Development and validation of a novel monocyte activation test with secreted luciferase-expressing cells

Pyrogen testing is essential for ensuring pharmaceutical product safety; however, current methods have limitations. The Bacterial Endotoxins Test cannot detect non-endotoxin pyrogens (NEPs), whereas the Rabbit Pyrogen Test was phased out for animal welfare reasons. Monocyte activation tests (MAT), using human whole blood samples or peripheral blood mononuclear cells, are effective but suffer from donor variability, biosafety concerns, and instability. We aimed to develop a sensitive, rapid, and simple alternative pyrogen test by devising a luciferase-based MAT using the Mono-Mac-6 (MM6) cell line. MM6 cells showed higher cytokine responses to endotoxin than THP-1 cells and exhibited comparable or greater sensitivity than human peripheral blood. Introducing an NF-κB-driven NanoLuc® reporter enabled endotoxin detection within 3 h; however, rapid degradation of intracellular luciferase was noted. To overcome this degradation issue, we generated MM6 cells expressing secreted NanoLuc (secNluc), allowing for stable detection between 3 and 12 h after stimulation. The MM6/NFκB-secNluc cells helped detect various pyrogens, including Pam3CSK4, peptidoglycan, poly(I:C), lipopolysaccharide, flagellin, macrophage-activating lipopeptide-2, and resiquimod. Adding recombinant human lipopolysaccharide-binding protein enabled establishing a serum-free MAT system, eliminating serum-derived endotoxin contamination. This reporter-based MAT provides a robust platform for rapid endotoxin and NEP detection, offering an improved alternative to conventional MATs.