Comparative analysis of common potency assays for assessing human TNF-alpha neutralising antibodies

Tumour necrosis factor-alpha (TNF-α) plays a central role in inflammation and autoimmune pathology, making it a key therapeutic target. This study systematically evaluated the performance of monoclonal anti-human TNF-α antibodies derived from hybridoma cells—originally developed during early TNF research—using two widely adopted in vitro potency assays: a cytotoxicity-based assay in L929 fibroblasts and an NF-κB reporter assay in HEK293 Blue cells. Antibody performance was assessed in terms of neutralisation efficiency, signal reproducibility, and assay sensitivity. While both assays yielded comparable IC₅₀ values (1.98 for L929 and 1.34 for HEK293 Blue), they differed in dynamic range, sensitivity, and biological relevance. The HEK293 Blue assay provided rapid, robust, and high-throughput-compatible detection of early TNF-α signalling events, with minimal background and superior reproducibility. In contrast, the L929 assay offered physiologically relevant insights into the later consequences of TNF-α signalling, such as apoptosis and metabolic disruption. ATP quantification using the CellTiter-Glo® assay proved to be a practical and sensitive method for viability assessment, though alternative metabolic and membrane integrity assays may complement interpretation. Overall, our findings highlight the complementary strengths of these assay platforms and support a dual-assay approach for comprehensive evaluation of anti-TNF-α antibodies.