Recombinant Human Thyroid Stimulating Hormone: Development of a Biotechnology Product for Detection of Metastatic Lesions of Thyroid Carcinoma

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH). rhTSH was expressed by co-transfecting a human -subunit cDNA with a human -subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be >99% by SDS polyacryl-amide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

1. Pierce, J.G. and Parsons, T.F. 1981. Glycoprotein hormones: structure and function. Ann. Rev. Biochemistry 50: 465−495. | Article | ISI | ChemPort |
2. Liao, T.H. and Pierce, J.G. 1971. The primary structure of bovine thyrotropin. II. The amino acid sequences of the reduced, S-carboxymethyl and chains. J. Biol. Chem. 246: 850−865. | PubMed  | ISI | ChemPort |
3. Lissitzky, S., Torresani, J., Carayon, P. and Amr, S. 1989. Physiology of the Thyroid, p. 512−540. In: DeGroot, L. J. (Ed. ). Endocrinology, 2nded. Vol. 1. Philadelphia, PA: W. B. Saunders Co.
4. Magner, J. A. 1990. Thyroid-stimulating hormone: biosynthesis, cell biology, and bioactivity. Endocrine Reviews 11: 354−385. | PubMed  | ISI | ChemPort |
5. DeGroot, L.J. and Sridama, V. 1989. Thyroid Neoplasia, p. 758−776. In: DeGroot, L. J. (Ed. ). Endocrinology, 2nd ed. Vol. 1. Philadelphia, PA: W. B. Saunders Co.
6. Sturgeon, C.T., Davis, F.E., Catz, B., Petit, D. and Starr, P. 1953. Treatment of thyroid cancer metastases with TSH and I¹³¹ during thyroid hormone medication. J. Clin. Endocrinol. Metab. 13: 1391−1407. | PubMed  | ISI | ChemPort |
7. Melmed, S., Harada, A., Hershman, J.M., Krishnamurthy, G.T. and Blahd, W.H. 1980. Neutralizing antibodies to bovine thyrotropin in immunized patients with thyroid cancer. J. Clin. Endocrinol. Metab. 51: 358−363. | PubMed  | ISI | ChemPort |
8. Frohman, L.A., Baron, M.A. and Schneider, A.B. 1982. Plasma immunoreactive TSH: Spurious elevation due to antibodies to bovine TSH which cross-react with human TSH. Metabolism 31: 834−840. | Article | PubMed  | ISI | ChemPort |
9. Law, A., Jack, G.W., Tellez, M. and Edmonds, C.J. 1986. In vivo studies of a human-thyrotropin preparation. J. Endocr. 110: 375−378. | PubMed  | ISI | ChemPort |
10. Kuku, S.F., Harsoulis, P., Kjeld, M. and Fraser, T.R. 1975. Human thyrotropic hormone kinetics and effects in euthyroid males. Horm. Metab. Res. 7: 54−59. | ISI | ChemPort |
11. Schneider, P.B., Robbins, J. and Condliffe, P.G. 1965. Thyroid response to human thyrotropin in man. J. Clin. Endocrinol. Metab. 25: 514−517. | PubMed  | ISI | ChemPort |
12. Will, R.G. 1991. An overview of Creutzfeldt-Jakob disease associated with the use of human pituitary growth hormone. Develop. Biol. Standard. 75: 85−86. | ChemPort |
13. Green, E.D. and Baenziger, J.U. 1988. Asparagine-linked oligosaccharides on lutropin, folitropin, and thyrotropin I. J. Biol. Chem. 263: 25−35. | PubMed  | ISI | ChemPort |
14. Green, E.D. and Baenziger, J.U. 1988. Asparagine-linked oligosaccharides on lutropin, folitropin, and thyrotropin II. J. Biol. Chem. 263: 36−44. | PubMed  | ISI | ChemPort |
15. Joshi, L.A. and Weintraub, B.D. 1983. Naturally occurring forms of thyrotropin with low bioactivity and altered carbohydrate content act as competitive antagonists to more bioactive forms. Endocrinology 113: 2145−2154. | PubMed  | ISI | ChemPort |
16. Matzuk, M.M., Kornmeier, C.M., Whitfield, G.K., Kourides, I.A. and Boime, I. 1988. The glycoprotein -subunit is critical for secretion and stability of the human thyrotropin -subunit. Molecular Endocrinology 2: 95−100. | PubMed  | ISI | ChemPort |
17. Kaetzel, D.M. and Nilson, J.H. 1988. Methotrexate-induced amplification of the bovine lutropin genes in Chinese hamster ovary cells. J. Biol. Chem. 263: 6344−6351. | PubMed  | ISI | ChemPort |
18. Corless, C.L., Matzuk, M.M., Ramabhadran, T.V., Krichevsky, A. and Boime, I.,1987. Gonadotropin subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells. J. cell. Biol. 104: 1173−1181. | Article | PubMed  | ISI | ChemPort |
19. Kashiwai, T., Ichihara, K., Endo, Y., Tamaki, H., Amino, N. and Miyai, K. 1991. Immunological and biological characteristics of recombinant human thyrotropin. J. Immunol. Methods 143: 25−30. | Article | PubMed  | ISI | ChemPort |
20. Takeuchi, M., Takasaki, S., Miyazaki, H., Kato, T., Hoshi, S., Kochibe, N. and Kobata, A. 1988. Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J. Biol. Chem. 263: 3657−3663. | PubMed  | ISI | ChemPort |
21. Yuen, C.-T., Carr, S.A. and Feizi, T. 1990. The spectrum of N-linked oligosaccharide structures detected by enzymatic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells. Eur. J. Biochem. 192: 523−528. | PubMed  | ISI | ChemPort |
22. Hiyama, J., Weisshaar, G. and Renwick, A.G.C. 1992. The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotropin. Glycobiology 2: 401−409. | PubMed  | ISI | ChemPort |
23. Meier, C.A., Braverman, L.E., Ebner, S.A., Daniels, G.H., Ross, D.S., Daraska, D., Valentine, M., DeGroot, L.J., Curran, P., McEllin, K., Reynolds, J., Robbins, J. and Weintraub, B.D.: The rhTSH Study Group. 1992. Diagnostic use of recombinant human thyrotropin in patients with thyroid carcinoma. Thyroid 2(suppl. 1): S−35.
