Immunogenicity of Biosimilars

Current Regulatory Expectations

The FDA’s latest guidance — Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Updated Recommendations for Assessing the Need for Comparative Efficacy Studies (October 2025) marks a pivotal shift in how biosimilarity can be demonstrated. Under this framework, a Comparative Efficacy Study (CES) may be waived when residual uncertainty about clinically meaningful differences has been resolved through a combination of analytical, pharmacokinetic, and immunogenicity data. This places immunogenicity characterization at the heart of the biosimilar development package. A comprehensive in vitro immunogenicity assessment directly strengthens the scientific justification that sponsors must provide to support a CES waiver. Far from being a supplementary exercise, in vitro immunogenicity data is now part of the evidentiary foundation upon which streamlined biosimilar approvals are built.

Demonstrating Biosimilarity in T Cell Antigenicity

ProScern® CFSE DC-T Cell Assays

CD4+ T cell responses are the primary driver of anti-drug antibody (ADA) production. Without helper CD4+ T cell activation, high-affinity ADA responses cannot mature. Demonstrating that your biosimilar does not elicit a greater CD4+ T cell response than the reference product is therefore central to any immunogenicity comparability argument.

This assay provides a direct cell-based comparative measure of this CD4+ T cell antigenicity. Monocyte-derived dendritic cells (DCs) are generated from HLA-diverse healthy donor panels, ensuring that T cell epitope recognition is assessed across a broad range of MHC class II genotypes. This maximises the likelihood of detecting immunogenic T cell epitopes that may be recognised across the wider human population. Your biosimilar and reference product are processed as independent test articles within the same assay run: DCs naturally internalise, process, and present protein-derived peptides on their surface via MHC class II, mirroring the pathway by which T cell responses are initiated in vivo. Co-incubation with CFSE-labelled PBMCs from the same donor enables precise quantification of CD4+ T cell proliferation by flow cytometry.

The result is a direct, side-by-side readout of T cell antigenicity between your biosimilar and reference product providing the comparative immunogenicity data needed to support your biosimilarity case and contribute to the scientific justification for a CES waiver.

Demonstrating Biosimilarity in Innate Immunogenicity

ProStorm® Cytokine Release Assay

Process-related impurities, including aggregates and host cell proteins, can activate innate immune pathways and act as adjuvants that potentiate downstream adaptive ADA responses. Detecting and comparing innate immune activation between your biosimilar and reference product is therefore an important dimension of a comprehensive immunogenicity comparability assessment.

This assay provides a sensitive, whole blood-based comparative measure of innate immune activation. Unlike cell-based systems that rely on isolated or cultured immune cells, the assay uses undiluted fresh whole blood, preserving the full complement of innate immune effector cells and plasma components present in the physiological state. Test articles are introduced within three hours of blood draw and incubated for 24 hours, after which plasma is isolated and a panel of key pro- and anti-inflammatory cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, TNFα and IFNγ) is quantified using a qualified and sensitive immunoassay.

This assay is supported by a Type V Drug Master File submitted to the US FDA, encompassing full validation data and SOPs, and has been independently referenced by the FDA in generic peptide ANDA submissions. While the biosimilar and generic peptide pathways are distinct, this recognition underscores the scientific validity and regulatory acceptability of the ProStorm® assay as a standardised immunogenicity assay.

The result is a direct, side-by-side comparison of cytokine release profiles between your biosimilar and reference product. Equivalent profiles provide evidence that your biosimilar does not carry a greater innate immunogenic risk, contributing to the broader immunogenicity comparability package needed to support your biosimilarity case and scientific justification for a CES waiver.

Demonstrating Biosimilarity in Antigen Presentation

ProPresent® MAPPS Antigen Presentation Assay

Despite sharing the same primary amino acid sequence as the reference product, a biosimilar can present a distinct repertoire of T cell epitopes. Differences in glycosylation, higher order structure, aggregation propensity, and process-related impurities can all influence how antigen presenting cells (APC) process and display peptide fragments via MHC class II. Empirically mapping the naturally processed and presented peptidome of your biosimilar and reference product in parallel is therefore a powerful approach to detecting immunogenic differences that sequence identity alone cannot reveal.

This assay directly identifies the peptides that APCs naturally process and present to T cells. Your biosimilar and reference product are processed as independent test articles within the same assay run by monocyte-derived DCs via endogenous antigen processing pathway. HLA-peptide complexes are recovered by immunoprecipitation, peptides are eluted and sequenced by LC-MS/MS. Identified sequences are subjected to rigorous analysis to confirm true positive epitopes with high confidence. The assay covers HLA-DR, DP and DQ, capturing the full breadth of MHC class II presentation relevant to CD4+ T cell activation, as well as HLA class I where CD8+ T cell responses are of interest. With a turnaround time of three to four weeks, the assay is designed to integrate efficiently into your development timeline.

Working with ProImmune

Building the immunogenicity assay platforms described here requires significant scientific infrastructure, specialised technical expertise, and time. ProImmune offers these capabilities as a dedicated service, working closely with biosimilar developers to design studies that are fit for purpose for their specific program without the burden of establishing these assays in-house.

Find out more from our customer’s testimonial here.

To find out more about how ProImmune can support your biosimilar immunogenicity program, reach out to us at enquiries@proimmune.com