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Abstract: Plasmids are small double stranded DNA molecules that are separate from the chromosome. Plasmids are present naturally in bacterial cells and are commonly used as raw materials in mRNA and gene therapy vaccines. These plasmids can be used as the gene template for mRNA vaccines, or to provide the necessary genes, such as rep/cap/gene of interest, in adeno-associated virus (AAV) processes. The current work outlines MilliporeSigma’s collaboration with Boston Institute of Biotechnology, to develop unit operations for the purification of a plasmid DNA used for AAV gene therapy application. Currently the purification of pDNA in a pure form ready for therapeutic use still presents various challenges starting from the lysis step. It also requires development of highly reproducible and scalable processing methods that meet regulatory manufacturing standards. The development here focuses on purification of the supercoiled isoform of pDNA and the use of scalable technologies on process steps of lysate clarification, ultrafiltration/diafiltration, capture using membrane ion exchange chromatography, and sterile filtration.