FDA Releases Final Guidance on Immunogenicity Testing for Biologics and Biosimilars

The FDA recently released final guidance about the use of therapeutic proteins in developing biologics and biosimilars. "Immunogenicity Testing of Therapeutic Protein Products—Developing and Validating Assays for Anti-Drug Antibody Detection" represents current FDA thinking about developing and validating assays for anti-drug antibody (ADA) detection.

The FDA recommends a multi-tiered testing approach, and the document spells out the development and validation of screening assays, confirmatory assays, titration assays, and neutralization assays. Screening assays, also known as binding antibody assays, are used to detect antibodies that bind to the therapeutic protein product. Confirmatory assays establish the specificity of ADAs for the therapeutic protein. Titration assays characterize the magnitude of the ADA response. Neutralization assays assess ADA for neutralizing activity.

The agency notes that setting the appropriate cut-point in assays “is critical to minimizing the risk of false-negative results.”

The document also discusses considerations for assay:

• Sensitivity: The FDA is recommending that screening and confirmatory IgG and IgM ADA assays achieve a sensitivity of at least 100 nanograms per milliliter (ng/mL), although a limit of sensitivity greater than 100 ng/mL may be acceptable depending on risk and prior knowledge. Previously, the agency recommended sensitivity of at least 250 to 500 ng/mL, but recent data suggest that concentrations as low as 100 ng/mL may be associated with clinical events.
• Specificity: A lack of assay specificity can lead to false-positive results; the FDA noted that there are challenges to demonstrating the specificity of antibody responses to mAb, Fc-fusion proteins, and Ig-fusion proteins, and gives guidance on managing those challenges.
• Selectivity: The FDA advised that assay results may be affected by interference from the matrix or onboard therapeutic protein product, and that failure to establish selectivity can contribute to a non-specific signal, obscuring a positive result.
• Precision: Results should be reproducible within and between assay runs; assay precision is critical to the assessment of ADA because assay variability is the basis for determining the cut-points and ensuring that low-positive samples are detected as positive.
• Reproducibility: A sponsor should establish the comparability of the data if multiple laboratories are running an assay in a study, including assay sensitivity, drug tolerance, and precision.
• Robustness and sample stability: The agency noted that the complexity of bioassays leaves them susceptible to variations in assay conditions; it is essential to evaluate and optimize parameters such as cell passage number, incubation times, and culture media components. The FDA also gave recommendations for managing short- and long-term stability in order to preserve antibody reactivity.
• Format: A sponsor should consider the pros and cons of each type of assay format, including throughput, sensitivity, selectivity, dynamic range, ability to detect various Ig isotypes, ability to detect rapidly dissociating antibodies, and availability of reagents. The number and vigor of washes, which can influence assay sensitivity, and epitope exposure is also important to consider.
• Reagents: If positive control antibodies, negative controls, and system suitability controls are generated specifically for the assay, it is important for sponsors to ensure ensuring consistent assay performance of critical reagents.
• Reporting results: Methods should be evaluated for appropriateness; the most common one is qualitative. The FDA gives guidance on reporting antibody results in ADA positive subjects.

There may be other considerations, such as the use of pre-existing antibodies, rheumatoid factor, monoclonal antibodies, and conjugated proteins, the document noted.

The FDA recommends that sponsors adopt a life-cycle management report, including these sections as the product moves through various stages:

• Immunogenicity risk assessment: This should be specific to the therapeutic protein and include information on quality and subject factors.
• Tiered bioanalytical strategy and assay validation summaries: Sponsors should summarize the assessment strategies for each phase of clinical development.
• Clinical study design and detailed immunogenicity sampling plans: Developers should include the sampling plans for all clinical studies that have an assessment performed.
• Clinical immunogenicity data analysis: Sponsors should include a summary of immunogenicity analyses for all clinical studies having an immunogenicity component.
• Conclusions and risk evaluation and mitigation strategies: Sponsors should provide a discussion of how immunogenicity affects the safety and efficacy of the protein product for the subject population, including how it will be monitored in the postmarketing phase.

On a case-by-case basis, the FDA said, the guidance may also apply to some peptides, oligonucleotides, and combination products.