Scalable Production of Transfection-Grade Plasmid DNA by Liquid–Liquid Extraction Without Chromatography: Application to CAR-T Vector Packaging

The growing demand for high-quality plasmid DNA (pDNA) in cell and gene therapy, including chimeric antigen receptor T-cell therapy (CAR-T) manufacturing, is constrained by the limited availability of transfection-grade plasmids. This study describes a practical, scalable, and cost-effective acidic phenol extraction method based on a modified alkaline lysis protocol. This approach efficiently removes endotoxins and residual genomic DNA without requiring chromatography or ultracentrifugation. When benchmarked against CsCl density gradient ultracentrifugation and a commercial midiprep kit, the acidic phenol method delivered comparable plasmid purity and functional performance while providing superior scalability, yielding 5–10 mg of pDNA per 500 mL culture. Notably, residual endotoxin levels were negligible for downstream applications such as transfection and lentiviral vector packaging. Lentiviral vectors produced with these plasmids reached titers exceeding 5 × 106 TU/mL, with transduction efficiencies statistically indistinguishable from those obtained with CsCl-purified DNA. The acidic phenol extraction method is a robust, scalable, and cost-effective alternative to industry-standard methods, matching them in yield and purity while being readily scalable, making it a practical tool for both academic research and preparative laboratory production.