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

Laura Riccetti; Samantha Sperduti; Clara Lazzaretti; Danièle Klett; Francesco De Pascali; Elia Paradiso; Silvia Limoncella; Francesco Potì; Simonetta Tagliavini; Tommaso Trenti; Eugenio Galano; Angelo Palmese; Abhijeet Satwekar; Jessica Daolio; Alessia Nicoli; Maria Teresa Villani; Lorenzo Aguzzoli; Eric Reiter; Manuela Simoni; Livio Casarini

doi:10.3389/fendo.2019.00503

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

Front Endocrinol (Lausanne). 2019 Jul 24:10:503. doi: 10.3389/fendo.2019.00503. eCollection 2019.

Authors

Laura Riccetti¹, Samantha Sperduti¹, Clara Lazzaretti^{1

2}, Danièle Klett³, Francesco De Pascali³, Elia Paradiso^{1

2}, Silvia Limoncella¹, Francesco Potì⁴, Simonetta Tagliavini⁵, Tommaso Trenti⁵, Eugenio Galano⁶, Angelo Palmese⁶, Abhijeet Satwekar⁶, Jessica Daolio⁷, Alessia Nicoli⁷, Maria Teresa Villani⁷, Lorenzo Aguzzoli⁷, Eric Reiter³, Manuela Simoni^{1

3

8

9}, Livio Casarini^{1

8}

Affiliations

¹ Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy.
² International PhD School in Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena, Italy.
³ PRC, INRA, CNRS, IFCE, Université de Tours, Nouzilly, France.
⁴ Unit of Neurosciences, Department of Medicine and Surgery, University of Parma, Parma, Italy.
⁵ Department of Laboratory Medicine and Pathological Anatomy, Azienda USL, NOCSAE, Modena, Italy.
⁶ Analytical Development Biotech Products, Merck Serono S.p.A. (an affiliate of Merck KGaA, Darmstadt, Germany), Rome, Italy.
⁷ Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Department of Obstetrics and Gynaecology, Fertility Center, ASMN, Reggio Emilia, Italy.
⁸ Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy.
⁹ Unit of Endocrinology, Department of Medical Specialties, Azienda Ospedaliero-Universitaria, Modena, Italy.

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 × 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.

Keywords: FSH; assisted reproduction (ART); bemfola; biosimilar; glycosylation; gonal-F; ovaleap.