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)

Abstract

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.

Biosimilarity and Interchangeability: Principles and Evidence: A Systematic Review

“…There are no well-designed investigations into the clinical implications of glycosylation differences that may occur in production of biosimilars [41, 116]. The terminal sugars of glycans attached to immunoglobulin heavy chain have been shown to be critical for safety and/or efficacy of therapeutic monoclonal antibodies.…”

“…NeuGc, a non-human sialic acid that is found on some therapeutic proteins, can reduce efficacy due to rapid clearance of the biotherapeutic, and has been reported to cause hypersensitivity. In summary, production processes can change the glycosylation profiles of product biosimilars [41], which may potentially affect safety and efficacy. To date, the clinical implications of these changes are not well understood.…”

“…Even when fully humanised and glycosylated, therapeutic monoclonal antibodies can be immunogenic because they form immune complexes with target proteins, undergo phagocytosis and potentially activate inflammation [126]. Even within the human population, genes encoding the constant domains of immunoglobulins are highly polymorphic (known as allotypes), and each therapeutic is made from only one variant, which, therefore, may be recognised as non-self in individuals who lack that polymorphism [40, 41, 127–129]. Therefore, tolerance to an originator biologic does not necessarily predict immunological tolerance to its biosimilar and vice versa.…”

Generation of site‐distinct N‐glycan variants for in vitro bioactivity testing

“…The cell culture supernatants and the glycoengineering samples were purified by a ProSep Ultra Plus column (0.6 ml, 0.5 × 3 cm, Merck Life Science, Darmstadt, Germany) and eluted with 20 mM acetic acid at pH 3.2. Subsequent pH adjustment with 1 M Tris to pH 5.0 and according to the previously reported method (Mastrangeli et al, ), the samples were suspended in the denaturation buffer containing 8 M guanidine‐HCl, 1 mM ethylenediaminetetraacetic acid, and 130 mM Tris pH 7.6. The reduction was performed at 37°C by dithiothreitol.…”

In vitro fertilisation/intracytoplasmic sperm injection beyond 2020

“…Low‐cost IVF focuses on affordable stimulation protocols, clinical judgment rather than excessive investigations, limited use of disposable materials, and clear and simple laboratory protocols, all of which ensure that low cost does not jeopardise quality . An option for affordable stimulation protocols involves use of cheaper biosimilar FSH preparations that are now emerging as patents expire on innovator products, although questions remain regarding their efficacy and safety profile . Other low‐cost protocols such as natural cycle IVF, low stimulation regimens, use of oral ovarian stimulants such as clomiphene citrate, and single embryo transfer (SET) can not obviate the necessity for a quality good laboratory .…”