ALTEX :: Alternatives to Animal Experiments

1998, Supplement

First Results in the Prevalidation of the Human Whole Blood Assay for Pyrogens in Biological Pharmaceuticals

Matthias Fischer¹, Katja Hartzsch¹, Thomas Hartung², and Thomas Montag-Lessing¹

¹Paul-Ehrlich-Institut, D-Langen; ²Biochemische Pharmakologie der Universitaet Konstanz, D-Konstanz

SUMMARY

When establishing the whole blood assay for pyrogens in our laboratory, we did several investigations on the standard pyrogen to be used. Because of the hydrophobicity of the lipopolysaccharid (LPS) it is difficult to get a homogeneous solution. Therefore, we used ultrasonic and biphenylhydroxylamine chloride for a better dispergation. Surprisingly the reactivity of the LPS in the whole blood assay for pyrogens dropped the more the LPS was dispergated. That's right inverse to the situation in the LAL. There are first experiences in the whole blood assay for pyrogens with biological pharmaceuticals. This includes immunoglobulins, vaccines, and coagulation factors. Except of the vaccines and albumin, the rabbit assay is the only way to test these pharmaceuticals for pyrogens. The albumins show a little amplification of the reactivity of LPS in the whole blood assay for pyrogens. The testing of albumin in the whole blood assay for pyrogens showed highly reproducible results and a detection limit under 50 pg/ml LPS. The immunoglobulins show an amplifying effect as well. Immunoglobulins spiked with LPS were detected as pyrogenic in all cases. Some vaccines show a slight pyrogenity in the whole blood assay which becomes visible when diluting the sample. Undiluted vaccines may cover a LPS contamination in the whole blood assay for pyrogens.

Keywords: pyrogen testing, whole blood model, endotoxin, interleukin, contamination of biological products