Burgmaier Luisa, Pölt Stefan, Avci-Adali Meltem, Reich Johannes
Microcoat Biotechnologie GmbH, Am Neuland 3, 82347, Bernried Am Starnberger See, Germany; University Hospital Tuebingen, Department of Thoracic and Cardiovascular Surgery, Calwerstr. 7/1, 72076, Tuebingen, Germany.
University of Applied Sciences Weihenstephan-Triesdorf, Am Hofgarten 4, 85354, Freising, Germany.
Biologicals. 2025 Feb;89:101808. doi: 10.1016/j.biologicals.2024.101808. Epub 2024 Nov 24.
Endotoxin masking poses a potential risk to patient safety by rendering endotoxin undetectable. While research often focuses on international endotoxin standards (RSE), the effects of LPS mutants on Low Endotoxin Recovery (LER) are poorly understood. Our study investigated S. minnesota and E. coli mutants with incomplete O-antigen chains (rough LPS) using Limulus amebocyte lysate (LAL), recombinant Factor C (rFC) and the monocyte activation test (MAT). All tested methods detected the mutants, with variations in activity observed. Measurements over time in a common drug formulation (10 mM sodium citrate and 0.05 % (w/v) polysorbate 20) showed different masking kinetics for the mutants using different methods. We were able to show that LAL and rFC have comparable kinetics, whereas MAT showed improved recovery of masked endotoxin. The study showed that the mutation of LPS have an effect on masking, independent of the assay system. We propose that polysaccharide length affects masking susceptibility, with lower hydrophilic/hydrophobic ratios caused by the shortened polysaccharide chain (rough LPS) reducing masking. In addition, the stronger negative charge of the rough mutants increases cation affinity and is suggested to contribute to the stabilisation of supramolecular structures, making the rough mutants less susceptible to masking than the smooth mutants.
内毒素掩盖通过使内毒素无法检测而对患者安全构成潜在风险。虽然研究通常侧重于国际内毒素标准（RSE），但脂多糖（LPS）突变体对低内毒素回收率（LER）的影响却知之甚少。我们的研究使用鲎试剂（LAL）、重组C因子（rFC）和单核细胞活化试验（MAT），对内毒素O抗原链不完整的明尼苏达沙门氏菌和大肠杆菌突变体（粗糙LPS）进行了研究。所有测试方法均能检测到这些突变体，但观察到活性存在差异。在一种常见药物制剂（10 mM柠檬酸钠和0.05%（w/v）聚山梨酯20）中随时间进行的测量显示，使用不同方法时，突变体的掩盖动力学不同。我们能够证明，LAL和rFC具有可比的动力学，而MAT显示出被掩盖内毒素的回收率有所提高。该研究表明，LPS的突变对掩盖有影响，且与检测系统无关。我们提出，多糖长度会影响掩盖敏感性，由缩短的多糖链（粗糙LPS）导致的较低亲水/疏水比会降低掩盖作用。此外，粗糙突变体更强的负电荷增加了阳离子亲和力，并被认为有助于超分子结构的稳定，使得粗糙突变体比光滑突变体更不易被掩盖。
