Comparison of anion-exchange chromatography matrices for purification of linear and supercoiled plasmid in a direct...

The efficient, large-scale purification of plasmid DNA is a critical yet challenging step in the development of various therapies, particularly with the increasing need for highly purified linear plasmids in mRNA-based therapeutic applications. In this study, we developed and optimized a direct lysate workflow which decreases the overall plasmid purification time compared to methods utilizing intermediate purification steps such as tangential flow or precipitation. An anion-exchange chromatography protocol was developed using widely available quaternary amine (Q) chromatography matrices including membrane, monolith, and resin. Plasmids were subjected to a two-step purification process involving initial isolation of supercoiled (sc) plasmid from crude, filtered Escherichia coli lysate, followed by enzymatic linearization and subsequent chromatographic purification. We evaluated plasmid recovery, impurity reduction, host cell proteins, and effectiveness for each matrix. After the first chromatographic step, sc plasmids exhibited average recoveries of 93 %, 99 %, and 54 %, average impurity reductions of 99 %, 97 %, and 84 %, and >99.9 % clearance of E. coli host cell proteins for membrane, monolith, and resin matrices, respectively. The linearized plasmid purification achieved step recoveries of 76 %, 89 %, and 61 %, and final purities of 85 %, 63 %, and 6 % for membrane, monolith, and resin, respectively. The functional integrity of the purified linear plasmids was confirmed by successful in vitro transcription and EGFP expression in CHO-K1 cells. These results demonstrate that membrane and monolith-based anion-exchange chromatography provides an effective and scalable approach for the high-purity preparation of linear plasmids suitable for mRNA-based therapeutic applications.