Mingren Wang, Shanshan Dong, Hong Shao, Can Wang* and Gang Chen* Pages 319 - 327 ( 9 )
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.
HL60-IL6 assay, pyrogen, monoclonal antibody, lipopolysaccharide, zymosan, lipoteichoic acid.
Shanghai Institute for Food and Drug Control, Shanghai 201203, Shanghai Institute for Food and Drug Control, Shanghai 201203, Shanghai Institute for Food and Drug Control, Shanghai 201203, Shanghai Institute for Food and Drug Control, Shanghai 201203, Shanghai Institute for Food and Drug Control, Shanghai 201203