24. Thotakura, N.R., Desai, R.K., Bates, L.G., Cole, E.S., Pratt, B.M. and Weintraub, B.D. 1991. Biological activity and metabolic clearance of a recombinant thyrotropin produced in Chinese hamster ovary cells. Endocrinology. 128: 341−348. | PubMed  | ISI | ChemPort |
25. Huber, G.K., Fong, P., Concepcion, E.S. and Davies, T.F. 1991. Recombinant human thyroid-stimulating hormone: Initial bioactivity assessment using human fetal thyroid cells. J. Clin. Endocrinol. Metab. 72: 1328−1331. | PubMed  | ISI | ChemPort |
26. Braverman, L.E., Pratt, B.M. and Longcope, C. 1992. Recombinant human thyrotropin stimulates thyroid function and radioactive iodine uptake in the Rhesus monkey. J. Clin. Endocrinol. Metab. 74: 1135−1139. | Article | PubMed  | ISI | ChemPort |
27. Williams, E.S., Ekins, R.P. and Ellis, S.M. 1969. Thyroid stimulation test with serum thyroxine concentration as index of thyroid response. Brit. Med. J. 4: 336−338. | PubMed  | ISI | ChemPort |
28. Uller, R.P., Van Herle, A.J. and Chopra, I.J. 1973. Comparison of alterations in circulating thyroglobulin, triiodothyronine and thyroxine in response to exogenous (bovine) and endogenous (human) thyrotropin. J. Clin. Endocrinol. Metab. 37: 741−745. | PubMed  | ISI | ChemPort |
29. Uller, R.P., Van Herle, A.J. and Chopra, I.J. 1977. Thyroidal response to graded doses of bovine thyrotropin. J. Clin. Endocrinol. Metab. 45: 312−318. | PubMed  | ISI | ChemPort |
30. Bakke, J., Lawrence, N. and Roy, S. 1962. Disappearance rate of exogenous thyroid-stimulating hormone (TSH) in man. J. Clin. Endocrinol. Metab. 22: 352−363. | PubMed  | ISI | ChemPort |
31. Odell, W.D., Utiger, R.D., Wilber, J.F. and Condliffe, P.G. 1967. Estimation of the secretion rate of thyrotropin in man. J. Clin. Invest. 46: 953−959. | PubMed  | ISI | ChemPort |
32. Hershman, J.M. and Edwards, C.L. 1972. Serum thyrotropin (TSH) levels after thyroid ablation compared with TSH levels after exogenous bovine TSH: Implications for 1311 treatment of thyroid carcinoma. J. Clin. Endocrinol. Metab. 34: 814−818. | PubMed  | ISI | ChemPort |
33. Friis, T. and Pedersen, L.R. 1987. Serum lipids in hyper-and hypothyroidism before and after treatment. Clin. Chim. Acta. 162: 155−163. | Article | PubMed  | ISI | ChemPort |
34. Nikkila, E.A. and Kekki, M. 1972. Plasma triglyceride metabolism in thyroid disease. J. Clin. Invest. 51: 2103−2114. | PubMed  | ISI | ChemPort |
35. Valdemarsson, S., Hansson, P., Hedner, P. and Nilsson-Ehle, P. 1983. Relations between thyroid function, hepatic and lipoprotein lipase activities, and plasma lipoprotein concentrations. Acta. Endocrinol. 104: 50−56. | PubMed  | ISI | ChemPort |
36. Hays, M.T., Solomon, D.H., Pierce, J.G. and Carsten, M.E. 1961. The effect of purified bovine thyroid-stimulating hormone in man. I. Dose-response characteristics with 1131. J. Clin. Endocrinol. Metab. 21: 1469−1474. | PubMed  | ISI | ChemPort |
37. Hays, M.T., Solomon, D.H. and Werner, S.C. 1961. The effect of purified bovine thyroid-stimulating hormone in man. II. Loss of effectiveness with prolonged administration. J. Clin. Endocrinol. Metab. 21: 1475−1482. | PubMed  | ISI | ChemPort |
38. Hays, M.T., Solomon, D.H. and Beall, G.N. 1967. Suppression of human thyroid function by antibodies to bovine thyrotropin. J. Clin. Endocrinol. Metab. 27: 1540−1549. | PubMed  | ISI | ChemPort |
39. Krishnamurthy, G.T. 1978. Human reaction to bovine TSH: Concise communication. J. Nucl. Med. 19: 284−286. | PubMed  | ISI | ChemPort |
