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首页> 外文期刊>Journal of Clinical Microbiology >Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.
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Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

机译：人类白细胞热原测试，用于检测重组大肠杆菌产生的生长激素中的热原物质。

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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.
机译：人生长激素是作为蛋氨酸的人生长激素（met-hGH）在大肠杆菌的重组菌株中生物合成产生的。从细菌培养物中纯化后，met-hGH在已建立的生长激素测定中具有生物活性。因此，在健康的成年人中进行了met-hGH的I期试验。然而，在第一次试验期间，观察到了对热原性药物有急性期反应的特征性体征，症状和临床实验室测试。在I期试验中使用的met-hGH制剂的先前测试未显示毒性证据，美国药典公约的兔热原测试以及Li变形细胞裂解液（LAL）测试均未检测到显着水平的外源热原。或内毒素。另外，添加内毒素的标准抑制研究表明，LAL试验没有抑制作用。当这种met-hGH制剂与人血单核细胞一起孵育时，白细胞热原（LP）释放到上清液中，表明该制剂含有热原物质。通过人类LP测定法测试了基于不同纯化和配制方法的各种met-hGH，以污染热原。这些测试的结果有助于鉴定在体外不诱导LP的met-hGH制剂的程序。因此，随后通过LP测试的大量met-hGH被用于重复的临床研究，未观察到炎症反应或热原反应。当使用LP测试时，实验表明原始的met-hGH被内毒素污染，而LAL或兔子热原测试中未检测到。在LAL试验和美国药典公约兔子热原试验中，甘氨酸磷酸盐缓冲液的冻干导致内毒素反应性降低了10到20倍。这些数据可能为在初始批次的met-hGH中引起急性期反应的LAL和兔热原测试的阴性结果提供了可能的解释。此外，这些研究表明从人细胞中释放LP是存在对人类有致热物质的可靠指示。

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