%0 Generic %A Riccetti, Laura %A Sperduti, Samantha %A Lazzaretti, Clara %A Klett, Danièle %A De Pascali, Francesco %A Paradiso, Elia %A Limoncella, Silvia %A Potì, Francesco %A Tagliavini, Simonetta %A Trenti, Tommaso %A Galano, Eugenio %A Palmese, Angelo %A Satwekar, Abhijeet %A Daolio, Jessica %A Nicoli, Alessia %A Villani, Maria Teresa %A Aguzzoli, Lorenzo %A Reiter, Eric %A Simoni, Manuela %A Casarini, Livio %D 2019 %T Table_3_Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars.docx %U https://frontiersin.figshare.com/articles/dataset/Table_3_Glycosylation_Pattern_and_in_vitro_Bioactivity_of_Reference_Follitropin_alfa_and_Biosimilars_docx/8988536 %R 10.3389/fendo.2019.00503.s007 %2 https://frontiersin.figshare.com/ndownloader/files/16475519 %K FSH %K biosimilar %K gonal-F %K bemfola %K ovaleap %K glycosylation %K assisted reproduction (ART) %X

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 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ 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 EC50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC50 range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ 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 EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

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