An improved monocyte activation test using cryopreserved pooled human mononuclear cells.

The monocyte activation test (MAT) is a promising replacement of the currently used rabbit pyrogen test to detect the presence of pyrogens in injectable drugs. In the MAT, drugs are incubated with a source of human monocytes and production of pyrogenic cytokines used as readout. The best results are obtained with human mononuclear cells (MNC). However, donor variation requires testing on four different donors, and for most laboratories access to fresh MNCs is a problem. The current study shows how to overcome these problems using frozen pooled MNCs. The MAT is performed by thawing pooled MNC and co-culture overnight with a test substance, LPS or non-endotoxin pyrogens, with IL-6 production as the readout. The study demonstrates that fresh and frozen pooled MNC have comparable sensitivity. The reproducibility of the MAT performed with different batches of frozen pooled MNC was excellent. Different non-endotoxin pyrogens induce IL-6, confirming the ability of the MAT to detect a variety of pyrogens. In conclusion, the MAT using frozen pooled MNC is a highly sensitive, specific and reproducible pyrogen test, able to detect and quantify endotoxin and non-endotoxin pyrogenic contaminations in parenteral pharmaceuticals.