As noted below, the comparison of absolute immunogenicity rates between different studies has its limitations; however, relative comparisons within any one given study are meaningful. All biosimilars showed numerically lower ADA and Nab rates than the reference product. These differences in ADA and Nab rates can be consistently observed in both PK studies and CES (see Figs. 1 and 2). This result highlights the main conclusion of our analysis, i.e., that the single-dose comparative PK studies are sensitive at detecting immunogenicity, a finding that in all instances is confirmed by the multidose CES in patients. Kurki et al. also reported consistent immunogenicity results between PK studies and CES for biosimilars in Europe [19]. This cumulative evidence supports the conclusion that in most cases comparative PK studies plus analytical data are sufficient for evaluating comparative clinical immunogenicity between proposed biosimilars and the corresponding reference product. Although single-dose PK studies in healthy volunteers may suffice, biologicals that cannot be compared in healthy volunteer studies for safety reasons may require comparative PK studies in patients, using the appropriate dosing schemes. We acknowledge situations wherein PK studies are not relevant, e.g., products with intraocular or intrathecal administration.

The immunogenicity data depends upon the performance characteristics of the respective assays, such as sensitivity, drug tolerance, and cut-off level to differentiate true signals from noise. Therefore, the actual numerical values typically vary between studies by different developers, which limits the comparison of absolute antibody rates between different products. Notably, the pivotal clinical studies of the reference product [35] showed lower post-baseline rates of ADAs (i.e., 9.5% and 6.8%, respectively) and Nabs (i.e., 4.7%) than were shown in any of the biosimilars studies (see Figs. 1, 2). It is apparent that the ADA and Nab assays conducted by biosimilar companies have higher sensitivity, leading to a higher detection rate of ADAs and Nabs. In the most pronounced case of high assay sensitivity in the Bmab 1200 studies, nearly all Bmab 1200 study participants showed positive ADA at least at one visit. The difference in ADA and Nab rates for the reference product between the biosimilar and the pivotal clinical studies is not new, as it has also been observed for other biological therapies over time [19]. The higher assay sensitivity in the later studies likely also reflects the evolving state-of-the-art in ADA and Nab assay technology.

The intrinsic immunogenicity of a biotherapeutic is a consequence of its quality attributes. It is governed first and foremost by the amino acid sequence, which determines the T cell epitopes that can be presented to and recognized by the immune system. In addition, certain process- and product-related impurities may further increase immunogenicity and must be controlled to sufficiently low levels.

Quality attributes that may be potential impurities that increase immunogenicity include aggregates [36] or any glycans not naturally occurring in the human body [37]. Certain non-human glycans include α-1,3 galactose (α-1,3 gal) and NGNA, both of which can be synthesized in small amounts in the mammalian cell lines used for the production of biotherapeutics. Evidence shows that α-1,3 gal has immunogenic potential; for example, it was linked with allergic reactions after ingestion of red meat and with anaphylaxis of cetuximab in sensitized patients [37, 38]. While cetuximab contains exposed α-1,3 gal epitopes in the fragment antigen-binding (Fab) part of the antibody, it has also been shown that more sterically shrouded α-1,3 gal epitopes in the fragment crystallizable (Fc) part, such as in ustekinumab, can be recognized by anti–α-1,3 gal antibodies and may therefore exhibit immunogenic potential [39].

The EPARs and FDA review information of the seven approved biosimilar ustekinumab products discussed herein reported similar levels of aggregates between the biosimilars and the reference product [25,26,27,28,29,30,31,32,33]. No quantitative information was published on process-related impurities such as host cell proteins. However, differences in non-human glycans were reported (see Table 1). Notably, the biosimilars show consistently lower levels of α-1,3 gal, and all but one biosimilar also showed lower levels of NGNA than the reference product, regardless of whether the biosimilar was made with CHO or SP2/0 cell lines. While these differences may partly explain the numerically lower immunogenicity rates of the biosimilars, they were deemed acceptable and with no clinically meaningful differences. As discussed, assay sensitivity and study design differences also contribute to the immunogenicity results in the clinical studies and should be considered in the interpretation of data. The ustekinumab case study is a particularly useful example, as we have a significant number of biosimilar candidates and their reviews are available to examine the sensitivity of a single-dose clinical PK study, when compared to a CES, for immunogenicity detection.

The EPARs and FDA review information include antibody rates over time data showing that both ADAs and Nabs develop quickly, often within days or weeks after the onset of the PK study, enabling a robust comparison of immunogenicity well within the study’s duration. Regarding the reported consequences, ADAs and Nabs slightly affect ustekinumab serum concentrations, but without ultimately impacting the PK assessment of the biosimilars. There were no adverse effects in safety or efficacy for patients treated with either product in the CES who developed ADAs and Nabs [25,26,27,28,29,30,31,32,33].

Overall, our study supports the view that in most cases a CES does not add new information with regards to the totality of evidence for establishment of biosimilarity over what is provided by the results of CAA and comparative PK studies. In addition, the ustekinumab case study supports the conclusion of the predictive nature of the analytical assessment for comparable immunogenicity, a principle that has been applied in the regulation of process manufacturing changes of biologics for over 3 decades [40].