Development of a Streamlined pDNA Purification Process Using an In‑Line Lysis Device

Abstract

The COVID-19 pandemic drew attention to mRNA vaccines, highlighting how they are a promising technology for treating infectious diseases. Plasmid DNA (pDNA) is essential for mRNA production, serving as the template for in vitro transcription and as a key component in other applications including viral vectors and DNA vaccines. A drawback of pDNA production is its labor-intensive purification process which currently requires two chromatography, and three ultrafiltration and diafiltration (UF/DF) steps. To address the need for a simplified procedure, we implemented improved capture feed conditions and a modified pDNA purification process that combines anion-exchange (AEX) monolithic chromatography, and hydrophobic interaction (HIC) monolith chromatography. Central to this approach is Sartorius’ Alkalizator, an in-line lysis device designed to reduce lysate impurities and enhance supercoiled DNA (scDNA) purity. Using this instrument, we achieved a scDNA purity of 52%, compared with 35% for traditional batch lysis. Following the upstream improvement, clarified lysate is directly loaded onto a CIM DEAE anion exchange monolith, eliminating the first UF/DF step. The eluate is then purified further using a C4 HLD HIC monolith column in place of the PlasmidSelect Xtra (PSX) resin column, removing the second UF/DF. With these changes, the modified process delivers yield and product quality comparable to the traditional workflow, with scDNA levels well above 90% and overall yields ranging from 49% to 60%, exceeding the approximate 40% yield reported for the original process. The modified process also offers key advantages such as a reduction greater than 50% in process time and approximate 19% reduction in cost of goods when evaluating comparable small-scale runs and controlling for specific consumables. Our results suggest that this modified process has the potential to be used for generating template pDNA faster and cheaper.