40. Melmed, S., Harada, A., Hershman, J.M., Krishnamurthy, G.T. and Blahd, W.H. 1980. Neutralizing antibodies to bovine thyrotropin in immunized patients with thyroid cancer. J. Clin. Endocrinol. Metab. 51: 358−363. | PubMed  | ISI | ChemPort |
41. Erwin, C.R., Croyle, M.L., Donelson, J.E. and Maurer, R.A. 1983. Nucleotide sequence of cloned complementary deoxyribonucleic acid for the subunit of bovine pituitary glycoprotein hormones. Biochemistry 22: 4856−4860. | PubMed  | ISI | ChemPort |
42. Devereux, J., Haeberli, P. and Smithies, O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12: 387−395. | PubMed  | ISI | ChemPort |
43. Maurer, R.A., Croyle, M.L. and Donelson, J.E. 1984. The sequence of a cloned cDNA for the subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions. J. Biol. Chem. 259: 5024−5027. | PubMed  | ISI | ChemPort |
44. Kirkpatrick, C.H., Meek, J.C. and Rich, R.R. 1973. Mechanism of allergy to components of commercial bovine thyrotropin. J. Allergy Clin. Immunol. 51: 296−302. | PubMed  | ISI | ChemPort |
45. Subramani, S., Mulligan, R. and Berg, P. 1981. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in Simian Virus 40 Vectors. Mol. cell. Biol. 1: 854−864. | PubMed  | ISI | ChemPort |
46. Fiddes, J.C. and Goodman, H.M. 1981. The gene encoding the common subunit of the four human glycoprotein hormones. J. Mol. Appl. Gen. 1: 3−18. | ChemPort |
47. Wondisford, F.E., Radovick, S., Moates, J.M., Usala, S.J. and Weintraub, B.D. 1988. Isolation and characterization of the human thyrotropin -subunit gene. J. Biol. Chem. 263: 12538−12542. | PubMed  | ISI | ChemPort |
48. Urlaub, G. and Chasin, L.A. 1980. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA 77: 4216−4220. | PubMed  | ChemPort |
49. Wigler, M., Silverstein, S., Lee, L.S., Pellicer, A., Cheng, V.C. and Axel, R. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11: 223−232. | Article | PubMed  | ISI | ChemPort |
50. Cole, E.S., Nichols, E.H., Lauziere, K., Edmunds, T. and McPherson, J.M. 1991. Characterization of the microheterogeneity of recombinant primate prolactin: Implications for posttranslational modifications of the hormone in vivo. Endocrinology 129: 2639−2646. | PubMed  | ISI | ChemPort |
51. Laemlli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T₄. Nature 227: 680−685. | PubMed  |
52. Burnette, W.N. 1981. "Western Blotting". Electrophoretic transfer of proteins from SDS-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112: 195−203. | PubMed  | ISI | ChemPort |
53. Hardy, M.R., Townsend, R.R. and Lee, Y.C. 1988. Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal. Biochem. 170: 54−62. | PubMed  | ISI | ChemPort |
54. Powell, L.D. and Hart, G.W. 1986. Quantitation of picomole levels of N-acetyl-and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay. Anal. Biochem. 157: 179−185 | PubMed  | ISI | ChemPort |
55. Levene, H. 1960. Robust tests for equality of variances, p. 278−292. In: Olkin, I., Ghurye, S. G., Hoefding, W, Madow W.G., Mann, H.B. (Eds.). Contributions to Probability and Statistics; Essays in Honor of Harold Hotelling. Stanford, California: Stanford University Press.
56. Winer, B.J. 1971. Design and analysis of single-factor experiments, p. 149−260. In: Statistical Principles in Experimental Design. 2nd ed. New York, NY: McGraw-Hill.
57. MicroMath Scientific Software. 1991. MINSQ, nonlinear parameter estimation and model development, MINSQ user handbook, rev. 7DBF., Salt Lake City, Utah: MicroMath Scientific Software.
58. MicroMath Scientific Software. 1990. Library of pharmacokinetic models for MINSQ. rev. 116DE., p. 87−90. Salt Lake City, Utah: MicroMath Scientific Software.

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