Next-generation therapeutics such as cell and gene therapies and mRNA vaccines hold significant promise for the treatment and prevention of a wide variety of diseases. Further developing these therapeutic technologies and working toward improved manufacturing processes will be vital to enhance their potential to help patients around the world.

One of the key materials involved in the production of these therapies are plasmids. Produced by bacteria, these small, circular fragments of DNA can be genetically engineered to contain a gene of interest. They can then be used to introduce therapeutic genes into cells for cell and gene therapy production, or to produce mRNA for mRNA vaccines.

As the cell and gene therapy and mRNA vaccine markets expand, the need for increased yields of high-quality plasmids is becoming more pressing [1]. Producing highly pure plasmid DNA (pDNA), free from any contaminants and animal-origin (AO) materials, is vital.

By improving plasmid yields, therapeutics can be produced more cost-effectively and at a larger scale, ultimately making it possible to treat more patients. Optimizing plasmid manufacturing methods will consequently be essential in advancing these promising modalities to their full potential.

A growing demand for DNA

The cell and gene therapy market is thriving, valued at $4.1 billion in 2021 and now predicted to grow to over $17.4 billion by 2026 [2]. With increasing numbers of cell and gene therapies progressing toward commercialization, and yet more entering the pipeline, the need for plasmids is soaring. The quantities of pDNA required to enable this expanding industry will put pressure on current capacity, driving efforts to significantly scale up plasmid manufacturing.

In addition to the demand generated by cell and gene therapies, plasmids are also required for the production of mRNA vaccines. Following the commercialization and continued widespread rollout of both the Moderna and Pfizer-BioNTech SARS-CoV-2 mRNA vaccines, there is an immediate demand for large quantities of pDNA. Moreover, as this technology becomes more widely used, particularly to update older vaccines, the demand for plasmids will only grow further. Reflecting these industry developments, the pDNA market—which was valued at $540 million in 2022—is forecast to grow to $1.5 billion by 2030 [3].

Beyond the sheer scale of demand, plasmid quality is also critical. Not only is high-quality pDNA necessary for efficient therapeutic production, it is also vital to protect patient safety. As investigational next-generation therapies progress from the lab to the clinic, and ultimately to the wider population, plasmids must be highly pure and safe for administration into patients. This means that they must be manufactured in accordance with cGMP guidelines, with critical quality parameters established ahead of scale-up. The combined requirements from innovative cell and gene therapies and mRNA vaccines therefore mean that the cGMP production of high-quality plasmids must be significantly increased.

The challenges of plasmid production

Plasmid manufacture is a relatively straightforward platform technology, in which the production process is the same for any gene of interest. pDNA is produced by the fermentation of genetically engineered bacteria in large, stainless steel or single-use bioreactors. Using Escherichia coli (E. coli) to produce plasmids is popular, due to its low cost, relatively fast growth rate, and good safety profile. Harvesting high plasmid yields can be challenging however, with the average amount of pDNA harvested from 1 kg of wet biomass ranging from 0.5–1.0 g, making the process inefficient when not optimized for commercial-scale production [4].

The bacterial fermentation process also requires continuous monitoring of key parameters—including pH, agitation, and temperature—to maintain an optimal environment, which can be expensive and time-consuming at large scale [4]. The nutritional needs of E. coli also must be considered, and the growth medium and supplementation used can impact plasmid quality and biomass. As a result, E. coli cultures require a specialized medium to maintain an appropriate environment and provide the necessary nutrients for maximum cell growth.

To enable high-capacity, high-quality pDNA production, medium choice is therefore critical. Unfortunately, media that have been conventionally used for the culture of E. coli, such as lysogeny/Luria-Bertani broth (LB), may pose challenges for cGMP manufacturing due to the presence of AO components [5]. These components may introduce variability and impact performance, or could even pose safety risks, such as the spread of bovine spongiform encephalopathy (BSE) and other transmissible spongiform encephalopathies (TSEs). Until recently, alternative options to conventional media have been limited, with few options available for achieving an optimized bacterial fermentation process.

Enhancing plasmid yields

To achieve highly pure pDNA and maximize yields while minimizing inconsistency and the risk of contaminants, there is a need for high-quality media and feeds optimized for microbial fermentation. Considerations around the nutritional needs and most productive environment for E. coli cultures are important, as is choosing products that can help reduce lot-to-lot variability.

Medium supplementation can be fundamental in supporting the microbial fermentation process, helping to provide necessary nutrients and promote rapid growth. Traditionally, peptones—protein hydrolysates derived from animal or plant proteins—have been used in LB to provide many of these essential nutrients. However, their origin may lead to inherent variability, which can hinder process consistency. Peptones can still be considered when choosing supplements however, as animal origin–free (AOF) options are available. Careful screening should be undertaken to identify the optimal peptone for a process, determining that it provides the necessary benefits and meets overall process requirements.

When sourcing supplements, it is important to choose suppliers that employ rigorous analytical monitoring to maximize lot-to-lot consistency. Thermo Fisher Scientific provides a range of high-quality Gibco™ supplements, designed to support consistent process performance. In addition, manufacturers can access easy-to-use samples to facilitate simple screening and help identify the optimal product for their process.

Selecting AOF media and supplements can help address contamination concerns and is often preferable for manufacturing under cGMP conditions. When using AOF peptones, identifying key drivers that impact the fermentation process and implementing control strategies to stabilize them can help provide process consistency. To further control the fermentation environment, chemically defined (CD) media may also be considered. These media can help enhance plasmid production, for example by reducing lot-to-lot performance variability due to their consistent formulation. Additionally, because CD media do not contain AO components, there is no risk of bacteriophage contamination or BSE/TSE spread [5].

Thermo Fisher offers a variety of Gibco™ products to support microbial fermentation, including peptones and a fully CD medium—Gibco™ Bacto™ CD Supreme Fermentation Production Medium (FPM). With an AOF and fully chemically defined formulation, this medium can help manufacturers achieve high-density E. coli cultures while reducing lot-to-lot variability. Furthermore, by building on knowledge of peptones, the medium can help provide similar nutritional support without the need for AO components. To help support the large-scale production of plasmids, the medium has also been developed with scalability in mind.

Enabling next-generation therapies

To meet the increasing demand for plasmid DNA, it is clear that plasmid production must be significantly scaled up. Careful choice of E. coli cell culture media and supplements can help improve yields and support the production of high-quality plasmids, while minimizing some of the challenges that can be present. To assist this further, manufacturers can choose advanced products designed to optimize the bacterial fermentation process for high-density plasmid production. By enabling advanced modalities as they gain increasing traction and supporting the industry to continue rapidly evolving, scaling up plasmid production could ultimately help accelerate life-saving therapeutics to patients.

For more information, please see Bacto™ CD Supreme