Recombinant Human Thyroid Stimulating Hormone: Development of a Biotechnology Product for Detection of Metastatic Lesions of Thyroid Carcinoma
| Autor: | Edward S. Cole, Karen Lee, Kevin Lauziere, Christine Kelton, Scott Chappel, Bruce Weintraub, Dawn Ferrara, Pamela Peterson, Rick Bernasconi, Tim Edmunds, Susan Richards, Lydia Dickrell, James M. Kleeman, John M. McPherson, Bruce M. Pratt |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Male
medicine.medical_specialty Molecular Sequence Data Biomedical Engineering Thyrotropin Bioengineering CHO Cells Biology Applied Microbiology and Biotechnology law.invention Thyroid carcinoma Mice In vivo law Cricetinae Internal medicine Cyclic AMP medicine Animals Humans Amino Acid Sequence Thyroid Neoplasms Neoplasm Metastasis Polyacrylamide gel electrophoresis Chromatography High Pressure Liquid Chromatography Chinese hamster ovary cell Thyroid Biological activity Recombinant Proteins Rats Kinetics Macaca fascicularis Endocrinology medicine.anatomical_structure Recombinant DNA Molecular Medicine Female Biotechnology Hormone |
| Zdroj: | Nature Biotechnology. 11:1014-1024 |
| ISSN: | 1546-1696 1087-0156 |
| DOI: | 10.1038/nbt0993-1014 |
| Popis: | 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 alpha-subunit cDNA with a human beta-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 polyacrylamide 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|