The rapid growth of the cell and gene therapy market is creating exciting opportunities in the advanced therapy sector. The ability to use the unique capabilities of RNA and DNA viruses to deliver functional genes in place of defective ones has led to the pursuit and development of a substantial number of cell and gene therapies. The vehicle for the delivery of these therapies is the viral vector, with adeno-associated viruses (AAV) being the most notable ones, as they have proven to be safe in humans while also exhibiting low immunogenicity.

AAV and lentiviral vectors are commonly produced through transfection with plasmid DNA containing the gene of interest. As products progress through clinical phases, there is a greater need for scalable plasmid DNA production, which must first be manufactured and purified in a GMP facility or high-quality plasmid DNA production areas. This has led to pressure on manufacturers to create the capacity and resources essential to meeting the rising demand for high quality plasmid DNA. CDMO Cobra Biologics recognized the need to address its own plasmid DNA capacity constraints for the rapidly increasing demand for viral vector production by not only increasing physical capacity but also by reexamining the purification process currently being used. Working with Cytiva, the companies developed a next-generation chromatography resin for the purification of plasmid DNA to reduce development costs and increase the manufacturing capacity through increased plasmid batch size and facility throughput. The resin was developed based on Cobra’s need but made available as product for others to order.

Supplier and bio-manufacturer relationships are often treated as transactional ones; yet, through this collaboration, Cobra and Cytiva were able to combine their expertise and experience and create a solution to address this manufacturing constraint. Their success demonstrates that mutual benefits can be achieved when suppliers and manufacturers work together to advance innovation and usher in a new era of drug development.

A need for change

When interest in plasmid DNA purification began over 20 years ago, Cytiva responded by developing the chromatography resin PlasmidSelect Xtra. It is a thiophilic aromatic adsorption chromatography resin with a selectivity that allows supercoiled covalently closed circular forms of plasmid DNA to be separated from open circular forms. However, PlasmidSelect Xtra’s binding capacity for plasmid DNA requires large column volumes to capture available quantities. It also offers limited flow rate due to high back pressures, which in turn restricts the size of column that can be packed and the batches sizes that can be processed.

Cobra knew these constraints would impact the scalability of its process, limiting its ability to meet the increasing demand for plasmid DNA from customers for late phase and in-market products.

Plasmid DNA is commonly used to produce viral vectors.

Fig 1. Plasmid DNA is commonly used to produce viral vectors.

In addition, as many plasmids are only manufactured in one or two batches for clinical trials, the effective resin costs can be high as only a fraction of the resin lifetime is utilized. Increasing binding capacity and throughput from the column was identified as essential to improve utilization rate and cost of goods as well as to increase total manufacturing capacity. There was an additional need for a resin that could be readily re-generated for late phase clinical trials and in-market clinical material.

Cobra and Cytiva knew they had to develop a solution that was not only scalable but could also maintain the resolution of the supercoiled and the open circle, which is required to meet customer specifications of more than 90 percent supercoiled plasmid DNA, while improving capacity and sustaining an acceptable cost of goods. The benefits of developing such a solution were valuable to both companies, as the opportunities in the gene therapy market were growing rapidly. Taking advantage of them, though, required investing in and developing products that could make these therapies a commercial reality.

A modern resin for plasmid DNA purification

At the time the project with Cobra began, Cytiva’s Capto resin platform had already demonstrated improved productivity over its legacy Sepharose resin through high binding capacity and high flow rates. Cytiva’s discussions with Cobra about what they had tried previously provided insight into what would potentially work better in terms of what specific Capto matrix would allow for improved productivity. This led to the development of several prototypes by the custom resin design team, where different existing base matrices and different ligand densities of the existing ligand were evaluated. The parameters considered were Capto base matrixes with different bead and pore size for improved throughput properties as well as variable ligand densities for optimization of selectivity and capacity.

A number of these prototype resins were sent to Cobra for evaluation with their plasmid DNA process. Testing was performed at Cobra with ÄKTA avant 150 system using pre-packed HiScreen columns, which allowed for automated screening of multiple resins without operator input between experiments. Prototype resins were evaluated based on yield, purity of supercoiled plasmid DNA, residual RNA, and residual host cell protein (HCP). The best prototype resin evaluated enabled up to 50 percent higher binding capacity and flow rate as compared to the current PlasmidSelect Xtra resin, with similar purification performance.

Custom column packing services were used to pack the prototype resin into HiScreen format columns, allowing for more rapid studies to assess performance. Upon selection of a suitable resin, Cytiva provided larger volumes of the most promising prototype resin, so Cobra could perform scale-up activities and identify the appropriate capacity for the column. Further experiments were performed utilizing higher resin loading levels reflecting real process conditions at large scale.

Based on the chosen resin prototype, Cytiva developed and commercialized the new resin named Capto PlasmidSelect. This resin was developed based primarily on Cobra´s needs but made available for other customers to order. The new resin enabled total resin volume in Cobra’s process to be reduced by up to 50 percent as compared to the existing plasmid DNA purification process. In addition, total buffer volume and the process time were reduced by up to 50 percent compared to existing processes using the legacy resin.

Criteria PlasmidSelect Xtra Capto PlasmidSelect
Quantity of Plasmid 70 g
Load Volume 650 L
Max Resin Capacity 2 mg mL -1 3 mg mL -1*
Max Bed Height 15 cm
Load/Wash Flow Rate 100 cm h -1 150 cm h -1
Elution Flow Rate 50 cm h -1 80 cm h -1

Table 1. Impact of new plasmid purification resin for large scale plasmid DNA purification;
* may be increased further for some plasmids.

With this new improved resin, Cobra now has the opportunity to address manufacturing capacity. It is currently planned to be implemented into the plasmid DNA manufacturing process within Cobra’s new manufacturing suites, enabling a 10-fold scale up of the plasmid DNA bioreactor from 50 liters up to 500 liters.

Addressing manufacturing constraints for novel therapies

As today’s portfolios become more diversified, open communication between biomanufacturers and suppliers is vital in order to head off industry constraints with novel therapies, such as Cobra’s need for a better resin to efficiently produce plasmid DNA. The bottlenecks in manufacturing supply may become the result of a lag between the realization that new technologies need to be developed and the response to actually invest in them. Identifying possible solutions earlier in manufacturing will help shorten timelines for early phase clinical trials as well as developing commercial rates for manufacturing. Utilizing the skills of a competent partner can facilitate these types of discussions and lead to the preemptive creation of meaningful solutions.

Often, though, partnerships fail because ambitious goals were set without the means to deliver them, not just from a technical perspective but also because of an inability to allocate the resources to actually achieve them. One of the many factors that contributed to the seamless collaboration between Cytiva and Cobra was not only the dedication of the necessary resources but also the shared commitment of both teams toward realistic goals and objectives. Each team brought a unique perspective to the table that they were willing to share openly with their partner: Cobra’s extensive experience with novel, early-stage clinical plasmid DNA and Cytiva’s expertise in biomanufacturing technologies and solutions. This type of united effort is what the industry needs to address many of the key challenges of manufacturing emerging therapeutics, such as gene therapy, in order to deliver these life-saving drugs to the patients who need them.


Janice Charlesworth, Downstream Processing Leader, Cobra Biologics

Tony Hitchcock, Technical Director, Cobra Biologics

Hazel Tharia, Head of Technical Co-ordination, Cobra Biologics

Henrik Ihre, Director CDM Cytiva

Åsa Hagner McWhirter, Senior Scientist Cytiva