title: N-glycosylation increases the circulatory half-life of human growth hormone.
abstract: Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.
authors.affiliation: Biopharm Research Unit, Novo Nordisk A/S, 2760 Måløv, Denmark.
authors.forename: Thomas V
authors.initials: TV
authors.lastname: Flintegaard
authors.forename: Peter
authors.initials: P
authors.lastname: Thygesen
authors.forename: Henrik
authors.initials: H
authors.lastname: Rahbek-Nielsen
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.forename: Claus
authors.initials: C
authors.lastname: Kristensen
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.forename: Gert
authors.initials: G
authors.lastname: Bolt
mesh_terms: D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006207:Half-Life; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D011134:Polysaccharides; D019382:Human Growth Hormone