Overcoming pDNA Supply Challenges for mRNA Manufacturing

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.

2024_side2_WA04

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.

2024_side3_WA04

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.

Wacker Biotech:
An Experienced, Stable, and Integrated pDNA and mRNA Outsourcing PartnerThrough its pDNA Center of Excellence in San Diego, California, Wacker Biotech has decades of experience in pDNA development and production. Our highly experienced team has been manufacturing GMP-compliant pDNA since 2003 and has released over 100 GMP batches.Wacker Biotech’s versatile plug-and-play PLASMITEC® platform leverages innovative, cutting-edge pDNA technologies and high-performing strains to produce excellent yields of supercoiled pDNA a consistent and reproducible manner. Even complex plasmids are produced efficiently, and the same process is leveraged to produce material of different grades in small to large volumes for seamless scaling.The proprietary platform process includes continuous lysis and pDNA quality control and enables efficient production of research, high-quality, and GMP grades of pDNA, including clinical and commercial material, for applications ranging from preclinical safety and tox studies to viral vector, mRNA, and injectable DNA product manufacturing.The combination of our expertise in pDNA and efficient production platform with extensive capacity enables Wacker Biotech customers to benefit from shortened lead times and reduced project turnaround times. Those clients with mRNA products can realize additional advantages by leveraging Wacker Biotech’s integrated mRNA development and manufacturing services, which include in vitro transcription, lipid nanoparticle production, and fill/finish operations from research to commercial scale.Wacker Biotech’s San Diego location provides pDNA production and has been manufacturing GMP-compliant pDNA since 2003.2024_side1_WA04

Originally published on PharmasAlmanac.com on April 1, 2024.

The Pivotal Role of Plasmid DNA

Plasmid DNA was key to the development of biologic drug manufacturing. Today, it plays a critical role in the production of next-generation cell and gene therapies and vaccines. With its plasmid DNA manufacturing expertise, Aldevron has helped facilitate the advance of these important therapeutics. The company continues to invest in additional capacity and novel capabilities to support biopharma manufacturers into the future.

Why Plasmids?

Plasmids are small extrachromosomal double-stranded DNA units that are typically circular in shape and are found across bacterial species. They behave independently of chromosomal DNA and are capable of self-replication. Generally, plasmids contain a few genes that encode proteins for cellular activities that are necessary for bacterial survival. Many are involved in establishing resistance to antibiotics, digesting foreign substances and killing other bacteria. Most notably, they can be picked up from the environment and transferred between bacteria via horizontal gene transfer (HGT), providing a unique nonsexual mechanism for the transfer of genetic information between individual bacteria and across species boundaries.

From Awareness to Understanding

While scientists were aware of the existence of independent strands of DNA in bacterial cells as early as the 1940s, the significance and function was not well understood. The term plasmid was coined in 1952 by Joshua Lederberg, who used it to refer to “any extrachromosomal hereditary element” when he observed virus particles picking up bacterial genes and transferring them to another host by a process he called transduction.

Once the double-helical structure of DNA was discovered and it was understood that DNA is the carrier of genetic information, the nature of plasmids as strands of DNA that can pass on traits became clear. Several plasmids were identified in the 1960s, including fertility and resistance (R) plasmids.

Easy to Manipulate

Plasmid DNA was first isolated in 1967, and researchers across disciplines have been manipulating it ever since. Plasmids are attractive as genetic engineering tools because they are stable and easy to genetically modify. They have 1,000–30,000 DNA base pairs, so they are relatively easy to handle. They do not degrade when cut and can return to their original shape, making it easy to insert new DNA sequences into existing plasmid backbones. And because they self-replicate in bacterial cells, growing large quantities of bacteria is an effective strategy to produce large amounts of DNA. 

Production of Therapeutic Proteins

The first pharmaceutical application of plasmids was achieved in the 1970s. Foreign recombinant DNA was introduced into bacteria, enabling them to produce therapeutic proteins. Production of human insulin via transgene-containing plasmids in Escherichia coli was first accomplished in 1978. By 1980, advances in expression vector and transfection techniques led to the use of plasmids for the expression of foreign genes in mammalian cells, enabling the manufacture of more complex proteins and other biomolecules.

Generation of Transgenic Animals and Plants

In 1981, plasmid DNA was used to generate the first transgenic animal. Mice expressing thymidine kinase from herpes simplex virus were created by injecting recombinant plasmid DNA into fertilized mouse embryos. Transgenic plants followed in 1983 — first to improve the properties of crops, but then as a means for enabling plant-based drug production.

Manufacture of Vaccines and Gene Therapies

By 1991, researchers were exploring the use of plasmid DNA for gene therapies. Compared with recombinant viruses, plasmids are attractive because they can deliver large quantities of DNA while presenting a low risk for oncogenesis and immunogenicity. They are also easier to manufacture in large quantities and tend to be fairly stable. Unfortunately, they are inefficient gene transfer vectors when used in vivo. Today, most gene therapies are delivered in the form of viral vectors, which are manufactured using several different plasmids.

Also in the early 1990s, scientists were learning about the ability of plasmid DNA to stimulate both humoral immunity (antibodies) and cell-mediated immunity (T cells) and thus the potential for plasmids to be used as vaccines. In this case, the plasmids are designed to produce specific proteins from the relevant pathogen and then purified. As with gene therapies, using plasmids avoids the need to work with or introduce infectious agents into the body, and they are stable and can be readily manufactured at large scale. However, they tend to induce weaker immune responses. DNA vaccines are under development for a number of different diseases, from infectious diseases, such as HIV-AIDS, Ebola, malaria and influenza, to various cancers and neurological diseases.

We can provide plasmid DNA for discovery research, clinical and commercial applications.

Gene Editing and Plasmid DNA

Integration of plasmid DNA into the genome allowed for further manipulation. Today, gene knock-outs, knock-ins, overexpression, disease models, conditional mutants and fluorescently labeled proteins are all facilitated by the use of plasmid DNA.

In 2002, plasmid DNA was engineered to synthesize small RNAs, enabling the suppression of gene expression in mammalian cells via RNA-mediated interference (RNAi). Gene editing within rodent cells was achieved in 2011 by coinjecting single-cell embryos with sequence-specific zinc-finger nucleases and relevant plasmid DNA. Mammalian cells transfected with plasmid DNA expressing Cas9 and guide RNA were produced in 2013, enabling the gene editing system CRISPR/Cas to rapidly and precisely edit genomes inside living cells.

Advancing Plasmid Manufacturing at Aldevron

Aldevron has been producing plasmid DNA since its inception in 1998. We began offering research-grade material only, and then expanded into custom manufacturing of plasmid DNA for preclinical, clinical and commercial applications.

We have proven experience producing plasmid DNA needed for viral vectors (AAV, lentivirus), DNA vaccines, CAR-T cell therapy, gene editing, cloning, DNA vaccines, personalized cell therapies and other applications. Companies pursuing truly novel products, such as Sarepta with its gene therapy treatment for Duchenne muscular dystrophy and Genprex with its plasmid DNA treatment for lung cancer, turn to Aldevron as their plasmid DNA supplier.

We can produce supercoiled, linearized, minicircle, nanoplasmid and bacmid plasmid DNA and have the capacity for thousands of small-scale plasmid preps per day and large-scale capacity for manufacturing hundreds of grams per lot. We can provide plasmid DNA for discovery research, clinical, and commercial applications.

Aldevron produces plasmids at different service levels to meet varying customer requirements. Research grade is the quickest option for high-quality plasmid DNA. GMP-Source™ is a cost-effective alternative to cGMP. Our cGMP service provides the highest quality oversight and process control and can support any application, including parenteral administration.

Recently, Aldevron also began offering bulk, off-the-shelf plasmids for the production of AAV and lentiviral vectors.1 The immediate availability of these plasmids significantly reduces the timeline and cost for production of AAV and LV vectors while also facilitating risk reduction through the reliable supply of consistent, high-quality product. The inventory plasmids are also offered in research grade, GMP-Source™ and cGMP service levels.

