Innate immunogenicity assessment is essential in peptide therapeutic development to ensure safety and efficacy. Peripheral blood mononuclear cells (PBMCs) provide a physiologically relevant in vitro platform due to their composition of antigen-presenting cells and innate immune receptors. This assay uses PBMCs from at least 20 HLA-diverse donors to capture immune variability and includes spiking with innate immune response-modulating impurities (IIRMIs) to simulate adjuvant effects. A ten-cytokine panel, measured via Luminex multiplex immunoassay, serves as the functional readout to detect innate immune activation. Methodological controls—such as cell viability thresholds (>85%), optimized drug concentrations, and validated positive/negative controls—ensure reproducibility and alignment with regulatory expectations. This platform supports critical quality attribute (CQA) specification setting by identifying immunogenic risks from both the drug substance and impurities. When appropriately validated, the PBMC-based assay meets FDA guidelines for innate immunogenicity risk assessment and offers a standardized, sensitive approach for detecting clinically relevant immunostimulatory contaminants.
Learning Objectives:
Understand how assay validation and control strategies support FDA-compliant specification setting for critical quality attributes (CQAs).
