Novel Real Time Polymerase Chain Reaction Approach for Rapid Detection of the Residual Escherichia coli Genomic DNA in Biopharmaceutical Products Establishment of Real Time Polymerase Chain Reaction to Detect Residual gDNA

Abstract

Background: Contamination of therapeutic recombinant proteins with residual host cell DNA must be controlled under the regulatory standards. Objectives: The current study established a new rapid, sensitive real time polymerase chain reaction (PCR) approach to measure the reliably of the residual Escherichia coli (E. coli) host cell genomic DNA in the recombinant streptokinase and alfa interferon preparations. Materials and Methods: In this assay, a specific primer pair was utilized to amplify a 115 base pair sequence inside the E. coli 16S rRNA using SYBR Green Chemistry. This method enabled the authors to detect a very small quantity of the residual genomic DNA, as low as 0.8 pg, in the protein-based drugs. This method can, therefore, offer a dependable way to quantitatively analyze the major contaminant of biopharmaceutical products, the host cell DNA, during the manufacturing process. Results: SYBR Green PCR master mix may contain a source of DNA contamination during its manufacturing process. Conclusions: The current study data showed that E. coli host cell DNA contamination in streptokinase and alfa interferon manufactured in the Pasteur institute of Iran is much lower than the safety limits suggested by the FDA.