Clinical Manufacturing of Plasmid DNA: Ensuring adequate supplies for gene and cell therapies
Gene therapy is not a new concept. Initial studies were done over 20 years ago. In the last few years, however, the gene therapy market has exploded beyond initial regenerative strategies using viral vectors at a reasonable Compound annual growth rate of 29.7% during the forecast period (2021-2027). The potential use of T cells modified with recombinant virus vectors to treat immunotherapy diseases is gaining more attention in the industry. These therapies can be based on either AAV (Adenoassociated virus) or lentivirus, with both vectors often generated using transient expression routes that use plasmid DNA to generate the starting material.
Each vector generated with two, three, or even four plasmids. Each vector contains two to three structural/helper genes, and one therapeutic transgene. These constructs used to create the viral vector of your choice.
It is well-known that Clinical Manufacturing of modified T cells on large scales for large patient populations poses many problems from a cost-of-goods, scientific and technical perspective. These products raise the complexity of biomanufacturing. This is evident in the large number of biological entities that produced.
As products progress into the late-stage clinical trials, and clinical success increases, there will be an increasing demand for commercial plasmid manufacturing at larger scales. As more people exposed to these products, there is a reasonable expectation that the manufacturing process will be subject to greater scrutiny. These clinical approaches continue to produce positive clinical outputs drug developers will need more “plans for success” and long-term manufacturing strategies to be able to carry through to later clinical phases and into in-market supply.
Many companies recognize the need to make significant improvements in manufacturing methods for cell therapies. This is also true for viral vector manufacturing processes. These changes are necessary to meet the needs of late-phase clinical trials as well as in-market requirements for products that have been successful. These changes require technical solutions.
There will certainly be an increase in the demand for plasmid-DNA production. Production platforms will need to scale up to meet increasing demand. With small-scale production platforms that produce 10g/batch, it may be possible to meet the niche therapy demands of approximately 10,000 patients. However, it is possible to predict that each plasmid vector will require 100-1,000g per year for a product on the market.
This increased material demand means that road maps needed to help guide changes in the process of manufacturing viral vectors or plasmid DNA. These guides must also be comparable in terms of safety and functionality.
This is a step further. As products move through the clinical phase, developers need to build strong supply chains. Good manufacturing practice (GMP), guidelines recommend that you establish backup suppliers to produce critical materials for late-phase or in-market products. This may be true for plasmid DNA. It could involve not only different sources and facilities but also different manufacturing processes.
Fermentation & Downstream Purification:
Fermentation processes are very similar. They usually operate in fed-batch mode. They will likely be antibiotic-free and use chemically defined media for larger-scale and later phases. The cell paste then harvested, and the plasmid by alkaline lysis. Manufacturers use proprietary cell lysis techniques to extract the plasmid DNA. This is where most process differences occur. It is not possible to determine how different methods affect the quality or functionality of a product. This information published in scientific literature.
Purification processes are usually a combination of ion-exchange (IEX) and hydrophobic-chromatography (HIC) steps, and in some cases include gel-permeation (size-exclusion) chromatography as well. These processes employ IEX to eliminate the endotoxin, residual RNA, as well as HIC to extract host DNA and to critically separate open- and close-circle plasmids. Precipitation steps used to reduce host RNA levels.
Specifications and Analytical Testing:
Most drug development companies currently base their specifications on FDA 2007 guidelines. However, the FDA Guidelines box explains closed-circle DNA level for injection DNA vaccines is 90% supercoil instead of 80%. While analytical methods can vary, accepted approaches to measuring residuals. Different methods used to quantify plasmid forms. Some groups prefer HPLC-based techniques while others prefer CE-based methods.
Measurement of functionality and potency, the only area not covered by those guidelines. Although plasmid supercoiled levels often used as a surrogate for plasmid stability and functionality, and transferability in the production of virus vectors, there is not enough evidence to support this. It is difficult to determine the critical quality attributes of plasmids that used to generate viral vectors. This is going to critical as future manufacturing processes evolve to meet potential demands. We also need to identify the less important attributes.
