Pyrogenicity of phage active pharmaceutical ingredients used for personalized therapy in Belgium

The assessment of pyrogenicity is critical for phage-based therapeutic products, especially when administered intravenously. However, current standard endotoxin tests, such as the Limulus Amoebocyte Lysate (LAL) and the recombinant Factor C (rFC) assay, capture only part of their immune-activating potential. Here, we compared the LAL test, the rFC assay, and the human cell-based Monocyte Activation Test (MAT) to evaluate the pyrogenic profile of phage active pharmaceutical ingredients (pAPIs). While LAL and rFC are rapid and cost-effective, MAT provides unique physiological relevance by capturing immune responses to both endotoxins and non-endotoxin pyrogens (NEPs), as well as potential interactions between phages and the human immune system. This study provides a first insight into the pyrogenic activity of pAPIs as perceived by human immune cells and highlights the complementary roles of endotoxin tests and MAT in ensuring product safety. The tested pAPIs generally exhibited low pyrogenicity, with most batches below 1000 EU/mL. Based on our experience, we propose a practical two-tiered quality control strategy using rFC as an initial screening tool for rapid and cost-effective endotoxin quantification, and MAT as a complement to assess pyrogenic potential as perceived by the human immune system. Importantly, MAT is not intended to overrule endotoxin detection by rFC, but rather to support the interpretation of complex pyrogenic signals. This approach provides a more physiologically relevant and comprehensive view of pyrogenicity, while supporting ethical testing practices aligned with the 3Rs principles (Replacement, Reduction, and Refinement of animal use).