Plasmids are critical raw materials for the production of viral vector-, mRNA-, and DNA-based therapeutics and vaccines. However, current capacity for their production is insufficient to meet growing demand. Capacity expansions will help, but increasing the efficiency and productivity of plasmid fermentation processes will also be essential. As greater understanding of reaction mechanisms increases, manufacturers are developing more sophisticated processes that overcome key manufacturing challenges. The application of design-of-experiment approaches is also leading to flexible, optimized platform processes that afford high yields of high-purity plasmid DNA across scales and for diverse constructs.

The Importance of Plasmids

Plasmids are small circular DNA molecules found in bacteria and some other microbes. Engineered plasmids were initially used to make bacteria capable of producing therapeutic proteins. Today, plasmids are critical raw materials for the production of viral vectors used in gene and gene-modified cell therapies and viral vector vaccines, as well as for the production of mRNA therapeutics and vaccines.¹ Plasmid DNA itself can be used in direct gene transfer therapy and in DNA-based vaccines.

Demand for plasmid DNA (pDNA) is rising rapidly as more of these next-generation products advance through the clinic, receive approval, and become commercialized. Not only is the number of candidates in the pipeline increasing, the indications they target are shifting from rare disorders with very small patient populations to much more prevalent diseases that affect larger numbers of people and require larger and sometimes multiple doses.²

As a consequence, the small-scale production platforms used to date to manufacture plasmids will not be sufficient to meet future demand. As much as 100–1,000 g of plasmid DNA could be required per year to meet the demand for biopharmaceutical products made from viral vectors.³ Additional quantities will be needed for mRNA products — more than 1 kg of pDNA per billion doses of a typical mRNA vaccine, according to one estimate.⁴

Reliance on Outsourcing for Plasmid Production

Owing to the specialized nature and unique set of skills required for pDNA manufacturing, most production takes place at contract development and manufacturing organizations (CDMOs).⁵ In fact, between 80% and 90% of plasmids used for biopharmaceutical applications are produced by CDMOs.³ Despite the expansion and/or addition of capacity by leading service providers, insufficient production space remains an issue. Sponsors seeking pDNA production support can wait longer than 12 months to gain access to GMP plasmid production capacity.⁶ This issue is only expected to increase, as developers are more and more frequently using GMP-grade plasmids for the production of therapeutic products not just for late-phase clinical trials and commercial production, but for early-phase studies.⁷

Becoming More Sophisticated

Most pDNA is produced via fermentation in Escherichia coli bacteria. As understanding of the mechanisms involved in plasmid production have increased, the design of fed-batch processes using carefully engineered E. coli strains that provide increased yields of pDNA has been possible.⁸ Advances in strategies around cell lysis— which is required to free the generated plasmid DNA from the E. coli cells — have also contributed to higher plasmid recoveries. Processes, as a result, are becoming increasingly sophisticated.

Many Manufacturing Challenges Remain

Despite the improvements in performance of plasmid fermentation processes, the process is complex and continues to pose difficulties for manufacturers. While yields have increased, plasmid titers are still much lower than those observed for recombinant proteins and monoclonal antibodies.⁸ Real-time monitoring of critical process parameters (CPPs) — such as agitation, pH, temperature, dissolved carbon dioxide, pressure, and foam formulation — is essential to ensure the highest possible yields.⁹ Issues remain with assuring plasmid stability and homogeneity. In addition, yields tend to decrease for plasmids that contain difficult genetic payloads in terms of size, base composition, toxicity, and other attributes. For instance, larger plasmids exert metabolic stresses on host cells.

The lysis step also remains a challenge. Under the conditions required for chemical lysis, the pDNA can degrade if processing times are too lengthy.¹ pDNA is also sensitive to degradation by nucleases and shear, particularly larger plasmids. In addition, lysates can be dense and viscous, making harvest and clarification difficult. Furthermore, clarified lysates generally contain less than 3% pDNA, which must be separated from a wide range of impurities, including RNA, genomic DNA, host-cell proteins, endotoxin, and unwanted plasmid isoforms.¹⁰ Finally, while the 2007 FDA guidance for DNA vaccines¹¹ requires that the supercoiled DNA content be at least 80%, the actual expectation today is for the percentage of supercoiled isoforms to be greater than 90%.²

