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Glycosylation of an N-Terminal Extension Prolongs the Half-Life and Increases the in Vivo Activity of Follicle Stimulating Hormone

Maxygen (S.P., B.v.d.H., J.C., S.G.-N., C.B.J., K.V.A., T.H., S.O., H.T.S.), DK-2970 Hoersholm; and Department of Gynecology and Obstetrics (S.P.), Hvidovre Hospital, 2650 Hvidovre, Copenhagen, Denmark

Address all correspondence and requests for reprints to: Dr. Hans T. Schambye, Maxygen, Agern Allé 1, DK-2970 Hoersholm, Denmark. E-mail: hs{at}maxygen.dk.

FSH is a key component in assisted reproductive technologies. Because of rapid clearance of the hormone, patients have to be treated with daily injections. To address this problem, a long-acting FSH mutein was created by introduction of additional N-linked glycosylation into the molecule. New glycosylation sites were introduced by two different approaches: structure-aided, site-directed introduction of sites within the FSH molecule and addition of N-terminal extensions. A mutein with the extension sequence ANITVNITV at the N terminus of the -chain (FSH1208) was efficiently glycosylated at both new sites. This resulted in a molecule with increased size and charge, factors known to reduce renal clearance of proteins. FSH1208 was found to have a 3- to 4-fold increased serum half-life, compared with wild-type recombinant FSH. Furthermore, in spite of a lower in vitro activity, FSH1208 had a markedly increased in vivo potency, as shown by increased ability to augment the ovarian weight and stimulate the serum estradiol levels in rats. These characteristics make FSH1208 a possible candidate for improved infertility treatment.

S.P. was supported by a Ph.D. fellowship from the Academia of Technical Science.

S.P. and B.v.d.H. contributed equally and should be considered co-first authors.

Present address for C.B.J.: Molecular Pharmacology, Novo Nordisk, DK-2760 Maaloev, Denmark; for B.v.d.H.: Høiberg A/S, DK-1264 Copenhagen K, Denmark; and for S.G.-N.: Lundbeck, DK-2500 Valby, Denmark.

Abbreviations: CG, Chorionic gonadotropin; CHO, Chinese hamster ovary; EPO, erythropoietin; FBS, fetal bovine serum; IEF, isoelectric focusing; h, human; rFSH, recombinant FSH.

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