Refined ADA testing strategy to overcome sRANKL interference in denosumab/FKS518 immunogenicity assessment
Year: 2025
Type: article
Abstract: BACKGROUND: Fresenius Kabi developed FKS518, a fully human monoclonal antibody biosimilar to denosumab, that inhibits osteoclast activation by targeting the receptor activator of nuclear factor kappa-Β ligand (RANKL). RANKL exists as a trimeric soluble protein (sRANKL) with high affinity for denosumab. Post-dose, sRANKL levels can rise due to drug-target complex accumulation, creating a risk of interference in bridging anti-drug antibodies (ADA) assays, particularly after acid dissociation. METHODS: We evaluated sRANKL interference in the ADA bridging assay and, to competitively block sRANKL, we introduced a specificity tier by adding osteoprotegerin (OPG). This approach enabled reanalysis of previously ADA-positive samples to confirm whether signals represented true ADA responses or artifacts caused by sRANKL interference. RESULTS: Acid dissociation significantly exacerbated target interference, resulting in ADA positivity rates of ~96-98% in clinical studies. Introducing a specificity tier corrected incidence to ≤3.9%. OPG incorporation did not change the minimum required dilution (MRD) of the assay and did not affect signals for negative/positive control, confirming assay integrity. DISCUSSION: These findings underscore the importance of early interference assessment and mitigation. A multi-tiered strategy, encompassing screening, confirmatory, and specificity tiers, provided a robust solution applicable to programs facing similar challenges.
Source: Bioanalysis
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: hybrid
OpenAlex ID: W7117484654