Fast and accurate method for quantitating E. coli host-cell DNA contamination in plasmid DNA preparations.

Plasmid DNA is being used successfully as a gene delivery vector in a variety of clinical applications. Similar to other pharmaceutical products for clinical use, the plasmid vectors must meet rigorous purity standards. One important contaminant is the DNA of the host cell used to produce the plasmids. We have developed a new method to accurately quantitate E. coli host-cell DNA in plasmid preparations. This method is based on kinetic PCR using the ABI PRISM 7700 with 23S rDNA as a target. This precise assay is significantly faster and has a lower limit of quantitation than the currently used Southern-based methods.

Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay.

The rising interest in gene therapy for the treatment of numerous disorders necessitates the need for the large-scale production of therapeutic biopharmaceuticals that meet stringent purity standards. Residual host cell DNA in recombinant pharmaceuticals has been identified as a potential risk factor that must be quantitated carefully both during the manufacturing process and in the final product. We describe a PCR method to quantitate contaminating levels of host cell DNA in clinical plasmid DNA preparations intended for human gene therapy. The quantitation is based on the coamplification of two similar templates, the target DNA and a synthetic competitor, and the quantitation of the resulting PCR products. The competitor is identical to the target DNA PCR product except for a 29-bp internal replacement. As a result, the two PCR products can easily be distinguished from each other. The competitive nature of the assay allows the use of the ratio of the target DNA PCR product to the competitor DNA PCR product to determine the original amount of target DNA in a sample. The primers used in this assay anneal to a conserved region of the E. coli 23S rRNA gene. One of the primers is biotinylated, allowing the PCR products to be detected colorimetrically after their capture on microtiter plates. The capture is accomplished by differential hybridization to target and competitor-specific probes covalently attached to wells of microtiter plates. The entire assay is performed in less than 2 hr postamplification. This method represents an attractive alternative to Southern blot analysis, which is the currently established method for DNA quantitation.