Newly reported roles of thyroid-stimulating hormone and follicle-stimulating hormone in bone remodelling.

Thyroid-stimulating hormone (TSH) and follicle-stimulating hormone (FSH) have both been recently implicated in bone remodelling. Clinical evidence, as well as data from TSH receptor and thyroid hormone receptor knockout mice, suggest that TSH has a direct effect on skeletal homeostasis, although some data are conflicting. Recently, the exogenous administration of TSH has been shown to positively impact bone in oophrectomised rats. These data, along with their potential implications for the treatment of severe osteoporosis, are discussed.

Development of an in vitro potency assay for therapeutic TGFbeta antagonists: the A549 cell bioassay.

A bioassay was developed to assess the potency of TGFbeta antagonists by measuring IL-11 production in TGFbeta-1 treated human lung epithelial cells (A549). The production of IL-11 by A549 cells, measured by ELISA, was shown to be proportional to the TGFbeta-1 concentration. The A549 cells were responsive to all three isoforms of TGFbeta in the range of 3.0 ng/mL to 1.4 pg/mL, with an 18 to 24 h exposure time found to be within the linear portion of the bioassay response range. The Effective Dose at 80% of the maximal response (ED80) of TGFbeta-1 determined for the assay was 0.3 ng/mL. With this level of TGFbeta-1, a human anti-TGFbeta-1 antibody (CAT-192) yielded an approximate median Inhibitory Concentration (IC50) value of 3 microg/mL. To investigate assay specificity, alternate members of the TGFbeta superfamily were evaluated. Recombinant human activin B, inhibin A and BMP-2 (Bone Morphogenetic Protein-2) did not elicit a significant IL-11 response from the A549 cell line. Bioassay qualification was performed to obtain estimates of precision and accuracy, as well as to establish plate validity criteria. Assay precision was estimated at 30%, while the accuracy was +/-13%. Additionally, the ability of the A549 cell potency assay to detect potency differences in structurally modified samples was investigated.

The effect of posttranslational modifications on the in vitro activity of recombinant human thyroid-stimulating hormone.

Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.

Comparison of two in vitro methods for the measurement of recombinant human TSH bioactivity.

Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint.