Abstract
Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions. In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baker R. S., Schmidtke J. R., Ross J. W., Smith W. C. Preliminary studies on the immunogenicity and amount of Escherichia coli polypeptides in biosynthetic human insulin produced by recombinant DNA technology. Lancet. 1981 Nov 21;2(8256):1139–1142. doi: 10.1016/s0140-6736(81)90589-4. [DOI] [PubMed] [Google Scholar]
- Bernheim H. A., Block L. H., Atkins E. Fever: pathogenesis, pathophysiology, and purpose. Ann Intern Med. 1979 Aug;91(2):261–270. doi: 10.7326/0003-4819-91-2-261. [DOI] [PubMed] [Google Scholar]
- Bodel P. Studies on the mechanism of endogenous pyrogen production. II. Role of cell products in the regulation of pyrogen release from blood leukocytes. Infect Immun. 1974 Sep;10(3):451–457. doi: 10.1128/iai.10.3.451-457.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dinarello C. A., Bendtzen K., Wolff S. M. Studies on the active site of human leukocytic pyrogen. Inflammation. 1982 Mar;6(1):63–78. doi: 10.1007/BF00910720. [DOI] [PubMed] [Google Scholar]
- Dinarello C. A., Goldin N. P., Wolff S. M. Demonstration and characterization of two distinct human leukocytic pyrogens. J Exp Med. 1974 Jun 1;139(6):1369–1381. doi: 10.1084/jem.139.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dinarello C. A. Interleukin-1. Rev Infect Dis. 1984 Jan-Feb;6(1):51–95. doi: 10.1093/clinids/6.1.51. [DOI] [PubMed] [Google Scholar]
- Dinarello C. A., Wolff S. M. Molecular basis of fever in humans. Am J Med. 1982 May;72(5):799–819. doi: 10.1016/0002-9343(82)90548-4. [DOI] [PubMed] [Google Scholar]
- Duff G. W., Atkins E. The inhibitory effect of polymyxin B on endotoxin-induced endogenous pyrogen production. J Immunol Methods. 1982 Aug 13;52(3):333–340. doi: 10.1016/0022-1759(82)90005-9. [DOI] [PubMed] [Google Scholar]
- Elin R. J., Wolff S. M., Finch C. A. Effect of induced fever on serum iron and ferritin concentrations in man. Blood. 1977 Jan;49(1):147–153. [PubMed] [Google Scholar]
- Elin R. J., Wolff S. M. Nonspecificity of the limulus amebocyte lysate test: positive reactions with polynucleotides and proteins. J Infect Dis. 1973 Sep;128(3):349–352. doi: 10.1093/infdis/128.3.349. [DOI] [PubMed] [Google Scholar]
- Olson K. C., Fenno J., Lin N., Harkins R. N., Snider C., Kohr W. H., Ross M. J., Fodge D., Prender G., Stebbing N. Purified human growth hormone from E. coli is biologically active. Nature. 1981 Oct 1;293(5831):408–411. doi: 10.1038/293408a0. [DOI] [PubMed] [Google Scholar]
- Rietschel E. T., Schade U., Jensen M., Wollenweber H. W., Lüderitz O., Greisman S. G. Bacterial endotoxins: chemical structure, biological activity and role in septicaemia. Scand J Infect Dis Suppl. 1982;31:8–21. [PubMed] [Google Scholar]
- Root R. K., Nordlund J. J., Wolff S. M. Factors affecting the quantitative production and assay of human leukocytic pyrogen. J Lab Clin Med. 1970 Apr;75(4):679–693. [PubMed] [Google Scholar]
- WOLFF S. M., MULHOLLAND J. H., WARD S. B. QUANTITATIVE ASPECTS OF THE PYROGENIC RESPONSE OF RABBITS TO ENDOTOXIN. J Lab Clin Med. 1965 Feb;65:268–275. [PubMed] [Google Scholar]
- van Miert A. S., van Duin C. T. Further studies on the antipyretic action of polymyxin B in pyrogen-induced fever. Arzneimittelforschung. 1978;28(12):2246–2251. [PubMed] [Google Scholar]