10.3389/fendo.2019.00503.s006
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_2_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_2_Glycosylation_Pattern_and_in_vitro_Bioactivity_of_Reference_Follitropin_alfa_and_Biosimilars_docx/8988533
<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>