Evaluation of bacterial DNA contamination in recombinant GCSF by Real-Time PCR

Abstract

Introduction: During the production of recombinant GCSF in the biological host, fragments of the host nucleic acid could enter the product. Legal entities have set a limit for the amount of host nucleic acid in the final product. Determining the amount of this DNA is one of the requirements for quality control of biological products. Aim: The aim of this study was to develop a sensitive method for investigating bacterial host DNA contamination in a recombinant GCSF drug model by Real-Time PCR. Material and methods: First, after designing specific primers and extracting DNA from the bacterial host model (E..coli BL21); Consecutive dilutions were prepared and then Real-Time PCR was optimized to draw the standard curve. Then, the efficiency of PCR in detecting DNA contamination in spiked samples was determined directly and indirectly after DNA extraction from GCSF using a standard curve. Agarose gel electrophoresis was performed on the samples to confirm the results. Results: The optimized PCR reaction does not show any nonspecific product and primer dimmers. PCR reaction also has the ability to detect and quantify DNA with LOD and LOQ values 0.09 pg / μl of 0.27 pg / μl respectively. Also, the results of gel electrophoresis and Real-Time PCR showed that DNA amplification was performed in the presence of the drug. Conclusion: The obtained results indicate that the Real-Time PCR technique developed in this study can be used as a practical method in order to detect DNA impurities in recombinant pharmaceutical products produced in E. Coli host such as GCSF.