The roles, rising demand and purification optimization of plasmid DNA: an interview with Christie Childers

In this interview, Christie Childers dives into the evolving roles of plasmid DNA (pDNA) in gene therapy, the growing demand for high-purity material and the purification standards shaping the field. She also shares insights into her work with novel nanofiber adsorbents designed to optimize pDNA purification.

Christie Childers is a Senior Scientist at Astrea Bioseparations (Cambridge, UK), leading the next-generation development of nucleotide purification solutions based on novel nanofiber technology. Her journey began in the field of molecular biology at Carleton University (Ottawa, Canada), where she received a PhD investigating enzyme purification and post-translational modification for metabolic regulation.

Christie now has four years of industry experience in downstream process development with a past focus on customer-based research and development (R&D), process scale-up and technical transfer of workflows at CPI (Darlington, UK). Christie now leads and develops nanofiber-based pDNA and mRNA processes at Astrea Bioseparations.

What roles do pDNA play in gene therapy product development and manufacturing?

pDNA plays a key role in nearly all gene therapy products. It serves as the starting template in raw materials for in vitro transcription (IVT) manufacturing, viral vectors and other gene therapies. Additionally, pDNA is being developed as a drug product in DNA-based therapies, such as vaccines.

How has the increasing demand for gene therapy products impacted the need for high-quality pDNA?

In 2020, the urgent need for high-capacity, high-quality pDNA production became very clear, as hundreds of biopharmaceutical companies transitioned from clinical development to commercial-scale manufacturing in response to COVID-19 [2]. The integrity of this starting material has been shown to be critical to ensuring the safety and efficacy of final therapies, and as such, its production is closely controlled. Across the industry, efforts are underway to reduce variability in pDNA (whether that template is E. coli generated or synthetic) while increasing analytical stringency wherever possible. As more companies turn to gene therapies for therapeutical applications, the demand for high-quality pDNA continues to grow.

What are the key challenges in pDNA manufacturing and how can they be addressed?

Many challenges in plasmid production can be addressed through strict upstream control, either through controlled fermentation processes or synthetic DNA production reactions. These approaches enable the production of high-quality, covalently closed circular plasmid (supercoiled plasmid) in an increasingly high titer, even before downstream clarification processes begin. This production control reduces target-related impurities, minimizing the need for very high-resolution capabilities.

Resolving product-related impurities can lead to low yields, particularly when separating target impurities with similar chemistries. Therefore, reducing this processing requirement supports yield maintenance. The rapid success in generating high-quality pDNA at high starting titers, however, has emphasized new challenges: downstream binding capacity, processing flow rates and pressure thresholds are increasingly becoming the rate-limiting factors in scaling production.

There is also a growing need for ultra-scaled-down methods to support screening of new plasmid targets and processes. Some groups require hundreds of R&D-scale plasmid purifications, which can be time-consuming and labor-intensive. Providing small-scale screening methods that can then be directly scaled up to manufacturing capacity will be an advantage moving forward.

What standard methods are used to assess pDNA purification throughout the manufacturing workflow?

Many methods are used to ensure pDNA products meet the critical quality attributes required for their intended application. While some small molecule methods that are well established under Good Manufacturing Practice conditions are appropriate, assessing larger biomolecules presents different challenges. For example, certain plasmids will become too large for standard HPLC columns, requiring larger flow paths to accommodate their size.

Restriction mapping and agarose gels are still considered the standard for confirming plasmid identity and purity. Other techniques such as capillary electrophoresis, HPLC and ELISA are used for isoform, host cell RNA, and host cell protein characterization. GMP-grade pDNA must also meet standard endotoxin, bioburden, sterility and mycoplasma requirements as outlined by the US Pharmacopeia (MD, USA) standards [5].

Can you share insights on optimizing scalable pDNA purification using novel nanofiber adsorbents?

Nanofiber adsorbents such as pDNAHERO® provide a more open flow path for pDNA production. The benefits of this design are two-fold: there are no dead ends in the membrane — often a source of fouling that limits yields — and nanofiber allows for larger plasmids as companies expand the size of their target sequences. Additionally, the reduced running pressures allow nanofiber-based products to operate at higher flow rates, even when fully loaded with plasmid, due to their high binding capacities. This low-pressure processing is critical for accelerating plasmid purification workflows and reducing costly time in GMP manufacturing suites.

AstreAdept® nanofiber is also a robust solution, enabling scaled-down methods to be cleaned in place and reused repeatedly for extensive R&D screening. This durability is especially valuable in smaller-scale production cycles and for companies that choose simulated moving beds for continuous processing as their means to increase production yields as needed. These strategies are typically explored for smaller production batches, such as those required for personalized medicine.

How do you see the industry progressing toward standardized quality control for plasmids used in cell and gene therapies?

There is growing momentum toward standardized and automated lysis methods, with the ultimate goal of reducing hands-on time for plasmid production. This is already becoming a viable approach with the introduction of technologies such as the alkalizer and other in-line mixing methods [3, 4], which can help reduce variability in the downstream starting materials prior to clarification and capture. There is also increasing interest in more stringent at-line monitoring and in silico process monitoring to improve downstream quality.

As the industry grows, a hurdle for manufacturers will be identifying robust, cyclable manufacturing methods. Pump flow rates are likely to become a primary limiting factor, making it essential to maximize capacity through rapid clean-in-place protocols and the reuse of large-scale devices.

The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of RegMedNet or Taylor & Francis Group.