Genomic DNA Reduction for Therapeutic DNA Manufacture. (2010)

Cai, Y., Bailey, D., Bazzani, R. P., Hebel, H. L., Hyde, S. C., Rodriguez, S. & Gill, D. R.

Molecular Therapy, 18, S288

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Plasmid DNA has been favored as a gene therapeutic entity over viral vectors owing to its high level of safety and low immunogenicity. However, standards for manufacture and quality control of plasmid DNA are typically below the requirements for other biological products. For example, the levels of host cellular impurities such as protein, RNA and genomic DNA (gDNA) are preferred < 1%. When plasmids are amplified in E. coli, preparations containing residual bacterial gDNA can act as a source of unmethylated CpGs, leading to undesirable inflammatory responses after gene transfer in vivo. When residual gDNA found in standard laboratory plasmid preparations is reduced 10-fold by enzymatic digestion, there is a concordant decrease in the pro-inflammatory cytokines IFN-g (5-fold; P=0.003) and IL12-p40 (2-fold; P=0.017). Therefore, it is of key importance that plasmid manufacturers develop effective techniques to minimize gDNA for therapeutic applications. Process development was conducted on a plasmid used for the treatment of the cystic fibrosis lung disease. The previous VGXI plasmid purification process still demonstrated superior product quality: % supercoiled (SC) plasmid >= 95%, host cell protein <= 0.1%, host cell RNA <= 0.5%, Endotoxin <= 1 EU/mg and gDNA ~ 0.3%. However to further improve the purities, a novel thiophilic aromatic chromatography step was integrated into the downstream process. Data from five purification batches indicated comparable %SC and endotoxin levels, with reduced protein and RNA contamination. In particular, gDNA levels, as quantified by two independent assays, could be reduced by up to 100 fold (~ 0.003%) in the final drug product. As a high concentration of plasmid was used (> 5 mg/ml), the low 0.003% gDNA level was at the limit of assay detection sensitivity. In conclusion, the ability to manufacture plasmid vector of high purity, with principally undetectable gDNA, is of paramount importance for clinical gene therapy.

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