Insight

With the advancement of vaccine technology, traditional infectious diseases such as poliomyelitis and diphtheria have been effectively controlled. Despite the preventive efficacy of vaccines worldwide, infectious diseases still claim millions of lives annually, largely attributed to the inherent limitations of conventional vaccination approaches. Influenza pandemics further highlight the urgent demand for the development of rapid-response vaccine technologies. Plasmid DNA (pDNA) vaccines have emerged as a highly promising alternative by virtue of their unique superiorities. Manufactured via bacterial fermentation, pDNA vaccines feature a simple and rapid production process, while the intrinsic stability of pDNA reduces cold-chain logistics requirements. At present, pDNA vaccines have achieved remarkable progress in multiple fields including breast cancer, leukemia, hepatitis, and Ebola prevention.

A pDNA vaccine consists of genetically engineered circular plasmid DNA encoding antigenic proteins of specific pathogens. Upon vaccination, host cells internalize the plasmid and express the target antigens, which are subsequently presented to trigger a dual immune response. This induces both humoral immunity mediated by B cells for antibody production and cellular immunity driven by T cells. Unlike conventional protein vaccines that only elicit antibody responses, pDNA vaccines confer comprehensive immune protection. Furthermore, sustained antigen expression facilitates the formation of long-term immune memory.

Plasmid DNA is an autonomously replicating element composed of covalently closed circular double-stranded DNA, whose structural characteristics directly govern its applications in gene therapy and vaccine development. DNA molecules consist of deoxyribonucleotides linked by phosphodiester bonds and carry net negative charges under physiological conditions. Two antiparallel strands form a canonical right-handed double helix via complementary base pairing, with major and minor grooves providing critical binding sites for ligands.

As naturally occurring extrachromosomal genetic elements in bacteria, pDNA belongs to mobile genetic components capable of horizontal gene transfer through bacterial conjugation. Its molecular size generally ranges from 1 to 1000 kbp, and the copy number within host cells varies from single copy to thousands of copies per cell. Plasmid DNA exists in multiple topological isoforms, among which the supercoiled conformation represents the most valuable nanoscale structure for clinical applications. Other common forms include open circular and linear conformations; linear plasmids are unsuitable for clinical use due to potential genomic integration risks. During preparation, alkaline conditions may induce local strand unwinding, forming loosely structured denatured supercoiled DNA. Distinct topological conformations exhibit significant differences in electrophoretic mobility, stability and biological activity, imposing specific requirements on the development of purification processes.

Plasmid DNA is predominantly produced by recombinant Escherichia coli fermentation. Small-scale laboratory pDNA preparation is straightforward, yet yields only 5–40 mg/L under non-optimized conditions, which fails to meet clinical demands. A production output of 10–100 mg is acceptable for research and development stages, while industrial manufacturing requires batch yields exceeding 50 g, even up to several kilograms annually. In large-scale pDNA production, chromatographic purification technology is critical for the preparation of high-purity plasmids.

The separation and purification of pDNA confront substantial technical challenges arising from its unique structural features and complex impurity profiles. As a high-molecular-weight, negatively charged supercoiled molecule, pDNA is susceptible to shear-induced degradation during purification. Meanwhile, it shares highly similar charge, molecular size and hydrophobicity characteristics with major impurities including RNA, genomic DNA (gDNA) and endotoxins. Supercoiled pDNA accounts for merely 1–3% of the total bacterial lysate in conventional fermentation broths, necessitating the development of highly specific and efficient purification workflows. The standard production workflow for pDNA vaccines mainly comprises fermentation culture, cell harvesting and lysis, solid-liquid separation, primary purification, polishing purification, and concentration.

The development of industrial-scale pDNA purification processes faces considerable technical challenges due to its specific physicochemical properties and complex impurity composition. As a critical therapeutic biologic, pDNA must comply with stringent regulatory standards. Additionally, the high molecular weight of pDNA increases solution viscosity and reduces diffusion coefficients, raising higher technical barriers for purification process design. At present, multi-step combined chromatographic processes are widely adopted in industrial production. Anion Exchange Chromatography (AEX) serves as the core step owing to its high selectivity and operational simplicity, enabling effective differentiation between supercoiled and open circular conformations. Hydrophobic Interaction Chromatography (HIC) achieves separation based on hydrophobicity differences of nucleic acids, while Size Exclusion Chromatography (SEC) is mainly applied to remove small-molecule impurities.

SEC achieves pDNA separation based on molecular size differences and can be used independently or coupled with AEX and other chromatographic methods. It is particularly suitable as a polishing step, with its separation mechanism relying on discrepancies in hydrodynamic radii among DNA molecules of different conformations. Typically, genomic DNA, with the largest molecular volume, elutes first, followed by relaxed plasmid conformations, while tightly compacted supercoiled pDNA elutes last. Small contaminants such as RNA, endotoxins and proteins can be efficiently removed simultaneously, yielding high-purity supercoiled pDNA with a recovery rate of approximately 70%, alongside concurrent buffer exchange. Despite limitations including low binding capacity and insufficient selectivity, which make it unsuitable for primary capture, SEC possesses unique advantages in final product polishing and effectively eliminates residual open circular pDNA, gDNA, RNA and endotoxin impurities.