Future Potential Manufacturing A Approaches
It will be more important to understand critical quality attributes. Manufacturers are looking for ways to improve plasmid production scales and manufacturing processes. This is because they want to find methods that allow them to achieve acceptable manufacturing scales and process flexibility while still maintaining the acceptable cost of goods (CoG). The current expectation is that material is purified according to specifications for injectable products. This is in direct contradiction to the approach taken by some companies to lentivirus manufacturing, where only a limited amount of purification done to remove host contaminants.
However, this is justified by strong demonstrations of cell therapy product clearance. Could similar methods used for the plasmid genome to simplify the purification process? Also, we need to consider the design of manufacturing plants. these products best produced in cleanrooms? Or can they made in smaller production areas that rely on closed-system methods, fixed or one-use?
We may also need to consider aspects of process validation when it comes to quality systems. Can we validate the platform to produce multiple products if plasmids created using platform-based methods, such as cleaning validation and risk assessment regarding extractable or leachable in single-use systems?
We also consider other approaches to plasmid production. It is possible to create viral vectors using synthetic manufacturing processes that make smaller DNA fragments, such as those developed by Touchlight (www.touchlight.com). They could be simplified in the very early stages of development. These small DNA fragments considered chemical entities (rather than biological entities) if they can fully characterized.
Supply Chains
To support the manufacturing of two to four plasmids per viral vector, each therapy that is going into clinical trials, and each product in the market, supply-chain management will become more important. With almost all plasmid DNA production outsourced to CMOs and specialist manufacturers, drug developers need to find long-term solutions to meet future market demands. This involves identifying risk-based manufacturing methods and supporting the development and investment in required capability and manufacturing capabilities. Flexible approaches required to ensure supply integrity. This is especially true for products in the market. Alternative approaches may possible to meet plasmid requirements in the future, which could allow for a reduction of CoG.
Making Promises a Reality
With the rapid emergence of cell and gene therapy products, we are living in exciting times. Companies are identifying manufacturing challenges as they seek to bring them to market. Solutions sought. We need to increase industry investment in plasmid manufacturing platforms. This includes novel approaches. It is also necessary to have clearer information about the quality systems that will support and direct these investments and development to speed up the production of life-changing therapies.
Plasmid Production Details
Multi-gram batches of cGMP for injection or ex-vivo (e.g. Applications of modified T-cells
With options to produce up to 120L per lot, the fermentor can hold 30L.
Scalable manufacturing process without the use of organic solvents or animal-derived products
Superior Quality Control Testing including HPLC/qPCR
Custom potency assays developed in-house
Production and testing of master/working cell banks
Access to plasmid backbones, fermentation methods, and other resources to increase process yield for difficult plasmids
For up to 2,000 doses, aseptically filled final product
Ultra-Pure Plasmid DNA
Waisman recently created an Ultra-Pure Plasmid DNA Purification Process for clinical applications, such as in infectious disease vaccines. It has very strict specifications regarding host contaminants. The Ultra-Pure step can improve plasmid DNA purity and quality beyond what is possible with standard manufacturing processes. Below is a table that shows the differences between our standard process and Ultra-Pure.
The plasmid should, in general, be as small as possible. A large plasmid can place a significant metabolic load on the E. A large plasmid can place a metabolic load on the E.coli host. However, the therapeutic approach may predetermine the size of the plasmid. Plasmids that code for one gene usually have a size of 5 kbp (monocistronic), whereas coding for multiple genes can be 8 kbp and larger (polycistronic). A gene-silencing approach based on RNAi results in plasmids that are quite small. Plasmids that code for expressed interfering RNA (eiRNA), rarely exceed 3.5kbp.
Another factor to consider is the selection marker of the plasmid. Most commonly, plasmids code for an antibiotic-resistance gene (e.g., against kanamycin) to allow the selection of plasmid-carrying clones. From a regulatory perspective, the therapeutic coadministration of a prokaryotic resistant gene and the use of antibiotics during manufacturing pose problems. Concepts for antibiotic-free selection of plasmids based on operator-repressor or the supplementation of an auxotrophic gene developed.
