Mitigating target interference challenges in bridging immunogenicity assay to detect anti-tocilizumab antibodies

<b>Aim:</b> An assay to detect anti-tocilizumab antibodies in the presence of high levels of circulating target and drug is needed for immunogenicity assessment in comparative clinical studies. <b>Methods:</b> An assay was developed and validated using a combination of blocking agents and dilutions to overcome target interference challenges. <b>Results:</b> No false-positive signal was detected in serum samples spiked with 350–500 ng/ml of IL-6 receptor. As low as 50 ng/ml of positive control antibodies could be detected in the presence of either 500 ng/ml of IL-6 or 250 μg/ml of the drug product. Assay also demonstrated high sensitivity, selectivity and precision. <b>Conclusion:</b> A robust, easy to perform immunogenicity assay was developed and validated for detecting anti-tocilizumab antibodies. The presence of soluble drug targets in biological samples can disrupt anti-drug antibody (ADA) assays. This interference occurs when the drug targets bridge the ADA assay reagents, causing higher background values. As a result, false positive results may occur. Reducing soluble target interference is crucial to obtain unbiased results in ADA assays. These results are necessary for accurately assessing the safety and efficacy of biopharmaceuticals. Previously published reports suggested that IL-6R levels peak around day 12–15 post single dose of tocilizumab and this corresponds to ADA sampling timepoint in clinical studies. However, none of the published reports acknowledge potential interference from IL-6R in bridging format to detect anti-tocilizumab antibodies. Meso Scale Discovery-based bridge assay was developed and validated with an aim to detect presence of anti-tocilizumab antibodies in human serum in presence of varying concentrations of IL-6R. Target interference was observed with 100 ng/ml of IL-6R in spiked samples. Increasing dilution of the samples along with introduction of high concentrations of IL-6 in the assay buffer enabled us to achieve high target (up to 500 ng/ml) and drug tolerance (250 μg/ml of drug in presence of 50 ng/ml of positive control). Despite increased dilution, assay retained its sensitivity, specificity and precision. This assay is easy to set up with minimal processing steps and ensures no false positives are reported due to presence of IL-6R in the samples.