(T1430-02-06) Immunogenicity Assessment of Both Anti-Drug Antibodies in Experimental Animals: Complexed with Drug or in Free Form Using an Automated Workflow

Purpose: Detection of induced immune response is an integral part of the safety determination of any new biotherapeutic. Although the detection of anti-drug antibodies (ADA) in preclinical species is not predictive of immunological responses in humans and is not always required (1,2), ADA assays remain a mainstay of preclinical bioanalytical evaluation. Generic ADA assays detecting immune complexes can accelerate analysis since they can be deployed without requiring reagent labeling or assay optimization. However, these assays aren’t always effective for the detection of immune complexes in the absence of circulating drug or without modification of the sample. In this study, modified methods were developed for a commercially available generic ADA that detects circulating immune complexes to also detect ADA in the absence of drug, detecting free and total ADA with low or no drug present.

Methods: Gyrolab^® Generic Rodent ADA Kit Reagents and Gyrolab xPand platform with Gyrolab Bioaffy 1000 microfluidic disc (Gyros Protein Technologies) were utilized to detect circulating immune complexes, free ADA, and total ADA based on three assay formats (Table 1). For all methods mouse and rat serum samples containing known concentrations of Gyrolab Generic Mouse ADA Positive Control (Mouse IgG-Human IgG dimer), and commercially available mouse and rat polyclonal anti-human antibodies were assessed after a 1-in-10 MRD for 10% matrix.

Results: Using the Gyrolab Generic ADA Kit method and samples where no drug is present (Figure 2a) the mean observed signal for the Gyrolab Generic Mouse ADA Positive Control increased as the concentration increased, however the response for the commercially available rat anti-human pAb ADA positive control (uncomplexed with drug) was unchanged. Figure 2b and 2c shows that if huIgG (drug) was introduced to the column with capture reagent prior to the addition of the free ADA sample, the rat free ADA control was detected. Figure 2b also shows when huIgG is introduced to the column of the Gyrolab Bioaffy 1000 disc prior to the Gyrolab Generic Mouse ADA Positive Control (covalently linked ADA complex), the signal is reduced. Figure 2c shows that a concentration-related response is achieved for both the free rat and the complexed Mouse ADA Positive Controls.

Conclusion: Three assay formats using the Gyrolab Generic Rodent ADA Kit Reagents were utilized to detect mouse ADA complex and free rat ADA positive controls representing immune complexes, free ADA, and both complexed and free ADA in the 3 sets of assay results. The technique of pre-loading the microfluidic disc affinity column with IgG drug, or a 4:1 mix of capture reagent and drug was effective in modifying the kit’s commercial approach of detecting immune complexes. This demonstrates the utility of the commercially available generic rodent ADA Kit for free and complexed ADA detection in preclinical animal studies.

References: 1. ICH S6 (R1) Preclinical safety evaluation of biotechnology-derived pharmaceuticals - Scientific guideline, European Medicines Agency (EMA), 1/3/1998
2. S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, US FDA Center for Drug Evaluation and Research (CDER), 11/1/1997


Gyrolab generic rodent ADA methods showing reagent and sample additions


Diagram of Gyrolab microfluidic generic ADA immunoassay


Mean response in Gyrolab generic ADA assay for concentrations of covalently tethered Gyrolab Generic Mouse ADA Positive Control or rat anti-human pAb free ADA control.