10.3389/fendo.2019.00503.s007 Laura Riccetti Laura Riccetti Samantha Sperduti Samantha Sperduti Clara Lazzaretti Clara Lazzaretti Danièle Klett Danièle Klett Francesco De Pascali Francesco De Pascali Elia Paradiso Elia Paradiso Silvia Limoncella Silvia Limoncella Francesco Potì Francesco Potì Simonetta Tagliavini Simonetta Tagliavini Tommaso Trenti Tommaso Trenti Eugenio Galano Eugenio Galano Angelo Palmese Angelo Palmese Abhijeet Satwekar Abhijeet Satwekar Jessica Daolio Jessica Daolio Alessia Nicoli Alessia Nicoli Maria Teresa Villani Maria Teresa Villani Lorenzo Aguzzoli Lorenzo Aguzzoli Eric Reiter Eric Reiter Manuela Simoni Manuela Simoni Livio Casarini Livio Casarini Table_3_Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars.docx Frontiers 2019 FSH biosimilar gonal-F bemfola ovaleap glycosylation assisted reproduction (ART) 2019-07-24 04:06:56 Dataset https://frontiersin.figshare.com/articles/dataset/Table_3_Glycosylation_Pattern_and_in_vitro_Bioactivity_of_Reference_Follitropin_alfa_and_Biosimilars_docx/8988536 <p>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<sup>−3</sup>-1 × 10<sup>3</sup> 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<sup>2+</sup> 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<sub>50</sub> range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC<sub>50</sub> range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca<sup>2+</sup> 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<sub>50</sub>s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca<sup>2+</sup> increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.</p>