Search

Background: To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. Results: Strategies were developed to overcome two major bioanalytical challenges: poor drug tolerance of the anti-drug antibodies assay and very low cut point observed in the screening and confirmatory assays. By using acid dissociation (400 mM glycine solution at pH 2.0), drug tolerance of 200 μg/ml drug was achieved for both the screening and confirmatory assays. Effects of biological matrix (disease state vs normal serum) and assay conditions (capture/detector reagent concentration, minimum required dilution, acid pretreatment) on assay cut points were systematically evaluated. Conclusion: A bridging immunogenicity assay for detecting anti-cabiralizumab antibodies in human serum has been successfully developed, validated and applied to clinical studies.

Immunogenicity testing is a mandatory and critical activity during the development of therapeutic proteins. Multiple regulatory guidelines provide clear recommendations on appropriate immunogenicity testing strategies and required bioanalytical assay performances. Unfortunately, it is still generally accepted that a comparison of the immunogenicity of different compounds is not possible due to apparent performance differences of the used bioanalytical methods. In this perspective, we propose the ‘cut-point anti-drug antibody–reagents complex’ (CP-ARC) concept for technical comparability of the bioanalytical methods. The feasibility and implementation in routine assay development is discussed as well as the potential improvement of reporting of bioanalytical immunogenicity data to allow comparison across drugs. Scientific sound comparability of the bioanalytical methods is the first step toward comparability of clinical immunogenicity.

Aim: Appropriateness of anti-drug antibody (ADA) assay is critical for immunogenicity assessment of biopharmaceuticals. Although cut point setting in ADA assay has a large impact on the results, a standard statistical approach for its setting has not been well established. Methodology: In this multi-laboratory study, to elucidate factors influencing the cut point setting, we compared the statistical approaches and calculated cut points for multiple datasets of ADA assays using the individual procedure employed at each laboratory. Conclusion: We showed that outlier exclusion, false-positive rate and investigating data distribution have the greatest impact on both screening and confirmatory cut points. Our results would be useful for industry researchers and regulators engaged in immunogenicity assessment of biopharmaceuticals.

Background: Currently, screening cut point (CP) calculated from an assay validation with replicates are applied to an immunogenicity study with nonreplicates, for which the antidrug antibodies rate is determined. IID treats the replicate of a sample as coming from another independent sample. AVE uses average results from each sample across runs but inter-assay variability is reduced. Therefore, we propose a random effect model (REM) for calculating CP. Materials & method: We investigate impact of noncompatibility design between validation and immunogenicity studies on CP and compare these methods. Conclusion: IID may not fit for use when replicates’ variability dominates all sources of uncertainty. REM considers covariance structure of repeated measurements. CP by REM is smaller than that by IID but larger than that by AVE.

Aim: Competitive inhibition with excess unlabeled drug is used to confirm the presence of antidrug antibodies (ADA) in study samples. We evaluated specific and nonspecific responses from both drug-naive and drug-treated subjects to identify conditions required by the confirmatory assay to make accurate ADA classifications. Results: Nonspecific signal measured in drug-naive samples used to determine assay cut points was uniformly low and close to the screening cut point. Confirmatory assays performed on incurred study samples with nonspecific responses significantly above the level observed during cut point determination resulted in incorrect ADA classifications. Conclusion: Intensity of confirmatory response should be proportional to the screening response and therefore, to ensure accurate ADA classifications, the confirmatory responses cannot be considered as independent but need to be evaluated in relation to the screening responses.

European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.