[BEBPA USA 2017] Development and Evaluation of a Novel Bioassay for Denosumab Activity

[BEBPA USA 2017] Development and Evaluation of a Novel Bioassay for Denosumab Activity

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Denosumab (Prolia®) is a fully human sequence derived monoclonal antibody used for the treatment in osteoporosis in menopausal woman via inhibition of the RANK (receptor activator of nuclear factor-kappa B) pathway. Companies developing a biosimilar are required to demonstrate that the proposed biosimilar is “highly similar” to the innovator material, using a mechanism-of-action (MOA)-based assay. The DiscoverX PathHunter® bioassay for Denosumab uses a U20S cell line expressing RANK and an IκB reporter protein that is tagged with one fragment of a split β-galactosidase system. When the tagged IκB is exposed to the other half of the split β-galactosidase protein, active β-galactosidase is formed which hydrolyses the substrate and produces a chemiluminescent signal. In the assay, RANKL binds to RANK on
the cell surface resulting in NF-κB signalling and IκB degradation, leading to a decrease in chemiluminescent signal. Denosumab inhibits RANKL-based activation of RANK, leading to an increase in chemiluminescence. The increase in chemiluminescence is directly proportional to
the functional activity of denosumab.

The data presented in this poster was obtained from an assay qualification performed at Sartorius Stedim BioOutsource. During the qualification study, data was obtained to demonstrate that the assay is accurate and precise over a linear range of 50% to 150% of reference standard. The assay also showed specificity for denosumab. The methodology is therefore deemed fit for the purposes of evaluating the functional comparability and potency of denosumab biosimilar and innovator material. The benefits of this commercially available assay are that it is easy-to-use, highly reproducible, and saves months of assay development time, translating into overall speed and cost savings in a biosimilar development program.