构建重组因子C测定以实现抗体开发中可持续的内毒素检测

Endotoxins, lipopolysaccharide components of the outer membrane of Gram-negative bacteria, are among the most potent pyrogens known. Even at trace levels, endotoxins can trigger severe inflammatory responses in humans, including fever, hypotension, septic shock, and multi-organ failure, particularly when introduced intravenously. Consequently, accurate detection and quantification of endotoxins are essential at every stage of biopharmaceutical development, from early-stage antibody discovery to large-scale manufacturing and final product release. In the context of antibody discovery, residual endotoxins in cell culture supernatants or during purification can confound in vitro bioassays, misrepresent functional activity, and affect downstream characterization. During manufacturing, endotoxin contamination poses a serious safety risk and regulatory non-compliance, necessitating rigorous quality control measures. Regulatory agencies globally, including the FDA and EMA, mandate strict endotoxin limits for injectable therapeutics, emphasizing the importance of robust and reliable endotoxin assays in maintaining both patient safety and product efficacy. Traditionally, the Limulus Amebocyte Lysate (LAL) assay has been the industry standard due to its high sensitivity, rapid turnaround, and broad adoption across biopharmaceutical laboratories. However, LAL relies on lysates derived from horseshoe crab blood, raising sustainability, ethical, and supply chain concerns. In response, recombinant Factor C (rFC) assays have emerged as a viable animal-free alternative. rFC assays leverage recombinant expression of Factor C, the key enzyme initiating the LAL coagulation cascade, with fluorescence-based detection of endotoxin binding. This study describes the development and validation of an rFC-based endotoxin detection method specifically optimized for antibody discovery and production workflows. The method was benchmarked against conventional LAL assays in accordance with established bioanalytical validation guidelines, demonstrating comparable sensitivity with a lower limit of quantitation (LLOQ) of 0.005 EU/mL. Notably, the absence of Factor G in rFC assays eliminates interference from beta-glucans, as verified by spiking experiments with 10 µg/mL beta-glucan, which produced no detectable non-specific signal. Extensive matrix testing across 25 common buffers and culture supernatants revealed recovery rates ranging from 75% to 109%, underscoring the assay's robustness and versatility. Overall, the rFC assay provides a reliable, sensitive, and ethically responsible approach to endotoxin monitoring, aligning with the 3Rs principles while maintaining rigorous analytical performance.