Background: The HL60-IL6 assay has been initially established, but the process of the assay and calculation was not simplified. And there are no reports on whether it can be applied to detect pyrogen contamination in the monoclonal antibody. Objective: The study aimed to improve the HL60/IL-6 assay and detect the pyrogens in the monoclonal antibody drug by HL60-IL6 assay. Methods: The human promyelocytic leukemia cell line (HL-60) was incubated with pyrogen standard solution, such as lipopolysaccharide (LPS), zymosan and lipoteichoic acid (LTA),or monoclonal antibody sample solution for 48 hours, and then cytokines interleukin-6 (IL-6),secreted from HL-60, were measured by ELISA. The study further described the standard curves on OD (Optical Density) value of IL-6 responding to pyrogen stimulation, and determined the content of pyrogen in the monoclonal antibody production after validation. In addition, the sensitivity of HL60 to three pyrogens was evaluated to establish one standard curve to determine endotoxin and non-endotoxin level. Then, the credibility of standard curves was evaluated. After improvement of the assay, 9 monoclonal antibody batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test (RPT) and HL60/IL-6 assay. Results: It was achieved that the standard curve between OD value of IL-6 and pyrogen concentration was established. Then, it was found that the sensitivity of HL60 responding to LPS was the weakest, as a result of which, only LPS standard curve needs to be described in each test for detection of pyrogens. Besides, to evaluate the credibility of standard curve, the parameters of the standard curve were restricted and the resulting interpretation was also specified. 3 Bevacizumab batches failed the RPT, which also showed pyrogenic contamination by the HL60/IL-6 assay. Conclusion: HL60-IL6 assay was improved and can be applied to pyrogen detection of monoclonal antibody.
Objective: To detect the pyrogen in CAR-T cells product employing the HL60-IL-6 assay. Method: 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 comparasion 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%. The results determined by HL60-IL-6 assay was consistent with that by the RPT. Conclusion: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test.
Background:: Pyrogens are fever-inducing substances and pyrogen detection is mandatory in parenteral pharmaceuticals. Succinylated Gelatin Injection (SGI) is a biopharmaceutical product containing multi-component and administrated parenterally. Objective: To assess pyrogen in SGI and to evaluate the feasibility of Monocyte Activation Test (MAT) for pyrogen detection in multi-component pharmaceutical product. Method: In the present study, the Bacterial Endotoxin Test (BET) and the Monocyte Activation Test (MAT) were employed to assess pyrogen in SGI. The MAT method was developed on the basis of HL-60/IL-6 assay. HL-60 cells were incubated with lipopolysaccharide (LPS) standards and sample solutions. The endotoxin produced by the incubation, interleukin-6 (IL-6), was measured by ELISA. The MAT method was validated and main parameters were investigated. Finally, the pyrogenicity of SGIs from two different enterprises was determined by the developed MAT method. Results: The BET failed in test for interfering factors and the MAT was proved suitable for the pyrogen detection of SGI. All the products examined showed negative results in pyrogen detection test. Conclusion: The MAT method is feasible in pyrogen detection of SGI. It can be applied in pyrogen detection for quality and safety control of multi-component biological products.
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