BIOLOGICAL AND IMMUNOCHEMICAL CHARACTERIZATION OF RECOMBINANT HUMAN THYROTROPIN

Recombinant human thyroid-stimulating hormone (recTSH) has recently be en engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogenei ty, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origi n (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amou nt of sialic acid and lack of N-acetylgalactosamine. Electrofocusing f ollowed by immunoblotting resolved recTSH into six glyco-forms with pI s ranging from 6.0 to 8.6, which were converted to a major species of pi 8.9 by sialidase treatment, pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to si alidase. Binding activity of both human TSHs to porcine thyroid membra ne receptors was found to be similar, but recTSH appeared to be 20% ac tive compared to pitTSH in eliciting cAMP production and cell growth i n rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was in vestigated using polyclonal and monoclonal antibodies raised against t he native hormone or synthetic peptide sequences of its subunits, Whil e rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti -beta 96-112 antisera bound recTSH to a greater extent than pitTSH, wh ile anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations. Inhibition assays showed that the alpha 1-25 and anti-alpha 26-51 regions contained at least two antigenic determi nants which are present in recTSH but absent in the pituitary hormone. It is therefore concluded that recTSH differs from pitTSH with respec t to several conformational features at the polypeptide surface, which are likely to be responsible for altered intrinsic bioactivity and ma y be potentially antigenic in patients repeatedly injected with the dr ug.