Read time: 5 minutes

Plasmid DNA serves a variety of purposes, from critical starting material for proteins, mRNA, viral vectors, and drug substances. Below, Dr. Emma Bjorgum, the Vice President of Client Services of the DNA Business Unit at Aldevron and an expert in plasmid manufacturing, provided insights into the process and an outlook on the future.

When working with plasmids, finding the right partner is essential for success. With 40-50% of CGT programs in Phase III facing disruptions – often due to CMC issues – minimizing risks from the start of your program is crucial. The whitepaper from Aldevron highlights unique challenges of developing cell and gene therapies, plus common pitfalls and avoidable costs that can impact long-term success. Practical considerations and solutions to help you navigate plasmid manufacture, quality assurance and regulatory issues from day one are highlighted.

What applications does Aldevron manufacture plasmid DNA for?

Emma Bjorgum: Aldevron manufactures plasmid DNA for a variety of end applications. Much of our experience and expertise is comprised of manufacturing plasmid DNA for cell and gene therapy applications. We also manufacture for all phases and stages of pipeline development from early discovery to commercial applications. Aldevron is among the first to offer plasmid DNA at full current Good Manufacturing Practice (cGMP) or clinical grade and has pioneered a mid-grade between research grade and full cGMP, called GMP-Source. This has allowed us to support an estimated 1500 clinical trials run by over 1000 clients. We thrive on supporting clients from the early stages of their clinical programs through commercialization.

How does your support vary by application?

Emma Bjorgum: We can provide plasmid DNA for various applications. Two specific examples include support of mRNA and AAV gene therapies and vaccines. For mRNA applications, we provide plasmid DNA as a linearized product and can perform the linearization with a client-designated enzyme. We also screen the plasmid construct prior to manufacturing to optimize conditions for both yield and stability of the poly(A) tail (if encoded). For AAV drugs, we optimize conditions for scale up by evaluating different host cell lines and temperature combinations for inverted terminal repeat (ITR) retention. A third example is our investment in next-generation plasmid technology, Nanoplasmid vectors. Nanoplasmids are comprised of very small, efficient backbones (~500 bases). Removal of bacterial and antibiotic resistance genes improve both safety and performance. One area where Nanoplasmids are showing particularly strong performance is as a homology-directed repair (HDR) donor template for CRISPR knock-in applications.

How do you ensure the quality of your plasmids at the different quality levels you offer?

Emma Bjorgum: Aldevron offers a comprehensive quality control testing panel of assays for release of plasmid DNA. Assays include various methods for identity, safety, bacterial host components and bioburden/sterility. Almost all our assays are conducted in-house, and methodologies are closely aligned for testing and release of RUO, GMP-Source and GMP methods.

How have the requirements for plasmids changed over the past 5-10 years?

Emma Bjorgum: In the earlier days of cell and gene therapy, there were hardly any references to plasmid manufacturing recommendations where plasmid DNA is utilized as a critical starting material or raw material. As cell and gene therapy has continued to see additional approvals, we have seen more recent considerations from the agency for CAR-T therapies with a recommendation to remove any unnecessary transgene in the vector such as antibiotic resistance markers. Aldevron’s Nanoplasmid technology ameliorates this concern as it utilizes a sucrose selection technology negating the need for any antibiotics in the manufacturing process.

What changes do you anticipate moving forward?

Emma Bjorgum: Moving forward, we are likely to see additional scrutiny on vector backbones and the removal of any extraneous sequences. We are also likely to see increased specificity on scale and how manufacturers can deliver exactly what is needed at the point in time of clinical development. Aldevron is focused on providing the ‘right sized’ scale for manufacturing and can meet both exact quantity and batch deliverables.

What innovation is helping to drive the industry forward and how will analytics need to evolve?

Emma Bjorgum: Newer vector technologies, such as nanoplasmid, can help address concerns with extraneous sequences in the plasmid backbone size since it consists of only 200 bp. Another innovation area is next-generation microbial cell lines to improve the yield and stability of plasmid DNA, such as the REVIVER cell line. Additionally, non-viral delivery systems are tackling challenges in the industry for payload delivery by lowering costs and delivering products without the constraints of a viral system. Additionally, innovation around the client experience is a key focus for us. Over the past 2 years we have been intensely focused on the client experience and have made incredible progress streamlining the new program on-boarding process, reducing lead times and eliminating deviations. For example, in 2023, we were able to reduce our lead time by up to 80% from construct selection through product release.

mRNA is driving industry growth. How does Aldevron support the mRNA modality from a plasmid perspective?

Emma Bjorgum: Aldevron can provide linear plasmid DNA at any scale and quality level (RUO, GMP-Source and GMP). Our processes allow for linearization with the client-selected enzyme, including a purification step post-linearization to ensure the product is free from any remaining enzyme. We can provide analytical testing for the final linearized product to confirm the percentage of linearized plasmid in addition to poly(A) tail length. Several of our clients get linear plasmid DNA from us and do the IVT and other reactions internally. Increasingly, clients are taking advantage of Aldevron’s broader RNA services, including linear plasmid, IVT and capping reactions, lipid nanoparticle encapsulation and sterile fill-finish services. That includes all the associated analytics, such as CGE, for instance.

What additional services are popular with those manufacturing plasmid for clinical applications?

Emma Bjorgum: Additional services often required to support plasmid DNA for clinical services include stability testing of both final plasmid DNA product and master cell banks. Commercialization support services such as process characterization and process validation are also often required in the late phases of clinical development. Additionally, regulatory services are often utilized to support Chemistry, Manufacturing and Control (CMC) sections of Investigational New Drug (IND) filings or Biologics License Applications (BLAs).