Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech

Abstract

Gonadotropin therapy plays an integral role in ovarian stimulation for infertility treatments. Efforts have been made over the last century to improve gonadotropin preparations. Undoubtedly, current gonadotropins have better quality and safety profiles as well as clinical efficacy than earlier ones. A major achievement has been introducing recombinant technology in the manufacturing processes for follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin. Recombinant gonadotropins are purer than urine-derived gonadotropins, and incorporating vial filling by mass virtually eliminated batch-to-batch variations and enabled accurate dosing. Recombinant and fill-by-mass technologies have been the driving forces for launching of prefilled pen devices for more patient-friendly ovarian stimulation. The most recent developments include the fixed combination of follitropin alfa + lutropin alfa, long-acting FSH gonadotropin, and a new family of prefilled pen injector devices for administration of recombinant gonadotropins. The next step would be the production of orally bioactive molecules with selective follicle-stimulating hormone and luteinizing hormone activity.

Figure 1

Gonadotropin Molecules. The alpha and beta subunits are represented by red and blue strands, respectively, whereas the carbohydrate chains are represented by light blue balls. A) Follicle-stimulating hormone. FSH is a glycoprotein composed of two subunits, the alpha subunit (red) and the beta subunit (blue). There are four carbohydrate attachment sites, two in each subunit. B) Luteinizing hormone. LH is a glycoprotein with two subunits, the alpha subunit (red), which is similar to FSH and hCG, with two carbohydrate attachment sites, and the beta subunit (blue) with only one carbohydrate attachment site. C) Human chorionic gonadotropin. hCG has structural attributes similar to LH. A notable exception is the presence of a long carboxy-terminal segment that is O-glycosylated (O-linked CHO), conferring a longer half-life to hCG.

Figure 2

Human Steroidogenesis. The starting point for steroid biosynthesis is the conversion of cholesterol in pregnenolone by P450scc. One route for pregnenolone metabolism is the delta-5 pathway (red arrows) through CYP17 (P450c17). Pregnenolone hydroxylation at the C17a position forms 17-hydroxypregnenolone, and subsequent removal of the acetyl group forms the androgen precursor dehydroepiandrosterone (DHEA). An additional route for pregnenolone metabolism is the delta-4 pathway (purple arrows), in which pregnenolone is converted to progesterone by 3b-HSD (an irreversible conversion). Progesterone is then converted to 17-hydroxyprogesterone by CYP17. In humans, 17-hydroxyprogesterone cannot be further metabolized. Importantly, CYP17 is exclusively located in thecal and interstitial cells in the ovary extrafollicular compartment, whereas CYP19 (aromatase), which converts androgens to estrogens, is expressed exclusively in GCs, which are in the intrafollicular compartment. Androgen aromatization to estrogens is a distinct activity that occurs in the granulosa layer, and it is induced by FSH via P450 aromatase (P450arom) gene activation.

Figure 3

Milestones in the Development of Gonadotropin Preparations. FSH was originally derived from animal (pregnant mare serum) or human (post-mortem pituitary glands) sources, but these preparations were abandoned due to safety concerns. Gonadotropins were first extracted from urine in the 1940s; human chorionic gonadotropin (hCG) in 1940; and then human menopausal gonadotropin (hMG) in 1949. Over a decade later, the first urinary forms of hCG and hMG became commercially available. Further improvements in purification methods yielded follicle-stimulating hormone (FSH)-only products in the 1980s and the subsequent development of highly purified FSH (HP-hFSH), which became available 10 years later, in 1993, and allows for subcutaneous injection. In the 1970s and 1980s, advances in DNA technology enabled the development of recombinant human FSH (rec-hFSH), which became commercially available in 1995. In 2000, recombinant human luteinizing hormone (rec-hLH) became available, and with the launch of recombinant human hCG (rec-hCG) in 2001, the full recombinant gonadotropin portfolio was available. The most recent developments include the filled-by-mass (FbM) follitropin alfa formulation, the fixed combination of follitropin alfa + lutropin alfa, long-acting FSH gonadotropin, and a new family of prefilled pen injector devices.

Figure 4

Recombinant gonadotropin technology. Chinese hamster ovary cells are first grown in T-flasks, then subcultured in roller bottles and allowed to expand for up to 36 days. Next, the cells are mixed with a microcarrier bead suspension and transferred to a bioreactor vessel continuously perfused with a growth-promoting medium for an average of 34 days. The cell culture supernatant medium containing ‘crude glycoprotein' is collected from the bioreactor and stored at 48°C until purification. The protein is purified by chromatography followed by ultrafiltration. The final product is released after extensive quality control testing over 7 weeks.