Gene knock-outs, knock-ins, overexpression, disease models, conditional mutants and fluorescently labeled proteins are all facilitated by the use of plasmid DNA.

Preparing for More Growth

Today, R&D and manufacturing take place at three locations in North Dakota, Wisconsin and Germany. All of our clinical and commercial production operations use disposable manufacturing equipment for reduced cycle times (no cleaning or cleaning validation) and reduced risk of carryover. We have been at the forefront of plasmid manufacturing technology, developing solutions that allow scaling of fermentation and purification processes from 10 L to 1,000 L.

In September 2018, Aldevron opened a new 70,000-ft2 manufacturing facility in Fargo, ND, the largest worldwide for plasmid DNA production, to ramp up capacity and to add capabilities for cGMP recombinant proteins, including CRISPR nucleases, and enzymatically synthesized mRNA. In August 2019, we broke ground on a 14-acre campus expansion at the same site that will include the addition of a second GMP manufacturing facility that more than quadruples the current capacity, with an Administration and Client Experience Center and a Research and Development and Training Center.

Earlier in 2019, we added approximately 4,300 ft2 of laboratory and production space to our Freiberg facility, expanding our capacity and adding a new Beacon Optofluidic platform to support rapid execution of species-agnostic single B cell antibody discovery. In April 2019, we added 1,000-L fermentation capacity to the Wisconsin plant, which will be online by the end of 2019, and plan to further expand the footprint of that site.

In July 2019, we were excited to partner with EQT, a leading private equity firm based in Europe. EQT is supporting Aldevron through investments in additional production capacity, R&D and growth initiatives and by leveraging its strong healthcare expertise, global presence and network of industrial advisors. EQT now owns a majority interest, with our founders, management and TA Associates retaining a minority stake.

What Lies Ahead for Plasmids?

Many of the therapies that rely on plasmid DNA are advancing rapidly through clinical development and will soon be commercialized. Newer technologies enabling non-viral delivery of gene therapies and vaccines, as well as personalized cell therapies, will further expand demand for plasmid DNA. In addition to scaling up to larger quantities, Aldevron is scaling out and investing in technologies to support personalized medicine, which requires a unique plasmid DNA product for each patient. As a result of these developments, plasmid manufacturing capacity will need to increase exponentially.

No one company will be able to support the entire market; multiple organizations are needed to supply the rapid growing field. In addition, breakthrough innovations in manufacturing technology will be necessary. Production processes and equipment — all originally developed for the manufacture of antibodies and proteins, which are much smaller than plasmids — need to be reengineered specifically for plasmid manufacturing to increase efficiency, productivity and scalability.

Aldevron intends to remain at the forefront, contributing to the development of novel systems and technologies designed specifically for plasmid DNA manufacturing.

Reference

  1. Brown, James. “Supporting AAV and Lentiviral Vector Development and Commercialization.” Pharma’s Almanac. 24 May 2019.

Originally published on PharmasAlmanac.com on December 6, 2019.

Translating Three Decades of Experience in Plasmid DNA Into Customer Success

With 30 years of experience manufacturing plasmid DNA (pDNA), Richter-Helm has gained an unparalleled amount of first-hand knowledge that they apply to each customer project, regardless of phase or scale. Richter-Helm’s Managing Director Kai Pohlmeyer, Ph.D., and Director of Business Development Thilo Kamphausen, Ph.D., discuss the company’s upcoming expansion in Bovenau, Germany, how pDNA manufacturing is differentiated from more conventional biomanufacturing, and how to ensure and test for the highest level of quality.

David Alvaro (DA): Can you give me a brief introduction to Richter-Helm BioLogics and the company’s history in the plasmid DNA space?

Thilo Kamphausen (TK): We are a versatile CDMO manufacturing a range of products using microbial fermentation — this not only includes plasmid DNA but also proteins and bacterial vaccines. Plasmid DNA consists of about 30–40% of our current business; we lead the industry in this technology, as we have almost 30 years of experience manufacturing it.

