Biosimilar SB16 Matches Denosumab in Exhaustive Analytical Comparison

Because biosimilars are made in living cells, regulators do not expect them to be molecularly identical to their reference products; instead, developers must demonstrate that any differences are unlikely to affect clinical performance. The new study lays out exactly how that demonstration was built for SB16, lot by lot and assay by assay.

Denosumab is a human immunoglobulin G2 monoclonal antibody that binds with high affinity and specificity to receptor activator of nuclear factor kappa-B ligand, known as RANKL. By preventing RANKL from activating its receptor on osteoclast precursors and osteoclasts, the antibody inhibits osteoclast formation and decreases bone resorption, which reduces the risk of fracture. The reference product is marketed in two dosage forms drawn from the same active drug substance: Prolia, a 60-mg pre-filled syringe for subcutaneous injection indicated for osteoporosis in postmenopausal women, glucocorticoid-induced osteoporosis and increasing bone mass in patients at high fracture risk undergoing hormone treatment for breast or prostate cancer; and Xgeva, a 120-mg single-use vial indicated for preventing skeletal-related events in patients with bone metastases from breast, prostate and other solid tumors and for treating giant cell bone tumors. The two presentations differ only in formulation details such as protein concentration and container closure systems.

The statistical backbone of the comparison was a quality range approach. The team tested up to 68 lots of EU-sourced Prolia, up to 35 lots of US-sourced Prolia, 29 lots of EU Xgeva and 17 lots of US Xgeva, purchased through local distributors and stored according to the manufacturer’s instructions, with residual shelf life ranging from 0 to 28 months at the time of testing. Similarity ranges were defined as mean plus or minus a multiple of the standard deviation derived from these reference lots, and SB16 batches were then evaluated against those pre-defined ranges or, where risk assessment dictated, by direct side-by-side comparison. The authors note that this extensive lot testing, spanning both EU- and US-sourced reference material at two different strengths, is one of the features distinguishing the dataset from earlier biosimilar analytical reports.

Primary structure came first. Peptide mapping by liquid chromatography-electrospray ionization-mass spectrometry was performed with three different endopeptidases, trypsin, Lys-C and Asp-N, whose overlapping peptide fragments confirmed 100 percent amino acid sequence coverage for both SB16 and the reference product. Tandem mass spectrometry confirmed identical amino acid sequences. Sequence variants such as N-terminal pyroglutamate and C-terminal lysine were qualitatively and quantitatively identical between the two products, and peptide mappings of oxidation and deamidation were also similar. The tryptic peak profiles showed comparable retention times with no differences, establishing that the two antibodies share the same molecular blueprint.

Higher order structure was assessed with four orthogonal techniques. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopy showed similar spectra in minima, maxima and residue molar ellipticity, indicating matching secondary and tertiary structure. Fourier transform infrared spectroscopy, run on samples at their original concentrations without buffer exchange, gave similar second-derivative amide I and amide II regions. Differential scanning calorimetry, heating samples from 10 to 100 degrees Celsius, confirmed similar thermal stability as measured by melting points. Most demanding of all, hydrogen/deuterium exchange-mass spectrometry tracked deuterium uptake over labeling timepoints from 10 seconds to 4 hours, and the uptake dynamics showed symmetry between SB16 and the reference product, probing conformational dynamics at peptide-level resolution.

Purity and impurity profiling followed. Size-exclusion high-performance liquid chromatography separated monomer from high molecular weight aggregates, and the relative quantities of both were similar between SB16 and the reference product. Capillary electrophoresis-sodium dodecyl sulfate, run under both non-reduced and reduced conditions, produced similar electrophoretic profiles, with comparable levels of immunoglobulin G species, light chain plus heavy chain, and the two-heavy-chain-one-light-chain impurity. The authors attribute a visible difference in the non-reduced immunoglobulin G peak pattern to IgG2 disulfide isoform heterogeneity, citing prior work showing that IgG2 isoforms shift in vivo toward the IgG2-B form after injection, which makes the observed difference not clinically meaningful.