AEX stands as the core technology for pDNA purification, with its separation mechanism relying on electrostatic interactions between phosphate groups on the pDNA backbone and positively charged functional groups on the stationary phase. Although topological isoforms carry similar total net charges, tightly folded supercoiled pDNA exhibits higher local charge density, resulting in longer retention times than open circular conformations during salt gradient elution. The separation of linear plasmids is further affected by conformational elasticity, while secondary interactions such as AT content and hydrophobicity may compromise separation selectivity.

To improve resolution, studies have adopted a mobile phase containing 10% isopropanol with temperature optimized to 35 °C, achieving baseline separation of three topological isoforms on DEAE weak anion exchange columns. In process development, novel monolithic column technology demonstrates prominent advantages. Polymethacrylate monoliths feature large pore sizes of 750 nm, overcoming diffusion limitations of traditional porous media via convective mass transfer and completing supercoiled pDNA separation within 3 minutes. Compared with particulate fillers, monolithic columns maintain low pressure drop at high flow rates of 100–800 cm/h, enabling high-recovery and high-purity separation. Notably, conical monolithic columns coupled with diethylamine systems deliver optimal cost-performance in the purification of 4.2 kbp plasmids, with the required elution salt concentration (~0.9 M NaCl) significantly lower than conventional methods.

For industrial manufacturing, single-step AEX using DEAE Sepharose FF resin enables efficient preparation of supercoiled pDNA, and complying with pharmacopoeial standards can be achieved by coupling with Sephacryl S-500 for desalting. Recent research reveals that quinine carbamate ligands specifically recognize pDNA topological isoforms with varying supercoiling degrees through hydrogen bond donor-acceptor interactions, providing a novel strategy for high-selectivity separation.

Reversed-Phase Chromatography (RPC) separates pDNA via interactions between hydrophobic regions of pDNA molecules and nonpolar ligands on the stationary phase, with elution achieved by organic solvent gradients. For the separation of polar nucleic acids, ion-pair reagents are required to form hydrophobic complexes. RPC has been successfully applied to supercoiled pDNA purification and can efficiently remove impurities such as RNA, small-fragment gDNA and linear plasmids. Nevertheless, it has obvious limitations: high-salt loading conditions (e.g., 3 M ammonium sulfate) are required to enhance pDNA hydrophobicity, and the use of organic solvents introduces toxicity and safety hazards, restricting its industrial application.

In contrast, HIC separates nucleic acids based on hydrophobicity differences between single-stranded and double-stranded structures. Although standalone HIC hardly meets pharmaceutical purity criteria, coupling with SEC can satisfy regulatory purity requirements.

Affinity chromatography is a high-selectivity purification technology based on specific biomolecular recognition, which separates target molecules via specific ligand-analyte interactions. It offers distinct advantages including simplified purification workflows, improved product yield and optimized process cost-efficiency. However, traditional affinity chromatography suffers from drawbacks such as poor stability and low binding capacity of natural ligands.

To address these limitations, multiple synthetic ligand systems have been developed, including Immobilized Metal Affinity Chromatography (IMAC), Tris-Helix Affinity Chromatography (THAC), as well as protein-DNA and amino acid-DNA affinity chromatography. IMAC achieves selective separation via metal ion chelation; among these systems, the Cu(II)-IDA complex specifically binds RNA and damaged DNA, allowing intact pDNA to be recovered in flow-through mode with a resin capacity exceeding 100 g/L. THAC realizes sequence-dependent purification based on nucleic acid triplex formation via designed specific oligonucleotide probes, yet it requires prior sequence modification of target plasmids. In amino acid affinity chromatography, arginine ligands exhibit specific recognition capability for supercoiled pDNA, achieving a recovery rate of 79% with excellent transfection efficiency. Protein-DNA affinity systems utilize DNA-binding proteins such as zinc finger proteins and lac repressors, among which the lac repressor system shows remarkable preference for supercoiled conformations.

These technologies feature respective merits: IMAC offers excellent scalability; THAC enables sequence-specific purification; amino acid-based systems operate under mild conditions with high biosafety; protein-based systems mimic natural molecular interaction mechanisms. Nonetheless, inherent limitations remain, including suboptimal selectivity of IMAC for intact pDNA, prerequisite plasmid modification for THAC, and potential genomic DNA residual in protein affinity systems.

The core challenge in pDNA vaccine production lies in establishing an efficient and scalable purification process. Current research demonstrates that chromatography, as a pivotal downstream purification tool, must simultaneously meet stringent requirements for high product purity, high homogeneity of supercoiled conformations, and economic feasibility. Convective interaction media monoliths exhibit prominent advantages due to unique mass transfer characteristics and high binding capacity, with uniform pore structures enabling direct scalability from laboratory scale to industrial production. Among various affinity chromatographic technologies, amino acid-based affinity systems integrate biological specificity and operational simplicity, holding promising development prospects.

Nevertheless, prevailing technologies still face common bottlenecks such as limited resin binding capacity and low molecular diffusion rates. Future research should focus on novel ligand design and technological integration to develop highly selective and cost-effective purification strategies, providing critical technical support for the industrial manufacturing of DNA vaccines.