Kai Pohlmeyer (KP): Richter-Helm was one of the first players in pDNA. The company started in 1997 and has continued to gain experience and expertise in pDNA manufacturing. We currently use three to four different processes and a range of scales to accommodate all products. For instance, we conduct feasibility studies at a scale of 1 L or smaller, and we manufacture at 200–1,000 L. Typically, the 200-L scale yields or leads to yields of 5–20 grams, while we can reach yields of more than 100 grams of pDNA with the 1,000-L scale. Different processes yield different scales for different purposes.

DA: What is the range of applications for the plasmids that you manufacture?

KP: Although we produce plasmids for many different applications, we originally began manufacturing plasmid DNA (pDNA) exclusively for vaccines as APIs. However, this has significantly changed over the last six years. We now produce plasmid DNA as critical raw materials for further processing. These plasmids are used as starting materials for virus production, cell and gene therapy, mRNA production, and cell-free expression systems, which are even newer. We also produce a high number of plasmids for API applications in vaccines, and, while none of those applications are currently registered, we’ve produced many batches for phase III and process validation applications.

Looking forward over the near term, I believe the greatest growth in demand will stem from late clinical trials into commercial applications, with the most potential in cell and gene therapy applications.

DA: From the point of manufacturing, are all plasmids fundamentally the same regardless of their ultimate application?

TK: The manufacturing process itself is mostly identical for all plasmids. In most cases, downstream processing involves two-step chromatography, hydrophobic interaction, and then anionic exchange. Both directions and combinations are possible, as the ultimate result is a pure plasmid. The challenge is to produce and identify the highest quality with high-resolution analytics. In addition to being GMP compliant, we’re able to judge the quality of our products with analytical methods that reveal isoforms of up to 98% purity.

KP: We see a lot of players producing high-quality, non-GMP pDNA, and, from the regulatory perspective, it’s possible to use that quality for phase I or phase II trials. Richter-Helm focuses on GMP production of plasmids and intermediates for phase III and commercial materials. Players are increasingly starting phase I and II trials using GMP quality, as we’ve seen a shift from high-quality DNA to GMP-grade DNA.

DA: Is Richter-Helm more focused on custom plasmids rather than more standard, off-the-shelf helper plasmids?

TK: We are fully focused on custom (or “customer”) plasmids. Though we do not have off-the-shelf plasmids, we will manufacture any plasmids a customer requests, regardless of size. This is critical in manufacturing, and we have several processes in place for complex products larger than 10–12 kilo-basepairs (kbp). This flexibility enables us to determine the best option for each specific plasmid. We offer cloning services once only the plasmid is available; we can also contribute cell lines, in most cases DH5α or DH10B. Highly specific cell lines are necessary if you have plasmids that tend to modify.

DA: Is plasmid manufacturing fully standard or are there ongoing challenges, particularly with these larger plasmids that you’re continuing to develop new solutions for?

TK: Our processes are fully scalable from 10 L to 1,000 L, and we select or suggest the right size depending on the corresponding plasmid demand. To assess that, we generally recommend a small-scale feasibility study. While there are variations from project to project, plasmids are generally much more standardized compared with proteins.

KP: We use our generic processes and analytics about 80% of the time for pDNA manufacture, though there are certain challenges, like very long poly-A tails or CG-rich sequences. In these outstanding cases, we optimize the process.

DA: A recurring theme in plasmid DNA for cell and gene therapy has been capacity challenges. Can you help explain why this is?

TK: Building up a facility for microbial manufacturing is complicated and takes time; there is also a high level of commitment needed to qualify for GMP. Demand massively increased due to the pandemic, as most providers decided to expand, which we had strategically done before 2020. I also think the ongoing crunch is a reaction to growing demand — the industry is attempting to compensate for a lack of manufacturing capacity.

KP: Biologics manufacturing was primarily focused on CHO and other mammalian cell culture over the last 30 years, and this accounts for approximately 75% of the existing capacity as that field has grown exponentially. Microbial technologies have always represented the more niche side of the business. Suddenly, however, because of CAR-T therapies, cell and gene therapies, and mRNA, the plasmid DNA market has experienced significant growth. Of course, when building facilities to support this capacity, it takes at least two to four years to become operational, which has created challenges for many.

