Insight

In the R&D and manufacturing of nucleic acid drugs, supercoiled plasmid DNA (scDNA) serves as the core raw material for mRNA vaccines, DNA vaccines and gene therapy vectors, as well as a critical substrate for recombinant viral vector packaging. The preparation quality of high-purity plasmid DNA directly determines the success rate of downstream processes and the safety of final products. Given that different application scenarios impose differentiated requirements on plasmid yield, purity and topological conformation, establishing a standardized and efficient extraction and purification system is of great importance.

Plasmid DNA naturally exists in bacterial cells in the form of double-stranded covalently closed circular structure with a supercoiled conformation. However, during extraction, processing and storage, mechanical shearing or chemical factors may cause single-strand breaks to form open circular DNA (ocDNA) or double-strand breaks to generate linear DNA (L-DNA). Therefore, optimizing extraction processes to maximally preserve the supercoiled configuration is the core technical challenge in plasmid production.

Escherichia coli host strains such as DH5α are commonly cultured in modified TB medium via fed-batch fermentation. With precise regulation of pH, dissolved oxygen and nutrient supply, the OD₆₀₀ value of bacterial liquid can reach 150–180 after 20–24 hours of cultivation. Cell harvesting adopts continuous flow centrifugation or tangential flow filtration (TFF, membrane pore size: 0.1–0.2 μm). TFF is especially applicable to large-scale production and enables simultaneous cell washing.

Alkaline lysis is the most widely used cell disruption technology, with its core lying in precise control of pH value and reaction time. The bacterial cell resuspension is mixed with lysis buffer containing 0.2 M NaOH and 1% SDS, followed by gentle stirring for 4–10 minutes to dissolve cell membranes, denature chromosomal DNA and release plasmids.

Key operational points for this step: add lysis buffer slowly and mix gently to avoid irreversible plasmid damage caused by local over-alkalinity or mechanical shearing; strictly control lysis duration to prevent plasmid fragmentation from over-lysis.

Upon completion of lysis, neutralization is immediately performed with 3 M potassium acetate (pH 5.5) for 30 minutes. The high-salt environment promotes the precipitation of denatured proteins, chromosomal DNA and SDS, while plasmid DNA remains in the aqueous phase. Cell debris and precipitates are subsequently removed by centrifugation combined with depth filtration or centrifugation coupled with tangential flow microfiltration to obtain clarified lysate.

The clarified lysate is subjected to ultrafiltration/diafiltration (UF/DF) for concentration and removal of partial small-molecule impurities, reducing the workload of subsequent chromatographic procedures.

The molecular weight cutoff (MWCO) of membrane cassettes is selected based on plasmid size: a 100 kDa MWCO membrane is recommended for plasmids of approximately 1 kb; a 500 kDa membrane is preferred for plasmids larger than 10 kb to prevent membrane fouling and ensure mass transfer efficiency. It is advisable to determine the optimal membrane pore size through lab-scale trials to balance recovery rate and flux.

Crude plasmid solution obtained from pre-treatment still contains impurities such as host cell protein (HCP), RNA, open-circular plasmid and endotoxins, which require fine separation via multi-stage chromatography. The classic process workflow of gel filtration chromatography – thiophilic affinity chromatography – anion exchange chromatography has been proven robust and rational.

Significant molecular weight differences exist between plasmid DNA (pDNA) and RNA. Meanwhile, RNA undergoes conformational contraction under high-concentration ammonium sulfate, reducing its hydrodynamic radius and further widening the size discrepancy with pDNA, which facilitates separation via gel filtration. Group separation mode is adopted with a loading volume of 0.2–0.3 column volume (CV). pDNA elutes first in the void volume of the chromatographic column, followed by RNA and other impurities.

scDNA features a double-stranded covalently closed circular structure, while single-strand breakage during fermentation and lysis relaxes the supercoiled structure to form ocDNA. ocDNA shares highly similar molecular properties with scDNA, differing only in higher base exposure and surface charge density of scDNA.

Thiophilic affinity chromatography leverages the subtle differences in base exposure and surface charge between scDNA and ocDNA. Under specific salt concentrations, scDNA is selectively bound to the medium, while ocDNA and linear DNA flow through or are eluted at varying salt concentrations, yielding high-purity supercoiled plasmid fractions.

Most HCP, RNA, ocDNA and endotoxins have been removed after the first two chromatographic steps. Trace residual endotoxins and HCP can be eliminated via high-resolution anion exchange chromatography media.

With high negative charge density, scDNA remains bound to anion exchange media at relatively high salt concentrations (e.g., 0.4 M NaCl). The electrostatic interaction between endotoxins and anion exchange media is significantly weakened under high-salt conditions, allowing endotoxins to be mostly removed in the flow-through fraction. scDNA is retained on the column and eluted with high-salt buffer to obtain high-purity, low-endotoxin plasmid samples.

Clarified pDNA solution can also be purified using mixed-mode chromatographic media. During sample loading, pDNA flows through in the void volume, while RNA, HCP, endotoxins and other impurities enter the core region of fillers and bind to the media via electrostatic and hydrophobic interactions for effective removal of RNA and endotoxins. Subsequent plasmid affinity chromatography is applied to eliminate ocDNA.

In addition, hydrophobic chromatography and salting-out methods can be adopted to remove RNA and ocDNA. Compared with the three-step chromatographic platform for plasmid purification, alternative workflows require more extensive optimization of binding and elution conditions.

The preparation of supercoiled plasmid DNA is a systematic project covering microbial fermentation, cell disruption, solid-liquid separation and multi-stage chromatographic purification. Only by strictly controlling operational parameters at each process unit and identifying and avoiding potential risk points can high-quality plasmid raw materials stably meet the production requirements of nucleic acid drugs.

With the rapid advancement of gene therapy and mRNA technology, plasmid DNA preparation processes will continue to be iterated and optimized, providing more solid technical support for the biopharmaceutical industry.