The stability of supercoiled plasmid DNA (sc DNA) is a core issue in the development of nucleic acid vaccines, directly determining the efficacy and safety of the vaccines. This study investigated the effects of ultrafiltration membrane packs with different molecular weight cut-offs on the concentration and purification of plasmid DNA, and the effects of different stabilizers on the stability of plasmid DNA during purification process and storage were evaluated. The results showed that as the molecular weight cut-off of the ultrafiltration membrane pack increased, the yield of sc DNA significantly decreased, the damage to the supercoiled structure was exacerbated, the melting temperature (Tm) dropped, and the degradation rate during storage accelerated. These findings suggested that larger pore sizes in the membrane packs may lead to increased physical or chemical damage to sc DNA, such as double-strand breaks or the formation of open circular structures. Adding trisodium citrate during ultrafiltration significantly enhanced the stability of sc DNA, reducing losses during the ultrafiltration purification and subsequent storage processes. Molecular dynamics simulations further revealed that trisodium citrate formed a protective layer around DNA molecules through hydrogen bonds, effectively reducing damage to sc DNA during purification and storage. Additionally, the addition of trisodium citrate significantly increased the yield of mRNA after in vitro transcription but had no significant effect on the content of by-products such as dsRNA. Therefore, using a membrane pack with a smaller molecular weight cut-off (e.g., 30 kDa) in combination with trisodium citrate as a stabilizer can significantly improve the purification efficiency and stability of sc DNA. This finding provides theoretical support for optimizing the purification and storage conditions of plasmid DNA and lays an important foundation for the development of more stable and efficient DNA and mRNA vaccines, which is crucial for advancing nucleic acid-based therapeutic strategies.