Several Approaches to Increasing Efficiency and Productivity

While plasmid production is complex, the numerous CPPs that can influence plasmid titer and quality provide opportunities for improving performance. Optimizing the sequences of the inserted gene of interest (GOI) and the plasmid itself are essential.⁵ Culture media and cell line selection, control of the bacterial growth rate, reaction temperature, pH and dissolved oxygen content, reducing cell stress, and avoiding DNase contamination are examples of means for impacting both yield and quality.^5,8 Appropriate fed-batch profiles that lower metabolic burden also contribute to higher pDNA production. Optimization of the lysis step, meanwhile, can boost plasmid recoveries. Continuously processing E. coli lysate has been shown to provide more control than traditional batch processing, resulting in reproducible production of high-quality pDNA across scales.¹

More extensive use of process analytical technologies (PAT) provides a means for achieving these goals. Automation is another important strategy for realizing process control.⁹

Platform Process Solutions Vital

Platform manufacturing solutions are well-known to provide significant benefits for recombinant protein and mAb production. Plasmid manufacturers (as well as producers of viral vectors, mRNA, and other next-generation biomolecules) are actively developing their own platform processes with the hope of realizing similar gains. The ultimate goal is to identify a single process that can be used to produce all plasmids at research to production plant scale and facilitate development and commercialization of cost-effective, high-quality plasmids.⁸

Platform pDNA processes should be robust and scalable and generate high-quality product that meets the highest regulatory expectations.¹ Developing such processes requires a thorough understanding of all aspects of upstream, mid-stream, and downstream unit operations and the influences that different E. coli strains and plasmids have on performance. The best strategy for establishing such a platform is to use a design-of-experiment (DoE) approach to fully characterize and identify all of the important CPPs for each step.

Maintaining Flexibility is Paramount

At this point, however, it is not yet possible to develop a single platform process that can be applied for every plasmid and GOI, as plasmid size and sequence affect process performance. Flexibility is therefore essential to enable the production of many different constructs.⁸ Establishing a flexible platform requires the exploration of different plasmid types and E. coli strains during process development in order to identify optimal solutions for different pDNA products.¹ Deployment of single-use technologies from research to GMP production is also important for achieving the necessary level of flexibility and scalability needed to meet growing pDNA demand.⁸ 

References

1. “Manufacture the Highest Quality Precision Plasmids™ with a Reliable Platform Process.” Center for Breakthrough Medicines. 7 Mar. 2023.
2. Ohlson, Jonny. “Plasmid manufacture is the bottleneck of the genetic medicine revolution.” Drug Discov. Today. 25: 1891–1893 (2020).
3. Hitchcock, Tony. “Manufacturing Plasmid DNA: Ensuring Adequate Supplies for Gene and Cell Therapies.” BioProcess International. 17 Oct. 2016.
4. Plasmid DNA Manufacturing Market Size Worth $1.51 Billion by 2030. Grand View Research. 5 Oct. 2022.
5. El-Hajjami, Nargisse and Laurens Vergauwen. “Plasmid DNA Production for Cell and Gene Therapy.” Cell Culture Dish. 30 Nov. 2020.
6. Becerra, Alejandro and Johannes F. Buyel. “Optimizing downstream purification of high-quality plasmid DNA for gene therapy and vaccine production.” Cell & Gene Therapy Insights. 7: 1217–1230 (2021).
7. Hocharoen, Lalintip Sarawuth Noppiboon, and Panit Kitsubun. “Process Characterization by Definitive Screening Design Approach on DNA Vaccine Production.” Bioeng. Biotechnol. 15 Oct. 2020. Sec. Bioprocess Engineering.
8. Challener, Cynthia A. “Achieving High Yields in Upstream Processing of Plasmids.” BioPharm Internationa 36: 10–13, 36 (2023).
9. Spivey, Kayla J. “Streamlining the upstream workflow for plasmid DNA manufacturing.” Thermo Fisher Scientific Technical Note. Accessed 27 Mar. 2023.
10. Carnes, Aaron E. and James A. Williams. “Low Metabolic Burden Plasmid Production.” Eng. News. 1 Mar. 2009.
11. Considerations for Plasmid DNA Vaccines for Infectious Disease Indications. S. Food and Drug Administration. Nov. 2007.