title: High-sensitivity hybridization assay for quantitation of residual E. coli DNA.
abstract: Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.
authors.affiliation: GlaxoSmithKline, King of Prussia, PA 19406, USA. shelley_ji-1@sbphrd.com
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Ji
authors.forename: Kwan
authors.initials: K
authors.lastname: Lee
authors.forename: Byron
authors.initials: B
authors.lastname: DiPaolo
mesh_terms: D000906:Antibodies; D002021:Buffers; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics*; D004076:Digoxigenin / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine
chemical_list: D000906:Antibodies; D002021:Buffers; D015342:DNA Probes; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D004076:Digoxigenin