Yi Sijia, Xu Jenny, Song Liping, Celeste Frank, Wang Christopher J, Whiteman Melissa C
Analytical Research and Development, Merck & Co., Inc., West Point, PA 19486, USA.
Biostatistics and Research Decision Sciences, Merck & Co., Inc., West Point, PA 19486, USA.
Vaccines (Basel). 2025 Sep 26;13(10):1009. doi: 10.3390/vaccines13101009.
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.
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.
The THP-1 reporter cells could detect a wide range of pyrogens via simple luminescence readouts. For OMPC testing, the MAT demonstrated strong linearity (R ≥ 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.
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.
背景/目的：热原是来自生物或环境源的致热物质，主要由人类单核细胞表达的Toll样受体（TLR）识别，是药品安全性的关键质量属性（CQA）。广泛用于热原评估的家兔热原试验（RPT）存在变异性高、准确性有限和重现性差的问题，特别是对于含有固有热原的疫苗，如基于外膜蛋白复合物（OMPC）的疫苗。现有的使用外周血单核细胞（PBMC）的体外替代方法受到供体间变异性和ELISA读数操作复杂性的挑战。为了支持3R原则（优化、减少、替代）并提供更可靠的质量控制（QC）方法，我们开发了一种适用于放行检测的基于报告细胞的单核细胞激活试验（MAT）。
我们筛选了用驱动发光报告基因的NFκB反应性启动子元件工程改造的人类单核细胞报告细胞系。报告细胞用不同的内毒素和非内毒素热原处理，并在刺激后对发光进行定量。选择源自THP-1的报告细胞来开发用于OMPC的MAT。按照验证指南评估测定性能：线性、准确性、精密度、分析范围（相对于参考批次）以及在故意参数变化下的稳健性。
THP-1报告细胞可以通过简单的发光读数检测多种热原。对于OMPC检测，MAT在25-300%范围内显示出很强的线性（R≥0.99）、相对偏差在±10.3%以内的准确性和高精度（总体%RSD≤6.9%）。测定参数的故意变化对性能没有实质性影响，表明其稳健性适用于常规放行检测。
基于报告细胞的MAT测定法的实施提高了评估药品热原性和安全性的一致性、可靠性和效率，支持全球减少动物试验的倡议，同时确保符合监管要求。
