Development and characterization of a long-acting recombinant hFSH agonist

Background: Fusion of the carboxyterminal peptide (CTP) of hCG to FSH results in a follitropin agonist with an extended half-life, presumably due to the four O-oligosaccharides on the CTP. Alternatively, an rhFSH analogue containing additional N-linked carbohydrate is described in this report. Methods: A DNA sequence containing two N-oligosaccharide signal sequences was ligated into a vector containing hFSHβ- and α-subunit encoding cDNA, and expressed in CHO-K1 cells. In-vitro bioactivity of the single-chain hormone was assessed in CHO cells expressing the hFSH receptor. Pharmacokinetic values were derived from serial serum assays of the analogue in immature female rats following a single i.v. injection. In-vivo bioactivity was assessed by measuring ovarian weight gain 3 days post-injection. Results: rhFSH-N2 and native rhFSH induced comparable levels of cAMP in vitro. t_1/2 for native rhFSH, rhFSH-CTP and rhFSH-N2 were 3.7, 7.1 and 7.3 h respectively. Rats receiving rhFSH-N2 had a mean ± SD ovarian weight 3 days post-i.v. injection (22 ± 3.6 mg) significantly greater than rats receiving rhFSH and saline (16.7 ± 1.5 and 15.3 ± 0.47 mg respectively, P < 0.05). Conclusions: rhFSH-N2 has prolonged half-life and increased bioactivity compared with native rhFSH. This rhFSH agonist, and other analogues containing additional N-oligosaccharides may have important clinical applications.