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Cobetter Pultrix™ PS Membrane Adsorbers for Efficient Plasmid Purification

2025.10.23 21

As a core material of mRNA vaccines and virus vectors, Plasmid DNA (pDNA) is at the forefront of the fight with cancer, cardiovascular diseases, immune system disorders, and infection diseases. The production of pDNA represents a key bottleneck - traditional methods not only has a low yield but also rely on complex purification processes, limiting the scalability and sustainability of the the overall manufacturing workflow.

Different Topological Morphologies of Plasmids

Supercoiled Plasmid (sc pDNA)

Also known as covalently closed circular DNA (cccDNA), this form is created when double-stranded DNA twists upon itself like a coiled rope, forming (negative) supercoils through the catalysis of the enzyme gyrase. Its compact structure facilitates the storage of vast amounts of genetic information within the confined space of a cell. sc pDNA is intact, stable, biologically active, and exhibits high transfection efficiency.

Open Circular Plasmid (oc pDNA)

This form results when one strand of the sc pDNA is broken ("nicked"). This can occur naturally during bacterial growth, or be induced by UV irradiation, nuclease activity, or mechanical damage during extraction, and is also influenced by storage temperature post-purification. The loss of the supercoiled structure makes it more relaxed. oc pDNA can be converted into linear pDNA through treatment with endonucleases.

Linear Plasmid (linear pDNA)

This form is generated when both strands of sc pDNA are broken at adjacent sites, or through specific cleavage by restriction endonucleases.

Oligomeric Concatemers

These can occur in any of the three structural forms mentioned above. Here, multiple plasmid units are linked together in a single chain, yet they behave identically to monomers in enzymatic digestion processes. Concatemers typically arise from homologous recombination after plasmid replication or during the replication process itself. Notably, the larger the inserted fragment in a plasmid, the higher the ratio of oligomers to monomers.

Process Challenges and Strategies

The purification of plasmid DNA (pDNA) presents several challenges: the target product account for only about 3% of the E. coli lysate, and most impurities share similar negative charges and molecular sizes with pDNA, making separation difficult. Additionally, the final supercoiled pDNA must meet regulatory purity requirements (>90%), demanding higher standards for process resolution and controllability.

pDNA is highly sensitive to shear forces and prone to structural damage during operations such as concentration, leading to alterations in topological forms and compromised product quality. Therefore, minimizing high-shear steps like tangential flow ultrafiltration helps preserve its structural integrity and high purity.

Traditional chromatography resins face limitations in pDNA purification: due to the significantly larger size of pDNA molecules compared to proteins, they struggle to enter the resin's micropores, resulting in low binding capacity and poor mass transfer efficiency. High feedstock viscosity and potential contamination can also lead to increased column pressure and extended processing times. Despite these shortcomings, large-scale purification still primarily relies on resin-based processes. To address this, a growing number of pDNA manufacturers are turning to convective media (such as membrane absorbers and monolithic columns) to enhance the efficiency and productivity of chromatography steps, thereby driving process upgrades.

Cobetter Pultrix™ PS innovatively employs a PES membrane as the chromatography matrix. The large-pore structure of the membrane material significantly increases the binding capacity for large plasmid molecules. Combined with the unique convective mass transfer mechanism of membrane chromatography, the mass transfer efficiency far surpasses that of traditional column chromatography. The purification step is reduced from approximately 2 hours per cycle in conventional methods to just 20 minutes, substantially improving the efficiency of both process development and scaled-up production. This offers a rapid, efficient, and scalable solution for plasmid purification.

SEM of Pultrix™ PES substrate film

Case Study of Cobetter Pultrix™ PS Membrane Absorbers

Material Information

Buffer Information

Sample Preparation

The eluate from the Pultrix™ XQ membrane was adjusted to a final concentration of 2.2 M (NH₄)₂SO₄ using Buffer D. Experiments were then conducted under the conditions specified in the table below, utilizing a Pultrix™ PS membrane (bed volume: 0.2 mL).

Since the volume of the small test membrane is small and the dead volume of the chromatography system has a greater impact, the volume is set to be relatively large. After peak collection, samples are taken for gel electrophoresis, concentration measurement, HPLC, and various impurity inspection items.

Experimental Results

The agarose gel electrophoresis results demonstrated that after the Pultrix™ XQ membrane eluate was processed through the Pultrix™ PS membrane, the open circular plasmid was substantially removed during the wash step, and the proportion of supercoiled plasmid in the eluate was significantly increased.

Impurities Residue

The Pultrix™ PS membrane adsorber (chromatography) achieves a polishing purification yield of approximately 70%, increasing the proportion of supercoiled pDNA from 89.1% to 96.4%, while effectively controlling the residuals of all impurities.

It is noteworthy that in head-to-head comparative tests across multiple projects, Cobetter's two-step plasmid membrane chromatography purification process resulted in lower impurity residuals in the final product compared to the traditional three-step method.