A new test for the detection of pyrogens in pharmaceutical products. Examinations for the validation of the human whole blood assay

Abstract

The human whole blood assay utilises the natural fever response to detect pyrogens by determination of the release of IL-1beta. In order to replace the official method, the rabbit pyrogen test, a validation of the whole blood assay is necessary. A comparison of the results obtained from many blood samples has revealed the following: 1) Blood not stimulated by LPS does not produce IL-1beta. 2) Stimulation by LPS induces a concentration-dependent release of IL-1( beginning at a concentration of between 2-5 pg/mL LPS. 3) The amount of IL-1beta released varies greatly between samples obtained from different individuals. 4) Storing blood samples results in a right shifted LPS/IL-1beta curve with a steeper gradient and higher maximum value of IL-1beta. In this paper we suggest an experimental method for the determination of pyrogens based on the established semi-quantitative LAL gelation method as detailed in the European Pharmacopeia. Using this methodology, we were able to show that the amount of endotoxin in a number of different infusion solutions was below the LAL-endotoxin limit concentration. LPS was quantitatively determined from spiked samples.