Comparative immunogenicity assessment Review
ate use, and the integrity of its immune epitopes must be highly ensured. Reagent preparation including purification, chemical labeling and any other modifi- cations to ensure consistent assay signal across differ- ent lots is important for assay performance during a long-term implementation. For the digoxigenin-based enzyme linked immunosorbent assay (DIG-ELISA) [21], optimal labeling condition for both DIG conju- gation and biotinylation of the therapeutic can help to retain its epitope integrity and thereby detect ADA more efficiently. The same principle applies to the elec- trochemiluminescence assay from Meso Scale Discov- ery where drugs are biotinylated and sulfo-tagged for ADA detection. For product-specific assay approach, the preparation of reagents and any required modifica- tion including chemical labeling should be conducted by following the same protocol for both proposed bio- similar product and the reference product in order to achieve assay performance equivalence and to have a scientifically sound and meaningful foundation for the comparison of data generated from these assays.
Assay controls Negative control Negative control is used to provide the base for comput- ing assay cut point for each individual assay run, and it can also serve as a ‘safe guard’ to monitor assay perfor- mance during the assay implementation. The negative control should be prepared from a pool of therapeutic- naive matrix samples from representative individuals who have not received the therapeutic product of inter- est. Regardless of one-assay or product-specific assay approach to be adopted, the same negative control shall be employed for evaluation of immunogenicity of both proposed biosimilar and reference products. Negative control should be considered as a critical reagent for an ADA assay, and if possible the same pool is highly recommended to apply for both method validation and sample analysis to ensure consistence of assay perfor- mance. If a new pool needs to be introduced during the sample analysis, it should be qualified against the pool used for the validation. Otherwise, a partial vali- dation needs to be conducted to ensure the new lot to be suitable for the study.
Positive controls ADA positive controls are used not only for evaluat- ing assay parameters such as relative sensitivity and therapeutic tolerance (aka drug tolerance), but also for monitoring assay performance during sample analy- sis. Positive controls should play a critical role in the cross-assessment of the assay performance when two product-specific assays are implemented, and demon- stration of the assays’ capabilities of detecting immune
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responses toward proposed biosimilar product and reference product for the one-assay approach. A set of monoclonal antibodies with well-defined epitope spec- ificity against the reference product and the proposed biosimilar product can be useful in the development of ADA assay. If available, such information is of value in the demonstration of immunological similarity or the identification of unique epitopes of the two products. Despite the sporadic use of monoclonal antibody in
the ADA assay, polyclonal antibodies are used more commonly as surrogate ADA positive control to mimic the reality of polyclonal antibody responses against biologics and other antigens in human. Since positive controls from human subjects are not often readily available, animal such as rabbit or monkey are usu- ally the main source to raise polyclonal antibodies for the positive controls. It should be noted that due to polyclonal nature of these antibody reagents (usually generated by hyperimmunized animals), even the same product can elicit different immune responses in dif- ferent animals. Composition of antibodies in different animals is most likely different. Therefore, an adequate number of animals should be immunized and their antisera be mixed to maximize the chance to generate representative ADA positive controls. Product-specific ADA positive controls should be considered for both proposed biosimilar product and reference product, and the generation and preparation of these positive controls should follow the same established proce- dures. Due to the difference in immune response to the therapeutic product of interest between animal and human being, the antibodies raised from animals may not correspond to all epitopes that would be rec- ognized by human immune system and detected in clinical samples. This limitation should be taken into consideration when interpreting data from the assay validation for the demonstration of assay equivalence. It is common that one positive control (usually anti-
bodies against the proposed biosimilar product) is used when the product-specific assay approach is adopted to allow meaningful cross-comparison of assay per- formance, while two positive controls for proposed biosimilar and reference product each are generally employed in the one-assay approach. For the one-assay approach, cross-reactivity of the positive control anti- body to a proposed biosimilar product and its reference product should be determined during the ADA assay development. Antibodies with equivalent binding pro- file including affinity toward the two products should be highly considered.
Screening & confirmatory assay cut points Various approaches to the cut point determination have been published [13,22]. Regardless of the assay format,
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