Immunogenicity Testing Assay Validations: Regulatory Perspective and Recommendations

FDA guidance for Immunogenicity Testing Assays: Development & Validation of Anti-drug Antibody Assays

The FDA has now finalized its guidance for the development and validation of assays for immunogenicity testing. This guidance supersedes other previous guidance for related to bioanalytical assay validation for immunogenicity testing. The objective of this guidance is to facilitate the industry’s development and validation of assays for assessment of the immunogenicity of therapeutic protein products during clinical trials. More specifically this guidance provides a stepwise approach to develop, validate the anti-drug antibody assay. The guidance helps to answer the following questions:
a) Why it is important to assess immunogenicity?
b) What is the ADA assay?
c) When is it appropriate to develop ADA assays and assess immunogenicity?
d) How to develop reliable, sensitive and specific ADA assays?

Why it is important to assess immunogenicity?
Immunogenicity is defined as the propensity of a therapeutic protein product to generate an immune response to itself and related proteins or to induce immunologically related adverse clinical events. The immunogenicity assessment allows an understanding of the potential impact of an immune response to pharmacokinetics, pharmacodynamics, safety, and efficacy. Therefore, understanding the relationship of ADA to PK/PD profile, adverse events and efficacy enable the sponsors to design a dosage regimen, prevent and mitigate life-threatening adverse events, formulate risk evaluation and mitigation strategy (REMS), etc. The immunogenicity assessments encompass the evaluation of:
a) incidence,
b) magnitude,
c) persistence and
d) effects such as neutralization activity, enhanced drug clearance, etc.

Figure 1: Immunogenicity Risk Assessment and Testing

What are the ADA assays?
Anti-Drug Antibody assays are designed to detect the drug-specific antibodies that could mediate unwanted biological or physiological consequences such as neutralizing activity or hypersensitive response. The propensity and type of immune response vary with the structural and molecular properties of the drug product. Therefore, the immunogenicity assessment is tailored to fit the immunogenicity risk of the product. Similarly, the assays designed to evaluate immunogenicity are not universal and a risk-based multi-tiered approach is used for evaluating immunogenicity. The sponsor is encouraged to discuss their immunogenicity testing plans to the FDA to better align the drug development programs with scientific and regulatory requirements.

The ADA assay may broadly be classified into two different assay categories:
a) Detection Assay
b) Characterization Assay

a) Detection Assay: The ADA detection assay is designed to identify the presence or absence of antibodies generated against the therapeutic candidate in the clinical samples. The detection assay includes screening and confirmation assay. Screening assay or binding assay is used to identify the potential positive antibodies. The confirmatory assay or specificity assay determines the specificity of the antibodies detected in the positive screening assay. The specificity of the antibodies may be demonstrated by utilizing a free therapeutic drug to competitively inhibit the assay signal response.

b) Characterization Assay: The characterization assays are designed to evaluate the parameters such as the magnitude of the ADA response, neutralizing capacity, cross-reactivity to endogenous counterparts, etc. The characterization of the antibodies allows evaluating the impact of ADA in PK, PD, safety, and efficacy of the drugs.

Table 1: Assay for Detection and Characterization of Anti-drug Antibodies 

Assay Type
Assay Tier
Screening (Tier 1)
-Detect potentially positive samples for anti-drug antibodies
Confirmatory (Tier 2)
-Determine specificity of antibodies against therapeutic candidate detected in the screening assay
Titer (Tier 3)
-Determine the magnitude of the drug-induced or drug boasted ADAs
-Evaluate whether the ADAs interfere the drug activity such as receptor binding,
Isotype Characterization1
-Evaluate IgG and IgM response against the therapeutic product and explore the impact on the effector function such as complement activation, immune complex formation.
-Evaluate IgA, when the route administration is mucosal
-Evaluate IgE isotype for the product that has a higher risk or incidence of anaphylaxis- such as PEGylated proteins
-Evaluate IgG4 sub-isotype in cases of chronic exposure of treatment
-Evaluate the cross-reactivity to the endogenous molecules when the drug product has close homology such as erythropoietin.
Domain Specificity1
-Characterization domain-specific antibodies for multiple domain therapeutics such as PEGylated proteins, bifunctional antibodies etc.
Epitope Mapping1
- Characterize predominant antigenic epitopes that may be mutated during manufacturing or process development.
1  These assay may consist of all three tiers.

Figure 2: Multi-Tier Approach To Anti-drug Antibodies

When to develop ADA assays?
The optimal time to design, develop and validate ADA assays during therapeutic protein product development depends on the risk assessment of the product. It is recommended to provide an immunogenicity risk assessment and the rationale for the immunogenicity testing paradigm with an investigational new drug. FDA considers that immunogenicity testing for phase 1 and 2 study may be sufficient with the qualified assay, but pivotal study should use a validated assay.

How to develop reliable, sensitive and specific ADA assays for immunogenicity testing? (Please click here)

- Immunogenicity testing is crucial for understanding the relationship of the immune response to pharmacokinetics, pharmacodynamics, safety, and efficacy.
- The immunogenicity testing paradigm is not universal for all the biological products and mainly driven by immunogenicity risk of the product
- The reliable, sensitivity, specific and valid ADA assay method development and validation require testing of key aspects of the assay such as cut point, drug tolerance, etc.
- FDA also expects the sponsor to conduct a systematic and methodological assay development and ensure that the assay has sufficient sensitivity to detect ADA that could mediate biological or physiological consequences.
- FDA expects the submission of the validation report with the biologic license application.
-Finally, the current document also provides the recommendation specific for neutralizing antibody assays, selection of assay format, positive controls, sample testing and documentation requirement that are/will be discussed in another subsequent post.

Immunogenicity Assessment for Therapeutic Protein Products: US FDA 2014
Guideline on Immunogenicity assessment of therapeutic proteins: EMA 2017
Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products
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