Demand for plasmid DNA (pDNA) is skyrocketing as mRNA and gene therapy candidates continue to progress from preclinical to early- and late-stage clinical studies. Sourcing challenges, quality issues, scaling difficulties, lack of standardization, and the need to meet specific and varying requirements for different applications are some of the hurdles that manufacturers must overcome if they are to meet that growing demand.
Plasmid DNA Plays a Critical Role in mRNA manufacturing
Production of messenger RNA (mRNA) begins with plasmid DNA (pDNA), which is a critical starting material for the process. Viral vector manufacturing also leverages pDNA as a critical starting material. With the dramatic increase in interest in both mRNA and viral vectors for gene and gene-modified cell therapies, the demand for pDNA has skyrocketed — particularly pDNA intended for use as a starting material rather than as a direct therapeutic.
Complexities of Sourcing pDNA
Capacity for the production of pDNA, despite significant expansions by existing players and the entry of many new suppliers, remains constrained. Indeed, it is often necessary to order GMP-quality, custom-engineered pDNA at least one year in advance. Raw material supplies also remain limited, particularly single-use equipment components. Fortunately, additional capacity is expected to alleviate this situation in 2024, but those manufacturers that have formed partnerships with suppliers tend to have more supply chain security.
Need for High-Quality pDNA
Regulatory expectations for critical raw materials, including for ancillary materials that do not end up in the final product, have been increasing in recent years. Suppliers have responded by establishing three different product grades: research, an intermediate “high-quality” version that approaches but does not fully achieve GMP standards, and GMP.
One challenge facing both manufacturers and customers for the high-quality grade is the lack of a standard definition and hence consistent standards. Each supplier has its own specifications, with high-quality defined differently by each supplier. In some cases, it is likely that customers must specify the attributes of the high-quality pDNA they seek to purchase rather than it truly being a universal quality grade like GMP-grade pDNA. Going forward, there is an expectation that the industry will continue to move first to high-quality pDNA but will ultimately use GMP-grade material for most development work.
Wacker Biotech’s pDNA production site in San Diego is equipped with 43-L single-use bioreactors and 650-L stainless-steel fermentation vessels, including continuous cell lysis.
Scaling Challenges
Most pDNA is produced via bacterial fermentation using Escherichia coli strains in fed-batch mode. Cell lysis frees the pDNA, which is then purified via tangential-flow filtration and typically two chromatography steps, followed by sterile filtration and fill/finish operations.
Traditional pDNA fermentation processes are slow, afford yields much lower than those obtained for recombinant proteins and antibodies, and suffer from frequent batch failures. Yields and quality are also affected by the size of the plasmid and the nature of the genetic payloads. Purification is, meanwhile, typically time-consuming and complicated by the size and high negative charge of pDNA, which lead to low flow rates and difficulties achieving sufficient concentrations — problems that are magnified at larger scale.
In addition, pDNA is shear-sensitive and can undergo topological changes, leading to higher levels of non-supercoiled isoforms, the risk of which rises as process scale increases. Furthermore, many impurities present after the lysis step have properties similar to those of the desired plasmid and can be quite difficult to remove without significant product losses.
Downstream processing of pDNA in Wacker Biotech’s San Diego facility is available to suit various customer needs along the manufacturing path.
A final complication is the wide variation in genetic payloads and other requirements of plasmids for different applications. Transient transfection to produce different viral vectors requires different plasmids with varying components and payloads, depending on the target virus. Those properties are different still from those of the pDNA required for the manufacture of mRNA vaccines and therapeutics, each of which will have a unique sequence. Furthermore, research has shown that careful engineering of plasmids to eliminate non-necessary genetic material can be highly beneficial.
Value of Platform Processes
To overcome these challenges, many manufacturers have focused on developing platform manufacturing processes for the production of pDNA. The key to success of these platform approaches, particularly for contract manufacturers, is the incorporation of sufficient flexibility to accommodate a wide range of pDNA molecules. Developing a platform for which the impacts of Escherichia coli strain characteristics and plasmid size and complexity on product yield and quality are understood eliminates the need to optimize individual processes for different plasmids, providing both cost and time advantages.
Benefits of Integration with End-Use Applications
For developers of novel drug products that leverage pDNA as a critical starting material, there can be tremendous benefits to partnering with a contract development and manufacturing organization (CDMO) that can support not only pDNA development and production but also development and manufacture of the ultimate therapeutic or vaccine product.
Combining capabilities in pDNA engineering, design, and production with mRNA manufacturing, for instance, ensures development of the optimal pDNA sequence for the specific mRNA therapeutic or vaccine product. In addition to simplification of planning and oversight of each process step, all materials are produced under the same quality, management, and regulatory systems. Furthermore, close collaboration between R&D, analytical, and production teams ensures alignment of all activities with reduced timelines, cost, and risk associated with transfer from pDNA to mRNA production.
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