ADDRESSING A MANUFACTURING BOTTLENECK IN CAR-T THERAPY: SCALABLE PRODUCTION OF TRANSFECTION-GRADE PLASMID DNA FOR CAR-T VECTOR MANUFACTURING

Abstract

The rapid advancing of cell and gene therapy has increased the demand for plasmid DNA used in viral vector manufacturing. The production of transfection-grade plasmid DNA remains a critical bottleneck, particularly for academic and emerging manufacturing facilities, due to the high cost and limited scalability of existing purification technologies. The aim of this study was to develop a practical and cost-effective approach for producing transfection-grade plasmid DNA suitable for lentiviral vector manufacturing. The described acidic phenol extraction method was evaluated using plasmids commonly employed in lentiviral packaging systems and compared with a reference method, CsCl density gradient ultracentrifugation. The resulting plasmid preparations showed efficient removal of genomic DNA and other impurities, including endotoxin – the acidic phenol method consistently reduced endotoxin levels to 1.4–3.8 EU/µg DNA. Moreover, the purified plasmid DNA was predominantly in the supercoiled (covalently closed circular) form. Functional validation in transfection assays confirmed that plasmids purified by the proposed method supported lentiviral vector production with efficiency comparable to the reference method, yielding titers exceeding 5×106 TU/mL, with no statistically significant differences in transduction efficiency between the groups. These findings confirm that the acidic phenol extraction method is a simple, scalable, and economical approach for isolating transfection-grade plasmid DNA for lentiviral vector production. The method represents a practical alternative to ultracentrifugation-based purification and may facilitate broader use of cell and gene therapy by reducing dependence on specialized equipment and costly commercial kits.