Advancing Pyrogen Testing for Vaccines with Inherent Pyrogenicity: Development of a Novel Reporter Cell-Based Monocyte Activation Test (MAT).

BACKGROUND/OBJECTIVES: Pyrogens, fever-inducing substances from biological or environmental sources, are recognized by Toll-like receptors (TLRs) predominantly expressed by human monocytes and represent a critical quality attribute (CQA) for pharmaceutical safety. The rabbit pyrogen test (RPT), widely used for pyrogen assessment, suffers from high variability, limited accuracy, and poor reproducibility, particularly for vaccines containing inherent pyrogens such as outer membrane protein complex (OMPC)-based vaccines. Existing in vitro alternatives using peripheral blood mononuclear cells (PBMCs) are challenged by donor-to-donor variability and the operational complexity of ELISA readouts. To support the 3Rs (Refinement, Reduction, Replacement) and provide a more reliable quality control (QC) method, we developed a reporter cell-based monocyte activation test (MAT) suitable for release testing. METHODS: We screened human monocytic reporter cell lines engineered with NFκB-responsive promoter elements driving a luminescent reporter. Reporter cells were treated with diverse endotoxin and non-endotoxin pyrogens and luminescence was quantified after stimulation. Selected THP-1-derived reporter cells were used to develop an MAT for OMPC. Assay performance was evaluated following validation guidelines: linearity, accuracy, precision, analytical range (relative to a reference lot), and robustness under deliberate parameter variations. RESULTS: The THP-1 reporter cells could detect a wide range of pyrogens via simple luminescence readouts. For OMPC testing, the MAT demonstrated strong linearity (R2 ≥ 0.99), accuracy with relative bias within ±10.3%, and high precision (overall %RSD ≤ 6.9%) across the 25-300% range. Deliberate variations in assay parameters did not materially affect performance, indicating robustness appropriate for routine release testing. CONCLUSIONS: The implementation of reporter cell-based MAT assays enhances consistency, reliability, and efficiency in evaluating the pyrogenicity and safety of drug products, supporting global initiatives to minimize animal testing while ensuring regulatory compliance.