TY - JOUR
T1 - N-glycosylation increases the circulatory half-life of human growth hormone
AU - Flintegaard, Thomas V
AU - Thygesen, Peter
AU - Rahbek-Nielsen, Henrik
AU - Levery, Steven B
AU - Kristensen, Claus
AU - Clausen, Henrik
AU - Bolt, Gert
N1 - Keywords: Animals; Cell Line; Enzyme-Linked Immunosorbent Assay; Glycosylation; Half-Life; Human Growth Hormone; Humans; Polysaccharides; Random Allocation; Rats; Rats, Sprague-Dawley
PY - 2010/11
Y1 - 2010/11
N2 - 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.
AB - 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.
U2 - 10.1210/en.2010-0574
DO - 10.1210/en.2010-0574
M3 - Journal article
C2 - 20826563
SN - 0013-7227
VL - 151
SP - 5326
EP - 5336
JO - Journal of Clinical Endocrinology and Metabolism
JF - Journal of Clinical Endocrinology and Metabolism
IS - 11
ER -