Comparison of assay formats for drug-tolerant immunogenicity testing.

BACKGROUND: Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development. RESULTS: Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(®) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive. CONCLUSIONS: Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.

An Affinity Capture Elution (ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug.

Monoclonal antibody therapeutics typically have relatively long half-lives and can be dosed at high levels. Although formation of anti-drug antibodies (ADA) is relatively rare, detection of these antibodies can be very difficult in the presence of high circulating levels of drug. Typically these ADA are detected by bridging ELISAs which can be very sensitive to even low levels of drug. We describe an ELISA method based on affinity capture of ADA on solid-phase drug followed by removal of excess free drug, release and transfer of bound ADA and subsequent detection using biotinylated drug. The assay is both sensitive and highly tolerant to free drug with detection of 500 ng/ml of ADA readily achieved in the presence of 500 mug/ml of drug.