3 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca 2+ increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8-and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC 50 range = 12 ± 0.9-24 ± 1.7 ng/ml; β-arrestin 2 EC 50 range = 140 ± 14.1-313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca 2+ increased upon cell treatment by 4 µg/ml Gonal-f ® , while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC 50 s were
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Laura Riccetti, Samantha Sperduti, Clara Lazzaretti, Danièle Klett, Francesco de Pascali, et al.. Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars. Frontiers in Endocrinology, Frontiers, 2019, 10, pp.503. ⟨10.3389/fendo.2019.00503⟩. ⟨hal-02501193⟩



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