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

The human whole blood assay utilises the natural fever response to detect p yrogens by determination of the release of IL-1 beta. In order to replace the official method the rabbit pyrogen test, a validati on of the whole blood assay is necessary. comparison of the results obtaine d from many blood samples has revealed the following: 1) Blood nob stimulated by LPS does not produce IL-IP. 2) Stimulation by LPS induces a concentration-dependent release of IL-1 bet a beginning at a concentration of between 2-5 pg/mL LPS. 3) The amount of IL-1 beta released varies greatly between samples obtained from different individuals. 4) Storing blood samples results in a right shifted LPS/IL-1 beta curve wit h a steeper gradient and higher maximum value of IL-1b. In this paper we su ggest an experimental method for the determination of pyrogens based an the established semiquantitative LAL gelation method as detailed in the Europe an Pharmacopeia. Using this methodology, we were able to show that the amount of endotoxin i n a number of different infusion solutions tvas below the LAL-endotoxin lim it concentration. LPS was quantitatively determined from spiked samples.