Investigation of the possibility of presence and the correlation between endotoxin test results and residual DNA in Sterile water for injection based on Real-Time PCR assay on Sterile water for injection

Abstract

In the process of Sterile Water for injection (SWFI) production, there is a possibility of Pyrogen contamination such as Endotoxins. Based on the FDA guideline, the highest endotoxin content must be lower than 0.25 IU in each injectable product. The LAL test is the standard method for evaluating the Endotoxin content. Since in Gram-negative bacteria such as E. coli, the genomic DNA is attached to its cell wall, analyzing the DNA using Real-Time (RT)-PCR method can evaluate the presence of the Endotoxin in injectable products. Aim: In this study, we aimed to investigate the possibility of endotoxin presence in SWFI and the correlation between endotoxin test results and residual DNA based on RT-PCR assay. Method: At first specific primers for the 16SrRNA region of E. coli have been designed. Afterward, two different samples of SWFI as templates were analyzed. To generate the standards DNA samples, we analyzed the serial dilution of extracted E. coli DNA by the RT-PCR method. Similarly, in order to generate Endotoxin positive standard samples, serial dilution of cultured E. coli was autoclaved, then RT-PCR and LAL methods were applied to evaluate the Endotoxin level. Results: The results indicated that the RT-PCR method could detect 0.984 ng/mL of residual DNA, equivalent to 104 CFU/mL E. coli, in the samples. Similarly, LAL method can detect the Endotoxin in the 103 CFU/mL. E. coli samples. Conclusion: Despite having correlation between RT-PCR and LAL test results, the LAL method has higher sensitivity rather than the RT-PCR method to detect the Endotoxin in SWFI samples.