Development and validation of an affinity capture elution bridging assay for the detection of anti-drug antibodies to romosozumab and its biosimilar SAL023

Introduction Romosozumab is a monoclonal antibody targeting sclerostin and is approved for the treatment of osteoporosis in postmenopausal women at high risk of fracture. SAL023 is a proposed biosimilar to romosozumab. For biosimilar development, a robust and unbiased anti-drug antibody (ADA) assay is required to support comparative immunogenicity assessment of the biosimilar and reference product. Methods An affinity capture elution bridging assay was developed as a one-assay approach for the detection of binding ADAs against both romosozumab and SAL023 using an electrochemiluminescence platform. The assay incorporated acid dissociation, affinity capture with immobilized SAL023, acidic elution, and bridging detection with biotin- and Ru(bpy)32+ (Ru)-labeled SAL023. Validation covered cut points, sensitivity, selectivity, precision, titer precision, hook effect, specificity, drug tolerance, antigenic equivalence, and sample stability, including comparisons of confirmatory inhibition and drug tolerance between SAL023 and Evenity. Results The screening cut point factor, confirmatory cut point, and titer cut point factor were 1.12, 14.17% inhibition, and 1.20, respectively. The screening and confirmatory assay sensitivities were 1.293 and 1.732 ng/mL, respectively, and the final low positive control concentration was set at 2.100 ng/mL. The assay showed acceptable selectivity in the 10 individual normal serum matrices tested, and all precision and titer precision results met predefined acceptance criteria. No high-dose hook effect was observed up to 20,000 ng/mL of positive control antibody. Drug-tolerance profiles, confirmatory inhibition responses, and antigenic equivalence results were comparable between SAL023 and the reference product Evenity. ADA-positive samples remained stable under the evaluated room-temperature, refrigerated, and freeze–thaw conditions. Conclusion The validation results support the use of the ACE-bridging assay for comparative binding ADA assessment of SAL023 and reference romosozumab, with limitations related to low-level ADA detection in the presence of drug and SOST-related interference.