Romosozumab Biosimilar Matches Reference in Functional Testing Despite Disulfide Differences

A romosozumab biosimilar in late-stage development showed no meaningful difference in sclerostin binding or bone-signaling potency compared with the reference product, Evenity (Amgen/UCB), despite carrying a measurably different disulfide isoform profile, according to a study recently published in Scientific Reports.1

Compared with the RMP, the biosimilar contained less immunoglobulin G2 subclass (IgG2)-B and higher proportions of IgG2-A/B and IgG2-A. The findings represent one of the first systematic evaluations showing that a structural quality attribute unique to IgG2 antibodies can differ between an originator biologic and a biosimilar candidate without affecting biological activity.

Why Disulfide Isoforms Matter for IgG2 Biosimilars

IgG2 antibodies contain 4 disulfide bonds in the hinge region that give rise to 3 naturally occurring structural variants known as IgG2-A, IgG2-A/B, and IgG2-B, which differ in how the light chains connect to the heavy-chain hinge. Secretory cells predominantly produce IgG2-A, which converts first to IgG2-A/B and then more gradually to IgG2-B once in circulation. Because these conformational differences can influence surface charge, hydrophobicity, and glycosylation, disulfide isoform distribution is increasingly considered a critical quality attribute for IgG2 biologics such as romosozumab, a sclerostin-neutralizing antibody used to treat osteoporosis.

Using reversed-phase high-performance liquid chromatography, investigators found the biosimilar contained 55.76% IgG2-B, 25.03% IgG2-A/B, and 19.21% IgG2-A compared with 62.12%, 20.22%, and 17.66%, respectively, in the RMP. A complementary electrophoresis method confirmed the biosimilar’s shift toward the more open, less compact isoforms. Accelerated, stress, and long-term stability studies also showed that the disulfide isoform profiles of both products remained stable over time, suggesting the differences arose during manufacturing rather than storage or circulation.

Assessing the Functional Impact of the Isoform Differences

To determine whether the altered isoform distribution affected function, investigators isolated individual isoforms from both products using 2 complementary methods: redox-based enrichment and low-pH cation-exchange chromatography. The isolated fractions were then evaluated for glycosylation patterns, sclerostin binding by surface plasmon resonance and ELISA, and bioactivity using a cell-based reporter assay measuring Wnt signaling reactivation.

Although the isoforms exhibited measurable physicochemical differences, including variations in high-mannose glycan content, these changes did not translate into functional differences. Fractions enriched for the more open isoforms contained higher levels of high-mannose glycans than the more compact IgG2-B-dominant fraction in both products. Because elevated high-mannose content has been associated with faster antibody clearance in other settings, the authors noted that it remains an important characteristic to monitor during biosimilar development.

Despite these structural distinctions, binding affinity and potency testing produced consistent results. Data from the enriched and fractionated isoforms, benchmarked against 10 RMP batches and 6 biosimilar batches, all fell within the reference product’s established quality range, demonstrating comparable sclerostin binding and Wnt pathway activation regardless of isoform composition.

What This Means for Other IgG2 Biosimilars

The authors noted that this is not the first report of differing disulfide isoform profiles among IgG2 biosimilars. Previous research on denosumab biosimilars identified shifts in disulfide isoform composition within the reference product over time; however, that study did not determine whether those differences affected biological activity. The current romosozumab analysis addresses that question directly by showing that the observed structural differences did not alter in vitro potency.

Still, the investigators cautioned against generalizing the findings to all IgG2 biosimilars. Because romosozumab neutralizes the soluble protein sclerostin, its mechanism differs from that of receptor-targeting IgG2 antibodies, whose biological activity can depend more heavily on antibody conformation. For IgG2 biosimilars that rely on receptor engagement or avidity-driven mechanisms, the authors recommend comprehensive evaluations of disulfide isoform profiles and their potential effects on physicochemical and biological properties.

The findings come as osteoporosis biosimilars continue to gain attention for their potential to expand access and lower costs. Denosumab biosimilars, for example, have been positioned as a way to improve access to treatment and ease costs associated with osteoporosis and cancer-related bone loss.2 Clinicians and payers have similarly noted that biosimilar competition in this space could reshape affordability and care delivery, provided rigorous analytical bridging supports claims of comparable efficacy and safety.3

Overall, the authors concluded that despite differences in surface charge, hydrophobicity, and high-mannose content, the romosozumab biosimilar's disulfide isoforms performed comparably to the RMP.1

“These findings suggest that the observed disulfide isoform profile differences do not compromise the potency of romosozumab under the conditions tested,” they wrote.

References

1. Zhao D, Liu Y, Wu C, et al. Similarity assessment of disulfide isoform profiles between the romosozumab biosimilar and the reference medicinal product. Sci Rep. 2026;16:21058. doi:10.1038/s41598-026-51578-9
2. Santoro C. Denosumab biosimilars approved with interchangeability to address osteoporosis, fracture risk. AJMC®. February 18, 2025. Accessed July 16, 2026. https://www.ajmc.com/view/denosumab-biosimilars-approved-with-interchangeability-to-address-osteoporosis-fracture-risk
3. Postmenopausal osteoporosis: clinical burden, treatment barriers, and the evolving role of denosumab biosimilars. AJMC. February 18, 2026. Accessed July 16, 2026. https://www.ajmc.com/view/postmenopausal-osteoporosis-clinical-burden-treatment-barriers-and-the-evolving-role-of-denosumab-biosimilars