Long-Acting Follicle-Stimulating Hormone Analogs Containing N-Linked Glycosylation Exhibited Increased Bioactivity Compared with O-Linked Analogs in Female Rats

Abstract

The effects of altering the number and type of additional carbohydrate moieties on the pharmacokinetic and pharmacodynamic properties of FSH were examined in this report. A series of single-chain follitropins, containing variable numbers of additional N- (or O-) linked carbohydrates, were designed and expressed in Chinese hamster ovary cells. Proper folding, efficient receptor binding, and signal transduction were confirmed by in vitro assays. Pharmacokinetic and pharmacodynamic parameters were evaluated in immature female Sprague Dawley rats. Increasing the number of glycosylation sites with either N- (or O-) linked moieties extended the elimination half-life as much as 2-fold compared with recombinant human FSH (rhFSH). However, there was a maximum elimination half-life such that further glycosylation provided no additional lengthening of the half-life. Conversely, biopotency, as assessed by inhibin A levels 74 h post injection, and follicle production were significantly higher for the N-linked analogs. Rats stimulated with the longest acting analogs (either N- or O-linked) showed significantly higher ovarian weights than rats receiving a single injection of rhFSH. The analog containing four additional N-linked sites (rhFSH-N4) had the greatest number of large, preovulatory follicles. Although the half-life of rhFSH-N4 displayed no further enhancement beyond the other longest acting analogs, this analog exhibited significantly increased biopotency in rats. This work provides the basis for the generation of a series of reagents potentially useful for therapeutic applications.

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

“…Glycosylated FSH analogs containing 2 or 4 N-glycosylation sites and a molecular mass of ϳ55 kDa exhibited a 2-4-fold increase of the AUC after i.v. injection into rats (20,21). Thus, for hyperglycosylated erythropoietin and glycosylated FSH a similar increase in half-lives was observed as for our glycosylated scDb variants.…”

“…In other studies, glycosylated analogs of follicle stimulating hormone (FSH) with prolonged circulation time and increased activity have been generated by adding N-glycans through an N-terminal extension of the ␣ subunit (20) or through a peptide linker joining the ␤-and the ␣-subunit (21), demonstrating the feasibility of this approach.…”

Efficacy of native and hyperglycosylated follicle-stimulating hormone analogs for promoting fertility in female mice

“…We also reported the production of a series of single-chain, long-acting FSH proteins produced by the addition of novel N-linked carbohydrate sequences (9). The consensus sequence for the addition of N-linked carbohydrates is known (Asn-X-Ser/Thr, where X represents any amino acid except Pro) while no clear sequence has been identified for O-linked glycosylation.…”

“…Therefore it is easier to manipulate N-linked glycosylation alterations. An additional benefit of N-linked carbohydrates is the increased number of sialic acid binding sites leading to reduced pI and longer in vivo half-life (9,10). Furthermore, N-linked carbohydrates are more complex which has been suggested to enhance the biological activity of a protein beyond simply increasing the protein half-life (11).…”

“…Furthermore, N-linked carbohydrates are more complex which has been suggested to enhance the biological activity of a protein beyond simply increasing the protein half-life (11). Previous evaluation of in vivo activity of rhFSH-CTP and rhFSH-N4 in rats demonstrated that both rhFSH-CTP and rhFSH-N4 enhanced protein half-life to a similar extent; but rhFSH-N4 increased biological activity as demonstrated by greater ovarian folliculogenesis as compared to rhFSH-CTP which could not be explained by increase of half-life alone (9). Furthermore, when rhFSH-N4 was injected into mice, it was found to have a similar bioactivity on ovarian folliculogenesis as the current gold standard for rodent hyperstimulation protocols PMSG (12).…”