Comprehensive Physico-chemical and Functional Similarity Assessment Study of RGB-14-P and RGB-14-X Drug Products as Proposed Biosimilars to Denosumab Reference Products

BioDrugs (2025) 39:697–724 https://doi.org/10.1007/s40259-025-00738-w ORIGINAL RESEARCH ARTICLE Comprehensive Physico‑chemical and Functional Similarity Assessment Study of RGB‑14‑P and RGB‑14‑X Drug Products as Proposed Biosimilars to Denosumab Reference Products Ágnes Szonja Garai1 · Dániel Hüse1 · Ádám Fizil1 · Zsolt Zólyomi1 · Gábor Lehoczki1 · Andrea Kis1 · Zsuzsanna Kálmán‑Szekeres1 · Viktor Háda1 Accepted: 28 July 2025 / Published online: 6 August 2025 © The Author(s) 2025 Abstract Background and Objective Denosumab is a fully human monoclonal antibody (IgG2) k subclass that targets and binds with high affinity and specificity to receptor activator of nuclear factor-κB ligand (RANKL). Gedeon Richter’s denosumab RGB-14-P and RGB-14-X are proposed biosimilar drug products to the reference medicinal products P rolia® and Xgeva® (marketing authorisation holder: Amgen Europe B.V. in the European Union [EU] and Amgen Inc. in USA, respectively). The present study demonstrates the structural, physico-chemical and functional similarity between RGB-14 and reference drug products marketed in the EU and US. Methods Using an extensive, state-of-the-art analytical and functional panel of 38 methods ensured the comprehensive characterisation of the biosimilar and reference drug products. To assess biosimilarity, physico-chemical and biological functional tests were performed using multiple orthogonal techniques, in addition to the in-depth comparison of the primary and higher-order structures of the therapeutic proteins. Results It has been demonstrated that the primary and higher order structures of RGB-14-P and RGB-14-X drug products are identical or highly similar to those of EU/US Prolia® and Xgeva®. The purity profiles of the biosimilar and reference products were similar. Only minor differences were observed in glycosylation patterns and charge variant profiles. A wide range of bioassays was used demonstrating similarity in terms of potency, ligand and receptor binding. Additionally, during comprehensive analysis of the reference product data as the function of expiry dates, shifts were revealed in certain quality parameters, although these did not impact the biological activity of the products. Conclusion The extensive analytical and functional similarity assessment study provides robust evidence that the structure and function of RGB-14-P and RGB-14-X are highly similar to those of EU/US Prolia® and Xgeva®. Key Points A comprehensive and detailed analytical and functional similarity study was carried out for Gedeon Richter’s biosimilar denosumab RGB-14-P and RGB-14-X products against the EU- and US-licensed reference drug products Prolia® and Xgeva®. On the basis of the comparative assessment, RGB-14-P versus Prolia® and RGB-14-X versus X geva® are highly similar drug products. * Viktor Háda 1 Biotechnology Process Development and Analytics, Biotechnology Business Unit, Gedeon Richter Plc., Gyömrői út 19‑21., Budapest 1103, Hungary 1 Introduction RGB-14-P and RGB-14-X are proposed biosimilar drug products to the denosumab reference medicinal products Prolia® and Xgeva®. Denosumab is a fully human monoclonal antibody (immunoglobulin [Ig]G2-subclass k-subclass) that binds to the receptor activator of nuclear factor-κB ligand (RANKL). RANKL induces osteoclastogenesis by binding with the receptor activator of nuclear factor-κB (RANK) expressed in bone marrow macrophages [1]. Denosumab inhibits osteoclastogenesis by decreasing RANKL/ RANK binding, thus reducing bone resorption [2]. Denosumab was approved by the European Medicines Agency and U.S. Food and Drug Administration as P rolia® [3, 4] for the treatment of postmenopausal osteoporosis in women, and as Xgeva® for the treatment of giant cell tumour [5, 6]. Vol.:(0123456789) 698 A biosimilar medicinal product is a highly similar version of the already authorised reference product in terms of structural, functional and clinical characteristics. The primary goal in biosimilar development is to ensure a high degree of similarity to the reference product, particularly in physicochemical and functional properties. A comprehensive similarity study covering all structural, physico-chemical and biological-functional critical quality attributes of the active pharmaceutical ingredient should be conducted to assess biosimilarity. The basic principles of biosimilar development should be compliant with the US [7] and EU [8–10] biosimilar guidelines. To align with the totality-of-evidence approach, it is essential to define Critical Quality Attributes (CQAs) in accordance with the requirements of ICH guidance documents (ICHQ8–11) [11–14], which outline the principles of pharmaceutical development relevant to Quality by Design (QbD). A fundamental aspect of biosimilar development involves the comprehensive characterisation of multiple batches of the reference products to establish quality ranges (QR). The current biosimilarity study focuses on the analytical and functional similarity evaluation of RGB-14-P (proposed biosimilar to P rolia®), RGB-14-X (proposed biosimilar to ® Xgeva ) and Prolia®, Xgeva® reference drug products. Additionally, the study includes an evaluation of the distribution of the reference product quality data as a function of expiry dates. 2 Materials and Methods 2.1 Evaluation Strategy for the Determination of Quality Ranges (QR) In the final similarity study, a total of 49 EU P rolia®, 34 US ® ® ® Prolia , 40 EU X geva and 25 US X geva batches were analysed alongside 8 RGB-14-P and 6 RGB-14-X drug product batches. These RGB-14 batches were produced from independent drug substance batches using the final commercial process and scale. QR were determined by measuring the reference product batches over several years using available analytical and functional methods. Compared with the biosimilarity literature [15–21], an outstanding number of methods was used. The descriptions of the major and critical methods and the obtained results are given in this publication, whilst the remaining descriptions and data are provided in the Supplementary Material in Table 1. For the quantitative results, the QR calculation was based on the formula mean ± X × SD (standard deviation of the method), determined from the results of the analysis of available EU and US P rolia®, Xgeva® batches. For the EU market, a fixed standard deviation multiplier X = 3 Á. S. Garai et al. was applied (i.e. mean ± 3 × SD) across all quantitative methods. In contrast, for US Prolia® and Xgeva®, a more rigorous range approach was used with parameter-specific standard deviation multipliers according to FDA requirements [7]. The value of ‘X’ was determined on the basis of the criticality of the quality attribute, the specificity of the parameter (how specific is the evaluated parameter for the attribute being considered) and the precision of the test method (RSD%). The ratings can be high, moderate or low, and scores between 2 and 3 (...truncated)