7.3.2 A single assay

Biosimilarity: The FDA Perspective

276 The platforms that are typically utilized for these assays are ligandbinding assays via ELISA and electrochemiluminescence methods; however, several emerging technologies can also be utilized, such as BiaCore or Octet, Luminex, and Gyrolab. Another critical component of all immunogenicity assays is the production of an appropriate PC and may include the use of commercially available polyclonal or antiidiotypic antibodies to the reference product, affinity-purified ADAs to the biosimilar and/or reference product, as well as immune serum from hyperimmunized animals dosed with the biosimilar and/or the reference product. It is important to collect serum from any animals evaluated during preclinical testing that could potentially provide an ADA PC for the immunogenicity assay in order to limit the number of animals required to procure this necessary reagent. The need for one or more PCs may also be influenced by the strategy that will be used for the assay(s) and the overall development. There are two predominant strategies for proceeding through an immunogenicity testing plan based on the utilization of a single assay or two separate assays. Using either strategy, the assays must have a high level of similarity for all parameters evaluated. A typical ELISA-based immunogenicity assay consists of the drug bound to a 96-well plate, which is used to capture any ADAs that are present in the serum or plasma samples when they are applied to the plate. This complex is then detected using an enzyme-labeled form of the drug in the case of a bridging assay. The levels of colorimetric or relative light units that are detected in the assay correspond to the level of ADAs present in the serum or plasma samples. The assay is evaluated based on sensitivity (the lowest amount of antibody that can be detected in the assay), precision, drug tolerance, specificity, selectivity, prozone, and accuracy of titration.

The single assay is developed for both products, and a limited set of parameters should be evaluated during validation including screening cut-point, specificity cut-point, precision, and drug tolerance for both products in order to ensure that the assay is capable of detecting antibodies from both the biosimilar and the reference product. This may involve the conjugation of a reporter molecule such as biotin or ruthenium to both the biosimilar and the reference product to facilitate the detection of antibodies when a bridging assay format is utilized. It is critical to ensure that the method of conjugation is tightly controlled and that these reagents are labeled in a reproducible manner in order to obtain comparable signals between the two reagents. Although the assay is essentially developed as two assays with respect to screening cut-point and precision, all other parameters should be evaluated on the background of the biosimilar assay (a plate coated with the biosimilar and detected with an enzyme-labeled biosimilar) where the biosimilar will be used