Biotherapeutics have proven to be sufficiently effective and have displayed an excellent targeting ability. However, their development and use can be constrained by the immune response from the body. Being large molecule drugs, they risk provoking an immune response in the shape of Anti-Drug Antibodies (ADA).
These antibodies can neutralize the therapeutic effects of the drug. In some cases, they may also cause more serious adverse effects. Immunogenicity testing thus plays a key role in the development of biotherapeutics. This testing involves using ADA assay to detect the presence of antibodies.
A tiered approach is used to see effects ranging from identification to characterization of the antibody response. A panel of immunogenicity assays can be used to get a full picture of the effects and the possible results.

Assay Validation And General Considerations

Validating an assay requires demonstrating that the performance characteristics of the ADA assay used are suitable for its intended use. The extent and scope of validation depend on how far the study has progressed. The second consideration is the risk it poses to subjects. For example, a full validation may not be possible in the preclinical phase. In such cases, a partial validation is acceptable.
There are some considerations that assay validation should include. These are, the cut-point, sensitivity, selectivity, precision, and drug tolerance. Other characteristics like robustness, reproducibility, and in-use stability may also be considered.

ADA Assay Development And Validation

ADA assay for screening is the first one employed for ADA detection. As such, sensitivity plays a key role. FDA recommendation is to pick an assay with a sensitivity of 100ng/mL. The assay should be able to detect low-affinity and high-affinity ADA. Establishing a cut-point usually takes a risk-based approach, where the cut point leads to 5% false positives. Sensitivity and cut point play a key role in the validation of the assay.

The drug tolerance limit is a key factor in evaluating and establishing assay sensitivity. Biotherapeutics have a long half-life and can be expected to be present in the sample matrix. An administered drug usually reaches its lowest concentration right before the next dose is administered. As this is the lowest concentration C(trough) the assay should have a drug tolerance higher than this level.

The ADA assay doesn’t often directly deal with background noise from the matrix. However, that is more the issue for quantitative assays. Therefore, it relies more on the cut point and thus a better statistical analysis is necessary for establishing a suitable cut point.

The method generally used for this assay is ELISA Assay method. However, it has its drawbacks. Therefore, other methods like Meso Scale Discovery (MSD) are used in conjunction with ELISA. Additional methods could be used where necessary.

It is important to note that the ADA assay is qualitative or quasi-quantitative. It detects the presence of binding antibodies but does not confirm if the antibodies are due to the specific biotherapeutic. Confirmation, titration, and neutralization assays are required to detect more information on the ADA. Put together, various immunogenicity assays help understand the full profile of the drug.