Table_6_Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars.docx

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.

重组促卵泡激素（FSH）（促卵泡激素α）及其生物类似物制剂已广泛应用于临床。这些制剂具有特定的FSH活性，其糖基化谱系取决于来源细胞。本研究旨在比较原研（参照）促卵泡激素α（戈那非®）与生物类似物制剂（贝姆福拉®和奥瓦莱普®）在体外诱导的细胞反应。通过ELISA结合素检测分析了促性腺激素N-糖基化谱系，揭示了制备特异的糖苷种类模式（Kruskal-Wallis检验；p < 0.05，n = 6），并通过糖基位点图谱分析。在体外，使用戈那非®或生物类似物（1 × 10−3-1 × 103 ng/ml）的递增浓度处理人初级黄体颗粒细胞（hGLC）和FSH受体（FSHR）转染的人胚胎肾293细胞。通过BRET、CREB和ERK1/2磷酸化检测评估了细胞内cAMP的产生、Ca2+的增加和β-arrestin 2的募集。通过实时PCR和免疫测定分别测量了12小时基因表达、8小时和24小时的孕酮和雌二醇合成。我们通过结合素检测发现了制备特异的糖基化模式（Kruskal-Wallis检验；p < 0.001；n = 6），并在FSHR转染的人胚胎肾293细胞中观察到相似的cAMP产生和β-arrestin 2募集（cAMP EC50范围=12 ± 0.9–24 ± 1.7 ng/ml；β-arrestin 2 EC50范围=140 ± 14.1–313 ± 18.7 ng/ml；Kruskal-Wallis检验；p ≥ 0.05；n = 4）。动力学分析显示，细胞经4 μg/ml戈那非®处理后，细胞内Ca2+增加，而等浓度的生物类似物未能诱导反应（Kruskal-Wallis检验；p < 0.05；n = 3）。所有制剂均能在hGLC中诱导8小时和24小时的孕酮和雌二醇合成，未观察到不同的EC50值（Kruskal-Wallis检验；p > 0.05；n = 5）。除制备特异的细胞内Ca2+增加在超生理激素剂量下实现外，所有化合物均诱导了相似的细胞内反应和类固醇生成，反映了相似的生物活性和整体结构同质性。