Analytical Characterization for Similarity Assessment Between Biosimilar SB16 and Denosumab Reference Product Using State-of-the-Art Analytical Method.

BACKGROUND AND OBJECTIVE: SB16 has been developed and recently approved as a biosimilar to denosumab reference product, which inhibits osteoclast formation by targeting receptor activator of nuclear factor kappa-B ligand. This study presents a comprehensive analytical characterization of SB16 denosumab biosimilar to demonstrate high similarity to the reference product (Prolia and Xgeva).

METHODS: Analytical similarity was assessed using side-by-side comparisons and similarity range approaches with state-of-the-art methods. Critical quality attributes evaluated included primary structure, post-translational modifications, higher order structure, purity, glycosylation, charge heterogeneity, and biological activities. Similarity ranges were established using extensive reference product lot testing (up to 68 EU Prolia, 35 US Prolia, 29 EU Xgeva, and 17 US Xgeva lots).

RESULTS: Peptide mapping confirmed 100% sequence coverage with identical amino acid sequences. Higher order structure analyses (circular dichroism, Fourier transform infrared, differential scanning calorimetry, hydrogen/deuterium exchange-mass spectrometry) demonstrated similar secondary and tertiary structures. Purity assessments (size exclusion-high performance liquid chromatography, Capillary electrophoresis-sodium dodecyl sulfate) showed similar monomer content and impurity profiles. N-glycan profiling revealed similar high mannose species; galactosylation differences were deemed not clinically meaningful. Charge heterogeneity (imaging capillary isoelectric focusing, cation exchange) demonstrated similar charge variant profiles. Fab-related activities (receptor activator of nuclear factor kappa-B ligand binding, receptor activator of nuclear factor kappa-B ligand neutralization, and anti-differentiation) and Fc-related activities (neonatal fragment crystallizable receptor binding, Fc-gamma receptor binding, C1q binding, antibody-dependent cell-mediated cytotoxicity, and complement-dependent cytotoxicity) showed similar results.

CONCLUSIONS: SB16 demonstrated high similarity to denosumab reference product across all structural, physicochemical, and biological properties, providing robust analytical evidence supporting biosimilarity. This dataset serves as valuable reference information for biosimilar development.