Review Liu, Zou, Sadhu, Shen & Nock
our recommendation is to use a floating cut point for the ADA screening assay to accommodate run-to-run assay variations. A cut point factor for the screening assay should be computed based on the signals from a panel of adequate (50 or more) therapeutic naive matrix samples from the targeted study population with the dataset generated from multiple assay runs performed by multiple analysts over a given period of time. For the confirmatory cut point determination, a panel of therapeutic-naive matrix samples (30 or more) from the targeted study population should be analyzed in the presence of an excess concentration of the thera- peutic of interest for binding competition. The signal reduction by the excess therapeutic product relative to the signal without the therapeutic is used to calculate the confirmatory cut point [13]. The outliers should be identified and thoroughly investigated. They should be excluded from the cut point determination with proper scientific justification. It must be noted that the cut point for the ADA
screening assay and the cut point (threshold) for the confirmatory assay are based on theraputic-naive sam- ples. Their assay signals reflect the magnitude of inher- ent interactions of matrix components with the assay reagents, that is, labeled therapeutics (either proposed biosimilar product or reference product). If the prod- uct-specific assay approach is chosen, the same panel of drug-naive samples for cut point determination should be used in both assays for proposed biosimilar product and the reference product. The cut points of the two assays should not be statistically different in order to meet the comparative purpose of the immunogenic- ity assessment of the two products. For assays to be considered comparable or equivalent, lack of statisti- cally significant difference in signals from the thera- peutic-naive samples must be achieved. Otherwise, the assay performance needs to be investigated and/or the similarity of the proposed biosimilar product to the reference product may be in question.
Relative sensitivity Relative sensitivity of an ADA assay is defined as the concentration of a given positive control at the assay cut point. For the determination of the assay sensi- tivity, a serial dilution of the positive control starting from an adequate concentration should be tested in the assay performed by different analysts in multiple runs. For the one-assay approach where different positive controls (one raised against the reference product and another against the proposed biosimilar product) are used, sensitivities of the assay determined from differ- ent positive control antibodies may not be the same if there are differences in affinity and other binding char- acteristics between the two antibodies due to the inher-
378 Bioanalysis (2015) 7(3)
ent variation of immune responses in the immunized animals. Therefore, choosing appropriate positive con- trols with the determination of their cross-reactivity is critical for the evaluation of assay relative sensitivity, which ensures the meaningfulness of immunogenicity comparison of the two products. For the product-specific assay approach, one positive
control will be used to determine the assay sensitivi- ties in separate methods which are developed specifi- cally for the respective products. If there is no differ- ence observed in the cut points of the two assays, the difference in assay relative sensitivities should not be expected. Relative sensitivity will be determined in multiple assay runs with a sufficient statistical power for comparison. Equivalent assay sensitivities dem- onstrate that these assays have similar capability of detecting antibodies against corresponding products. No statistically significant differences in relative sensi- tivity should be detected between the two assays when the product-specific assay approach is chosen.
Therapeutic tolerance & target interference Therapeutic tolerance (aka drug tolerance) is defined as the highest concentration of the therapeutic in the presence of which a sample containing ADA at a given level is still detected as a positive. The assay should have an adequate degree of therapeutic tolerance that ensures detection of potential antibody in the samples with the presence of a certain level of therapeutic from the clinical study. Obviously, Therapeutic tolerance is relevant to the type(s) and levels of the ADA present in the sample. Due to different approaches implemented for
the comparative immunogenicity assessment of
biosimilars among the developers, the practice of therapeutic tolerance determination remains incon- sistent regarding the choice of positive antibodies and drugs (i.e., proposed biosimilar product or reference product). For the one-assay approach, when two positive con-
trols are chosen, we recommend therapeutic tolerance determination should be based on each positive con- trol antibody and its respective therapeutic product. For the product-specific assay approach, where one positive control antibody is employed for different product-specific assays, we recommend the therapeutic tolerance determination should be conducted with the same positive control in each assay using the respective product. If the therapeutic product binds to a soluble ligand,
truncated receptor or binding protein in the blood cir- culation, such a binding target may interfere the detec- tion of ADA and subsequently impact the comparison of immunogenicity of the products. Caution should be seriously taken on the circulating level of such a soluble
future science group
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