A biosimilar is defined as a biologic product, highly similar to a reference or originator, with no clinically meaningful differences in terms of safety, purity, and potency. Biosimilars offer a cost-effective alternative without compromising safety or efficacy. Their development hinges on demonstrating comparability, not de novo efficacy, across analytical, functional, and clinical domains. There are 4 key requirements for biosimilarity:
- Utilisation of the same mechanism of action, as far as it is understood.
- Indication for the same condition of use as the reference product.
- Identical route of administration, dosage form, and strength.
- Demonstrated analytical similarity through physicochemical and functional testing.
The overarching goal is not to replicate every study conducted for the originator, but to provide a scientifically justified package that proves the biosimilar behaves equivalently.
Analytical Strategy: A Foundation of Comparability
Analytical comparability is the cornerstone of biosimilar development. Assays must be capable of detecting subtle differences in structure, binding kinetics, and functional activity. Ligand binding assays (LBAs) are frequently used to evaluate binding affinity, specificity, and biological activity. Absorbance and electrochemiluminescence platforms such as MSD, can provide high sensitivity and throughput for comparative testing.
One of the primary decisions in assay development is whether to adopt a single-assay or two-assay approach. EMA and FDA generally prefer single-assay formats, provided they are validated to detect both products with equal sensitivity and specificity.
- Single-assay approach: Utilises a universal method to measure both biosimilar and reference products under identical conditions. This is cost-effective and aligned with regulatory preference, provided analytical performance is equivalent across both products.
- Two-assay approach: Employs separate methods for each product, allowing greater customisation but increasing complexity and cost. This route requires full justification and is generally reserved for cases where a single method fails to provide adequate comparability due to differing binding properties.
Validation of these assays requires careful assessment of calibration range, precision, accuracy, and stability, using materials derived from both the biosimilar and the reference product.
Practical Implementation: Tailoring Assay Design
Effective assay development begins with understanding the reference product including its mechanism of action, known impurities, and immunogenicity profile and securing high-quality materials for development and validation. Lot-to-lot variation, market exclusivity, and sourcing constraints can pose logistical challenges, particularly for originator products no longer in active production.
Impurity profiling, potency assays, and functional bioassays must be rigorously qualified to detect meaningful differences in product attributes. Sensitivity, reproducibility, and regulatory compliance are essential throughout.
Pharmacokinetics (PK): Confirming Exposure Equivalence
PK studies are central to biosimilarity evaluation, aiming to demonstrate equivalent systemic exposure between the biosimilar and the reference product. These studies typically involve healthy volunteers and are designed with sufficient power to detect differences in key PK parameters. Regulators expect 90% confidence intervals for key PK parameters to fall within the equivalence range (typically 80–125%) to establish exposure similarity.
A validated bioanalytical method must accurately quantify both products within the same assay system. When feasible, the same product should be used to generate both calibration standards and quality controls. However, if cross-reactivity or assay performance diverges, alternate approaches using both products may be needed.
Equivalence in calibration response, precision, and accuracy across products must be demonstrated statistically. Where differences exceed acceptable thresholds, full validation with both products is required.
In scenarios where CES is waived, extended PK studies may play a dual role — not only assessing exposure equivalence but also generating longitudinal immunogenicity data. This is essential when:
- Anti-drug antibodies (ADAs) may emerge late
- Neutralising antibodies could impact efficacy
- Hypersensitivity or infusion reactions are a known risk
Sponsors should consider multiple dosing and prolonged follow-up to capture the full ADA profile, especially for biologics with immunogenic potential.
Immunogenicity: Assessing Anti-Drug Antibodies (ADAs)
Immunogenicity remains a critical safety consideration in biosimilar development. ADA assays must detect antibodies generated in response to either the biosimilar or the reference product.
ADA assessment typically follows a tiered structure:
- Screening assay to identify potential responders.
- Confirmatory assay to determine ADA specificity.
- Titer and neutralising antibody (NAb) assays, as applicable, to quantify response and assess functional impact of ADAs.
Key validation parameters include antigenic equivalence (comparable inhibition of signal), confirmatory cut-points established for both products, and drug tolerance, which measures the ability of the assay to detect ADAs in the presence of circulating drug. The ADA-positive control must exhibit equivalent binding to both the biosimilar and the reference compound.
A two-assay design may be used in rare cases where antibodies are highly specific to one product. However, this increases assay and data interpretation complexity and is generally discouraged without clear justification.
Pharmacodynamics (PD): Optional but Insightful
PD endpoints, while not required in most biosimilar submissions, can provide valuable supportive evidence of biosimilarity. The inclusion of PD biomarkers is most appropriate when they are sensitive to the drug’s mechanism of action and display well-characterised kinetics and dynamic range. For example, absolute neutrophil count has served as a sensitive PD marker in G-CSF biosimilar programs, while CTX is a well-established biomarker of bone resorption that can provide supportive evidence of biological activity in denosumab programs.
Validation of PD assays must reflect the intended context of use, ensuring that at a minimum, parameters such as linearity and precision are addressed. While only a minority of approved biosimilars to date include PD data, their strategic use can bolster similarity claims, especially in cases where clinical efficacy trials are waived.
When is Clinical Efficacy No Longer Required?
As regulatory science matures, agencies like the EMA and FDA increasingly recognise that high-quality analytical and PK comparability can obviate the need for large-scale confirmatory efficacy trials. This is especially true when:
- The mechanism of action (MoA) is well understood
- Critical quality attributes (CQAs) are thoroughly characterised and matched
- Functional similarity is demonstrated through in vitro assays
- A robust comparative PK study confirms similar exposure
EMA’s 2025 draft reflection paper reinforces this evolution, stating that confirmatory efficacy studies (CES) add limited value when analytical, PK, and PD data (where available) are robust. In such cases, tailored clinical programs focusing on PK and immunogenicity may be sufficient.
Conclusion
Biosimilar development is a scientifically demanding process requiring a strategic blend of analytical precision, regulatory alignment, and clinical insight. As the field evolves, regulatory agencies are increasingly open to reducing clinical burden when analytical and PK data are compelling. The focus, therefore, remains on developing fit-for-purpose, highly sensitive assays that can robustly confirm biosimilarity across all critical domains, helping bring affordable therapies to patients faster.
With well-planned assay strategies and an evidence-based mindset, biosimilar programs can achieve both scientific credibility and commercial success in this growing sector.