Glycosylation and charge heterogeneity revealed the study’s most interesting nuances. N-glycan profiling by hydrophilic interaction-ultra performance liquid chromatography with 2-aminobenzamide labeling showed that the M9 high-mannose peak was detected in SB16 but not in the reference product; however, the total relative amount of high mannose species was similar between the two. Relative galactosylation content of SB16 was lower than that of the reference product. The authors judged this difference not clinically meaningful on three grounds: denosumab’s mechanism of action involves no Fc effector function; SB16 showed no detectable antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity in functional assays; and published evidence indicates that galactosylation has minimal impact on pharmacokinetics and safety compared with mannose and sialic acids. This justification was established before clinical trials and subsequently confirmed by phase I and phase III results showing similar pharmacokinetics, safety, efficacy and immunogenicity.

Charge variants told a similar story of differences that could be explained. All SB16 batches fell within the pre-defined EU and US similarity ranges, yet mean values of acidic and basic variants differed between the products, and the reference product showed higher lot-to-lot variability, contributing to wide similarity ranges. Imaging capillary isoelectric focusing revealed an SB16-specific peak at approximately pI 8.98, mirrored by a corresponding peak at roughly 25 to 26 minutes in cation exchange chromatograms. Fractionation and peptide mapping of individual charge variant peaks traced the differences to known modifications: denosumab reference product showed higher deamidation at Asn385, Asn390 or Asn391 in acidic fractions, while SB16 carried more C-terminal lysine variants and more C-terminal alpha-amidation, a modification well documented in CHO-cell-produced antibodies. C-terminal lysine variants have been shown not to affect antibody structure and function, alpha-amidation does not affect effector function or binding affinity, and biological activity assays across all chromatography fractions matched the starting material.

Biological activity was then tested across the full functional repertoire. Fab-related assays covering RANKL binding by enzyme-linked immunosorbent assay, RANKL neutralization potency in a RANKL-overexpressing engineered cell line, and anti-differentiation potency in Raw 264.7 cells all showed no meaningful differences, with every tested SB16 batch within the EU and US similarity ranges. Fc-related assays covering neonatal Fc receptor binding by surface plasmon resonance, binding to Fc-gamma receptors IIa, IIb and IIIa, and C1q binding likewise showed similarity. Notably, neither SB16 nor the reference product showed any Fc-gamma receptor Ia binding response, antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, a finding consistent with denosumab’s mechanism of action and reinforcing the argument that the galactosylation difference carries no functional consequence.

The authors conclude that SB16 demonstrated high similarity to the denosumab reference product across all structural, physicochemical and biological properties, and that this analytical dataset forms part of the totality of evidence supporting biosimilarity, complementing the previously reported phase I and phase III clinical trials. They position the work as reference information for the scientific community, illustrating how a tiered, risk-based quality attribute strategy combined with extensive reference lot testing and state-of-the-art orthogonal methods can document analytical similarity. As with any analytical characterization study, the findings describe molecular and in vitro comparisons rather than new clinical outcomes; the clinical evidence cited comes from the earlier trials. The article contains no studies with human participants or animals performed by the authors, and the dataset is presented as a template for how biosimilar developers can assemble the analytical foundation that regulators expect before clinical data are even considered.

Subject of Research: Analytical similarity assessment of the denosumab biosimilar SB16 against its reference products Prolia and Xgeva

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

Article References: Jung, E., Kim, J., Do, M., Kim, J., Kweon, H., Lee, J., Lee, J., Yoo, T., & Yang, S.-Y. (2026). Analytical Characterization for Similarity Assessment Between Biosimilar SB16 and Denosumab Reference Product Using State-of-the-Art Analytical Method. Drugs in R&D. https://doi.org/10.1007/s40268-026-00555-8

Image Credits: AI Generated

DOI: 10.1007/s40268-026-00555-8

Keywords: biosimilar, SB16, denosumab, Prolia, Xgeva, RANKL, analytical characterization, peptide mapping, glycosylation, charge heterogeneity, biological activity, mass spectrometry