Target interference is a well-recognized challenge in immunogenicity testing for biotherapeutics, particularly monoclonal antibody (mAb) therapeutics, when soluble targets are present at sufficiently high concentrations as dimers, or when a substantial portion of anti-drug antibodies (ADAs) target epitopes overlapping the drug–target interface. The standard practice to assess target interference involves spiking soluble target and mitigating its effects using one of several reagents—typically competitive anti-target antibodies, soluble ligands, or decoy receptors. While effective, these reagents are often costly and introduce additional complexity as critical assay components. In this presentation, we present case studies and propose a new strategy.

Immunogenicity assessment remains challenging for complex antibody-based modalities. This presentation discusses ADA troubleshooting across ADCs, T-cell engagers, and multi-domain molecules, highlighting assay interference, baseline reactivity, and data interpretation challenges. Practical case examples illustrate modality-specific strategies, integrated assay designs, and cross-functional approaches to support risk-based, fit-for-purpose immunogenicity assessment in drug development. 

Recent advancements in xRNA therapies (ASO, siRNA, mRNA) are expanding therapeutic opportunities across multiple diseases. This expansion increases modality complexity and manufacturing challenges. Individual components and combinations may activate innate or adaptive immunity, with potential safety impact. We will present pre-clinical assessments to identify immunogenicity risks and evaluate potential immune responses to xRNA during drug development and provide a framework to design xRNA with reduced clinical immunogenicity and improved safety.

Heterogeneity in regulatory submissions highlights the need for continued harmonization in evaluating immunogenicity and assessing clinical impact. Clinical pharmacology data have emerged as a sensitive measure for evaluating potential impacts on efficacy. The FDA's Office of Clinical Pharmacology is facilitating harmonization across data submission, analysis, and reporting. Recent reviews show substantial increases in labeling communication of immunogenicity risk and clinical impact communication for newly approved products. This presentation will share ongoing efforts and future collaboration opportunities.

Drug tolerance in immunogenicity assays requires a mechanism-informed, modality-specific approach, as circulating drug and target-mediated interference can compromise ADA detection. Optimized dissociation strategies, including acid dissociation and high ionic strength dissociation (HISDA), improve drug tolerance, with combined approaches enhancing performance in high-drug or high-affinity settings. Case examples across multiple modalities highlight the importance of tailoring strategies to exposure and interference to enable reliable ADA assessment.

Neutralizing antibody (NAb) assays for biotherapeutics are often limited by poor drug tolerance, often driving the need for more complex sample treatment strategies such as biotin-drug extraction and acid dissociation (BEAD). This presentation describes a case study in which critical sample treatment and assay conditions were identified through screening DOE and refined using optimization DOE to balance sensitivity, precision, and drug tolerance. The validated assay achieved sub-100 ng/mL sensitivity and =100 µg/mL drug tolerance, meeting study expectations.

Historically, health authorities have required highly sensitive assays (100 ng/mL) to detect ADA, even when samples contain drug at trough levels. However, for most protein therapeutics, clinical experience has shown that the important question is whether sufficient ADA is present to alter clinical exposure, safety or efficacy. This talk reviews regulatory guidance, explains ADA-to-drug stoichiometry, and proposes an exposure-based, flexible sensitivity target tied to trough concentrations of recently approved mAbs.