Ji Xiaoying, Lee Kwan, DiPaolo Byron
GlaxoSmithKline, King of Prussia, PA 19406, USA.
Biotechniques. 2002 May;32(5):1162-7. doi: 10.2144/02325dd06.
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.
重组蛋白治疗药物的杂质检测对于确保批次间的一致性以及满足美国食品药品监督管理局（FDA）对特征明确的生物制药的标准至关重要。对于残留宿主细胞DNA的测定，传统上一直使用利用从宿主细胞基因组DNA制备的放射性标记DNA探针的膜杂交试验，来检测源自细菌表达系统的产品，以获得所需的低皮克灵敏度。非放射性方法虽然有望消除放射性废物处理和安全问题，但通常灵敏度较差且背景较高。我们报告了一种通过狭缝印迹杂交法来定量纯化蛋白药物中残留大肠杆菌DNA水平的灵敏度适宜的非放射性检测方法的开发。该检测方法使用地高辛标记的大肠杆菌DNA探针和超敏化学发光底物。优化后的化学发光杂交试验具有低背景和高灵敏度，能够常规检测到2.5 pg的大肠杆菌DNA。该方法可针对检测/定量大肠杆菌或其他细菌表达系统中表达的重组蛋白产品中的DNA污染进行调整。
