Abstract
Compare the glycosylation properties of follitropin delta, from a human cell line, with those of natural human derived FSH and with follitropin alfa from a hamster cell line. Follitropin delta (Rekovelle®) produced in PER.C6® human derived cell line, follitropin alfa (Gonal f®) produced in CHO derived cell line and natural human urinary derived FSH (Bravelle®) were used in the study. Isoelectric Focusing (IEF), was used to resolve and profile the isoforms present in FSH preparations. IEF was carried out using a gel with a pH range between pH 3-7. Glycans were released from FSH preparations by enzymatic digestion with PNGase and derivatized with 2-aminobenzamide (2AB). Glycans were sequenced with specific exoglycosidases and analysed using normal phase HPLC and weak anion exchange chromatography. A more acidic isoform profile was obtained by IEF for follitropin delta and human derived FSH in comparison to follitropin alfa. Compared to follitropin alfa, the released glycans from follitropin delta and natural human FSH both show higher abundance of tri- and tetra-antennary forms and a lower abundance of the di-antennary forms. Sialylation of glycans on follitropin delta and natural human FSH comprises both α2,3 linked and α2,6 linked sialic acid, whereas follitropin alfa exhibits only the α2,3 linked form. The resemblance of follitropin delta glycans to the human derived FSH was further supported by the presence of N-acetylgalactosamine (GalNAc) and bisecting N-acetylglucosamine (GlcNAc) which are absent in CHO-derived FSH (Table). Follitropin delta glycosylation is richer and more complex compared to follitropin alfa with a higher proportion of tri- and tetra-antennary and charged glycans, as well as by the presence of α2,6-linked sialic acid, GalNAc residues and bisecting GlcNAc. In these ways follitropin delta shares numerous glycosylation features and more resembles natural human derived FSH.
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