Biological Assay to Determine Gonadotropin Potency: From In Vivo to In Vitro Sustainable Method

Abstract

Various preparations of follicle-stimulating hormone (FSH) are commercially available; however, they differ in glycoforms composition and purity owing to their respective sources. Additional chemical/physical changes can also be introduced during manufacturing and can impact their biological activity (biopotency), which is routinely assessed using an in vivo bioassay (Steelman–Pohley). This study aimed to determine whether an in vitro bioassay could assess biopotency by distinguishing between r-hFSH chemical/physical variants with similar ability to the in vivo bioassay. The specific activity (units of biological activity per mg of product) of variants of r-hFSH generated through enrichment (acidic/basic), stress (oxidative/acidic pH) and enzymatic treatment (desialylation and desialylation/degalactosylation) was compared using the in vivo and in vitro bioassays. The in vitro bioassay reliably detected potential chemical/physical modifications in r-hFSH variants that may impact biopotency. Overall, the methods demonstrated a comparable ability to detect changes in specific activities due to chemical/physical differences in r-hFSH variants. These data indicate that the in vitro bioassay is suitable to replace the in vivo bioassay.

Follicle-Stimulating Hormone Biological Products: Does Potency Predict Clinical Efficacy?

“…The accepted coefficient of variation for each determination is between 10% and 20%, meaning that, considering the maximum variation (20%), by injecting 100 IU of the FSH product, the actual bioactivity found in the assay can be any value between 80 and 120 IU [50]. Recently, however, an in vitro bioassay demonstrated similar ability to the Steelman-Pohley in vivo bioassay to detect chemical/physical differences in r-hFSH variants that strongly impact biopotency [51,52]. Notably, the in vitro bioassay was recently approved by the European Medicines Agency (EMA) to replace the in vivo bioassay after obtaining a positive opinion from the Committee for Medicinal Products for Human use (CHMP) on 27 October 2022 for originator follitropin alfa (GONAL-f ® ) and follitropin alfa/lutropin alfa (Pergoveris ® ) [53].…”

“…By contrast, follitropin alfa is highly purified and has a high batch-to-batch consistency. The potency of follitropin alfa can be determined using either the Steelman-Pohley in vivo bioassay or a recently developed in vitro bioassay [51,52]. The FSH content can be quantified using chromotagraphic techniques, where analysis of the protein mass considers the entire protein, including the oligosaccharide side chains [50].…”