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.
Conclusion: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test.
Keywords: CAR-T; HL60-IL-6 assay; LOD; limit of quantitation; pyrogen; the RPT; the recovery rate.
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