Biological Activity and Metabolic Clearance of a Recombinant Human Thyrotropin Produced in Chinese Hamster Ovary Cells

Abstract

The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.

The effects of recombinant human thyrotropin, in normal subjects and patients with goitre

“…(1991) demonstrated that rhTSH is able to activate the TSH receptor, as reflected by the accumulation of cAMP, the induction of Tg secretion and thyroid epithelial cell multiplication. Although rhTSH has an amino acid structure identical to hTSH (Cole et al ., 1993), its glycosylation is different with higher sialic acid content (Thotakura et al ., 1991;Szkudlinski et al ., 1993). As a consequence, rhTSH exhibits lower immunological activity, lower TSH receptor binding affinity, and lower in vitro bioactivity than hTSH.…”

“…As a consequence, rhTSH exhibits lower immunological activity, lower TSH receptor binding affinity, and lower in vitro bioactivity than hTSH. The metabolic clearance rate (MCR) is significantly lower for rhTSH than for hTSH (Thotakura et al ., 1991) and due to this difference in MCR it may turn out that rhTSH in vivo is equivalent to or maybe more potent than hTSH, despite the lower in vitro bioactivity (Thotakura et al ., 1991). Estimated by immunoassays the specific activity of rhTSH is between 5·51 and 7·63 IU/mg based on the second International Reference Preparation (80/558) of hTSH as the standard (Rafferty & Gaines, 1999).…”

Clinical use of recombinant human TSH in thyroid cancer patients

“…Once the b-subunit of the human TSH gene had been cloned (11), the encoded protein could be overexpressed in a cell system (Chinese hamster ovary cells), by transfection with the human a-and bsubunits of complementary or genomic DNA. With this technique, large quantities of highly puri®ed rhTSH can be obtained (12). Its bioactivity and metabolic clearance seem to be affected by the pattern of posttranslational glycosylation.…”

Truncated equine LH beta and asparagine(56)-deglycosylated equine LH alpha combine to produce a potent FSH antagonist

“…The Asn 52/56 oligosaccharide masks the secondary role of Asn 13 requiring that the former be eliminated before the impact of the latter can be demonstrated (Matzuk et al 1989). The subunit oligosaccharides exert their primary influence on hormone activity by determining the circulatory half-life of glycoprotein hormones (Rosa et al 1984, Bishop et al 1995, which is an important factor determining in vivo biological activity (Thotakura et al 1991).…”