title: Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach.
abstract: BACKGROUND
  Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK) , and alpha interferon (IFN-α) preparations.
  
  
  METHODS
  A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.
  
  
  RESULTS
  The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.
  
  
  CONCLUSIONS
  Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers' minds on refining drugs, and provides consumers with safer biopharmaceuticals.
authors.affiliation: Dept. of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Babak
authors.initials: B
authors.lastname: Mamnoon
authors.affiliation: Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.
authors.forename: Taghi
authors.initials: T
authors.lastname: Naserpour Farivar
authors.affiliation: 3Dept. of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Kamyab
authors.affiliation: Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.
authors.forename: Dariush
authors.initials: D
authors.lastname: Ilghari
authors.affiliation: Center of Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Khamesipour
authors.affiliation: Dept. of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Karimi Arzenani
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D055772:Green Chemistry Technology / Q000592:standards* / Q000639:trends; D006801:Humans; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards* / Q000639:trends; D011994:Recombinant Proteins / Q000235:genetics* / Q000627:therapeutic use
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins
keywords: DNA contamination; Interferon-alpha (IFN-α); Real-time PCR; Streptokinase