Impact of positive control binding properties on anti-drug antibody assay performance

Monitoring anti-drug antibodies (ADAs) is a critical component of recombinant protein drug development. Positive controls in ADA assays are essential for evaluating assay quality, yet the influence of their binding properties on assay performance is not well understood. We evaluated a panel of surrogate positive controls with varying binding characteristics to investigate how their affinity and kinetic parameters impact ADA assay performance. Binding properties were measured using Bio-Layer Interferometry (BLI), and assays were evaluated for relative sensitivity and drug tolerance. This was a laboratory-based study; no human or animal subjects were involved. We observed a correlation between higher affinity (lower K_D (equilibrium dissociation constant)) and lower k_off (off-rate constant) with increased relative assay sensitivity. However, no consistent relationship was found between these binding parameters and drug tolerance. These findings suggest that binding kinetics of the positive control can significantly influence sensitivity but may not predict drug tolerance. Understanding the relationship between positive control binding properties and ADA assay performance can support the selection of reagents that optimize sensitivity. Limitations include the use of surrogate positive controls, which may not fully replicate the complexity of clinical ADA responses. When developing protein-based drugs, it’s important to test for anti-drug antibodies (ADAs), which can interfere with how well a drug works. These tests use positive control reagents to ensure the assay is working as expected. However, it’s not fully understood how the binding characteristics of these controls affect test results. In this study, we tested a group of lab-generated positive controls with different binding properties, including how tightly they bind to the drug and how quickly they bind and fall off. Using a technique called Bio-Layer Interferometry, we measured the strength and speed of these interactions. We then evaluated how these binding properties impacted the sensitivity of the ADA test and its ability to function in the presence of the drug (drug tolerance). We found that tighter binding and slower dissociation were linked to greater test sensitivity. However, there was no clear relationship between the binding properties and drug tolerance. This suggests that while the binding strength and kinetics of the positive controls can influence test sensitivity, they may not predict how the test performs when drug is present. These findings could help improve assay design, though it’s important to note that our use of lab-made controls may not fully replicate the complexity of actual patient antibodies.