目的:采用HL60-IL-6测定法检测CAR-T细胞产物中的热原。
方法:将HL60细胞与CAR-T细胞注射或内毒素标准一起孵育48小时。在那之后,通过ELISA测定HL60细胞分泌的细胞因子白细胞介素-6(IL-6)。根据光密度(OD)值对应的IL-6和内毒素标准浓度拟合的四参数逻辑曲线,可以测量CAR-T细胞产物中热原含量的内毒素当量。然后,该方法得到了验证,包括检测限(LOD),定量极限,回收率以及HL60-IL-6与常规热原试验测定结果的比较,兔热原试验(RPT)。
结果:用于CAR-T细胞产品热原检测的HL60-IL-6检测方法已经建立和验证,LOD为0.03EU/mL,LOQ为0.07EU/mL,回收率分别为121.4%和94.5%。HL60-IL-6测定的结果与RPT测定的结果一致。
结论:HL60-IL-6测定可用于CAR-T细胞产物的体外热原测试。
OBJECTIVE: To detect the pyrogen in CAR-T cells product employing the HL60-IL-6 assay.
METHODS: The HL60 cells were incubated with CAR-T cells injection or endotoxin standard for 48 hours. After then, the secreted cytokine interleukin-6 (IL-6) from HL60 cells was determined by ELISA. According to the four-parameter logistic curve fitted by Optical Density (OD) value corresponding to IL-6 and endotoxin standard concentration, the endotoxin equivalents of pyrogen content in the CAR-T cells products can be measured. Then, the method was validated, including the limit of detection (
LOD), limit of quantitation, the recovery rate and the comparison of the determined results by HL60-IL-6 assay with that by the conventional pyrogen test, the Rabbit Pyrogen Test (RPT).
RESULTS: The HL60-IL-6 assay applied to pyrogen test in CAR-T cells products has been established and validated, The
LOD was 0.03 EU/mL while the LOQ was 0.07 EU/mL, the recovery rates were 121.4% and 94.5% respectively. The results determined by HL60-IL-6 assay were consistent with that by the RPT.
CONCLUSIONS: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test.