In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f).

Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated

Glycan and antennarity and sialylation distribution at Asn52.

<p><b>A)</b> Comparison of glycan distribution at Asn52 between Bemfola and GONAL-f (individual species) <b>B)</b> Comparison of antennarity and sialylation at Asn52 between Bemfola and GONAL-f. Asn, asparagine.</p

Bemfola and GONAL-f batches tested.

<p>Bemfola and GONAL-f batches tested.</p

Antennarity, fucoslylation and sialylation at α chain Asn52.

<p>Antennarity, fucoslylation and sialylation at α chain Asn52.</p

Extracted ion chromatograms of the <i>N</i>-glycan distribution at Asn52 for Bemfola and GONAL-f.

<p>Fig was constructed using MassLynx 4.1 (Waters, Milford MA, USA) from the respective XIC of the glycopeptides extracted from only Asn52. Asn, asparagine. Blue square, GlcNAc. Green circle, mannose. Yellow circle, galactose. Red triangle. fucose. Purple diamond, sialic acid NeuNAc. Glycan naming: F at the start of the abbreviation indicates a core a(1–6) fucose linked to the inner GlcNAc. Ax indicates the number of antenna (GlcNAc) on trimannosyl core. A2 indicates bi-antennary with both GlcNAcs as b(1–2) linked. A3 indicates tri-antennary with a GlcNAc linked b1-2 to both mannose and a third GlcNAc linked b(1–4) to the a(1–3) linked mannose. A4 indicates tetra-antennary with GlcNAcs linked as A3 with additional GlcNAc b(1–6) linked to a(1–6) mannose. Gx indicates the number (x) of b1-4 linked galactose on the antenna. Sx indicates the number (x) of sialic acids linked to galactose.</p