Immunogenicity Testing: FDA Finalizes Guidance on Assay Development and Validation

Regulatory NewsRegulatory News
| 23 January 2019 | By Zachary Brennan 

The US Food and Drug Administration (FDA) on Wednesday finalized guidance to help industry develop and validate immune assays for the assessment of the immunogenicity of therapeutic protein products during clinical trials. 

The 33-page guidance features updated information on the development and validation of screening assays, confirmatory assays, titration assays and neutralization assays, as well as an additional discussion of immunogenicity risk assessment.

The recommendations for assay development and validation provided in this document apply to assays for the detection of one or more anti-drug antibodies (ADAs), the guidance says.

The guidance finalizes the April 2016 revised draft guidance for industry entitled "Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products" and includes a revised title.


Information on immune responses observed during clinical trials, particularly the incidence of ADA induction or any implications of ADA responses affecting pharmacokinetics, pharmacodynamics, safety or efficacy, is crucial for any therapeutic protein product development program, FDA explains. Accordingly, the agency says that such information, if applicable, should be included in the prescribing information as a subsection of the ADVERSE REACTIONS section entitled “Immunogenicity.”

And for the purposes of this guidance, immunogenicity is defined as the propensity of a therapeutic protein product to generate immune responses to itself and to related proteins or to induce immunologically related adverse clinical events.

Assay Design and Testing Strategy

FDA says it recommends a multi-tiered ADA testing approach, where a sensitive screening assay is initially used to assess clinical samples.

“To gain a more accurate understanding of the natural history of the ADA response, the screening assay should be sensitive and designed to detect low levels of low- and high-affinity ADA; for example, by minimizing wash steps. However, in most cases it is not necessary to empirically determine the affinity of antibodies that are detected by the initial screening assay,” FDA says.

Samples testing positive in the screening assay are then subjected to a confirmatory assay to demonstrate that ADAs are specific for the therapeutic protein product.

“For example, a competition assay could confirm that an antibody is specifically binding to the therapeutic protein product and that the positive finding in the screening assay is not a result of non-specific interactions of the test serum or detection reagent with other materials in the assay milieu such as plastic or other proteins,” the guidance says.

And samples identified as positive in the confirmatory assay should be further characterized, FDA says, in other assays, such as titration and neutralization assays, while in some cases, assays to detect cross-reactivity to other proteins may be necessary.

The guidance then goes into greater detail on immunoglobulin isotypes or subtypes, domain specificity, assay cut-point, sensitivity, specificity, selectivity, precision and reproducibility. Selection of reagents, how to report results for qualitative and quasi-quantitative assays as well as other considerations are also included in the guidance.

Sections on assay development, validation, the implementation of assay testing and documentation are also discussed.

Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection Guidance for Industry


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