(T1030-01-06) Generic ADA Method Development for Preclinical Study

Purpose: According to current FDA guidelines, monitoring anti-drug antibody (ADA) formation is required in preclinical studies, as animals frequently develop immune responses to biologic therapeutic products, which can affect drug exposure as determined by toxicokinetic analysis. However, immunogenicity of biologics observed in animals does not reliably predict human immunogenicity. This study aims to identify a broadly applicable positive control for the majority of preclinical ADA assays, define a range for optimizing conjugate concentrations, evaluate blocking buffers, and provide guidance on evaluating the necessity of sample pre-treatment. In addition, the study explores the use of a singlicate analysis and signal to noise (S/N) ratio from the screening assay to replace the titer value calculated from the titration assay. The ultimate objective is to develop a streamlined, user-friendly workflow that enables team members to complete method development efficiently, using the simplest and most robust approach. Furthermore, several of these strategies may also serve as valuable references for clinical ADA method development.

Methods: This method development served as the foundation for designing an effective, workflow applicable to multiple preclinical studies. Polyclonal and monoclonal anti-human IgG were evaluated side by side as positive controls (PCs) to compare sensitivity and prozone effects. Various concentrations of biotin- and sulfo-tag-conjugated drugs (challenge ratio 1:10) along with different storage buffers were tested. A variety of blocking buffers were tested to assess their ability to minimize non-specific binding. Individual monkey serum and drug tolerance samples have also been used to evaluate the necessity of pre-treatment for method development. The simplest and most robust method based on compatibility with multiple types of therapeutic antibodies was selected. Finally, applying the parameters above, the S/N ratios obtained from the screening tier were compared with the corresponding titer values of ADA-positive samples across multiple studies was evaluated to determine the feasibility of using S/N ratios to replace titer values.

Results: This study identified the polyclonal anti-human IgG as a suitable PC for most preclinical ADA methods across different species, and three drug modalities: naked, bispecific and fusion IgGs with different Fc mutations, demonstrating broad compatibility, a wide dynamic range (9–50,000 ng/mL) without prozone effect, and adequate sensitivity (10 ng/mL or lower).  A recommended range of conjugate concentrations (0.2–1.0 µg/mL) was established for evaluation during method development, along with practical guidance on when and how to apply sample pre-treatment. A specific storage buffer was identified that demonstrated the highest sensitivity (~3 ng/mL) and the greatest drug tolerance ( >1000 µg/mL of drug in the presence of 500 ng/mL PC), significantly outperforming 1XDPBS. Furthermore, the use of casein blocking buffer was identified as effective to reduce non-specific binding for most of drug modalities. Across multiple studies, most results demonstrated a positive correlation between the S/N ratio from the screening assays and the corresponding sample titer values.

Conclusion: This study established a streamlined workflow for preclinical ADA method development, addressing key challenges such as positive control selection, conjugate optimization, buffer evaluation, as well as sample pre-treatment. The polyclonal anti-human IgG was identified as a suitable positive control across multiple studies and species, offering high sensitivity ( < 10 ng/mL), and a wide dynamic range (9–50,000 ng/mL). It also demonstrated that the absence of prozone effects led to the possibility to eliminate the need for a titration assay. The findings emphasized the importance of storage buffer of conjugates on assay performance beyond stability, including sensitivity and drug tolerance. In our case, optimizing the storage buffer resulted in a two-fold increase in both assay sensitivity and drug tolerance. Moreover, the strategies outlined here may serve as valuable references for corresponding clinical ADA assay development.