The robust assays are crucial for facilitating the understanding of immunogenicity and their impact on pharmacokinetics, pharmacodynamics, safety, and efficacy of therapeutic protein products. During development and validation, assay sensitivity, specificity, drug tolerance, precision, critical reagent stability are assessed.
Table 2: FDA guidance for Immunogenicity Testing Assays:Summary of the key assay parameters for developing and validating ADA assays
-The level of response of the assay that defines the sample response as positive and negative.
-Screening, confirmation, and titer cut points are used for three tiers of the assay.
- Cut-points are critical to minimize the risk of false-negative
- Drug naïve individuals from the target population should be used to derive cut point, if feasible.
- Normal healthy individuals obtained from commercial vendors during development and confirmation by study samples when it becomes available.
- The statistical method for deriving cut points is the preferred method.
Sensitivity and Surrogate Positive Controls
-The lowest concentration at which the antibody preparation consistently produces either positive results.
- TAb, IgG, and IgM less than 100 ng/ml.
- IgE assay is less than 10ng/ml.
- Positive control- Affinity-purified antibodies should be used.
- Assay sensitivity may be assessed by testing serial dilution of positive control antibody of known concentration, using individual or pooled matrix from treatment.
- Interpolate values may be used.
The assessment of the sensitivity of the assay in the presence of the expected level of interfering therapeutic protein product.
- Drug tolerance may be assessed by deliberately adding different quantities of the therapeutic protein product
The ability of a method to exclusively detect the target analyte. Lack of specificity leads to false-positive results, obscuring the ADA –PK/PD relationship.
-Drug tolerance may be assessed by deliberately adding closely related proteins to ADAs, including isotype controls.
The ability of the assay to identify ADAs specific to the therapeutic protein product in the presence of other components in the samples
-Matrix interference and minimum required dilution should be assessed by spiking the positive controls in the drug naïve sample.
-Should be assessed using drug naïve samples from clinical subjects
Assay Robustness is an ability to remain unaffected with a small variation on the temperature, incubation times, buffer characteristics, freeze-thaw cycle etc.
Robustness may be assessed by deliberately introducing the small variation of method and instrument performance
Precision is a measure of the variability in a series of measurements for the same material run in a method.
Assessed by evaluating the imprecision (%CV) of low, intermediate and high concentration quality controls (HQC, Intermediate QC, and LQC)
Critical Reagent Stability and lot-lot variation
Critical reagents such as capture and detection reagents, positive control antibodies, cell lines, and passages
Assessed by the ability to remain unchanged with tested storage conditions and durations, lot-to-lot variations, etc.
- 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