DA: Can you provide details on the E. coli strains that you work with? What considerations go into selecting the optimal strain for a given plasmid project?

KP: We are using several processes to produce pDNA, each of which has a preferred strain. We can use all the strains that the customer provides us with, though we start each project with a strain selection or strain development approach and compare the yield and the quality of each. Our experience with respect to quality and yield indicates that DH5α and the DH10B are often most optimal, though we do use many others. More complex systems, like the StblTM system, are used to produce more complex sequences. We always start by testing different strains to determine the best process for the existing expression system.

DA: Is development for downstream processing and analytics more straightforward for plasmids than proteins because every plasmid is fundamentally similar?

TK: The analytics themselves aren’t easier, but they can be applied to a broad range of plasmids once developed since the plasmids are very similar. Proteins have to be specifically tested and adapted in more detail. We have a generically validated set of analytical methods that can be easily applied with one test to most of the plasmids in use.

Ion exchange and hydrophobic interaction appear in all plasmid DNA processes, and, compared with proteins, there are three, sometimes four chromatography steps. Impurities are removed after two steps; this is a standardized downstream process that can be used for all plasmids.

KP: Our analytical testing is outstanding, as we have a panel of methods that are all qualified or validated and can be applied easily to every new project — this saves time and ensures that our plasmids meet the highest quality standards.

DA: Throughout the 30 years that you’ve been working in plasmids, have there been any transformative innovations that have completely changed your approach, or have improvements happened gradually and iteratively?

KP: There have been many gradual improvements. We learn from every project we take on at Richter-Helm, and we made some relevant changes, especially when we began intermediate manufacturing. Before that, we only manufactured APIs; our documentation was too oversized to produce or deliver intermediates or critical raw materials. We were able to reduce this to the necessary minimum, which was a big improvement. Over time, we developed our own manufacturing process, which took five or six years. We have reaped the benefits of this work, as we now have our own process available that we can offer to our customers.

TK: We’ve also made improvements in fermentation technology leading to higher plasmid DNA yields.

DA: Do you foresee any disruptive technologies on the horizon?

TK: I believe there will always be progress in development. In terms of disruptive technologies, we have an eye on the synthetic manufacturing of linear DNA, which could be sufficient for mRNA manufacturing. However, we doubt that it will truly compete with API plasmids.

DA: Among these obviously constant improvements, what are the latest developments at Richter-Helm?

TK: We are expanding and adding new capacity in two areas. We’ve identified that a 200-L fermenter is sometimes too large for certain plasmids. To address this, we are evaluating the installation of a 50-L fermenter to meet the needs of smaller quantity requirements. Even more importantly, we are constructing a new manufacturing facility in Bovenau, Germany. This will enable us to triplicate our capacity by adding two more fermenter lines; we already have the space and the supportive technologies needed to add a third line. This expansion is ongoing, and our goal is to be fully operational by the end of 2023.

DA: Over the longer term, is it relatively likely that you will continue to grow capacity to meet the ongoing demand?

KP: Our plan is to grow via our operations, not through acquisitions or mergers. We are in constant discussions with our customers to ensure we have sufficient capacity available to meet their demands for product launches, and we plan to continue to grow our capacity over the long term.

DA: Were supply chain bottlenecks that were caused by the pandemic felt acutely in pDNA manufacturing?

TK: The shutdown in China impacted all, as masks, cleanroom equipment, and disinfectants were in short supply. Luckily, we did not lose any batches because of this, and the energy to manufacture continuously was very high. Based on the delays in acquiring materials needed for a process, you could figure out what other businesses were using, which is why certain lead times increased in a very apparent way. We reacted by increasing our warehouse backup material and quite openly communicating that we needed longer planning times for the batches to ensure manufacturing could be done.

DA: Has that permanently changed how you approach the supply chain?

KP: To get ahead of a potential shortage, we increased our warehouses and storage capacity. Although this has had a financial impact, we now have a huge stock of materials.

DA: What do you view as the most critical differentiators for Richter-Helm in the pDNA market?

KP: We think there are three main selling points at Richter-Helm. The first is our rich history and experience spanning 30 years. Over that time, we have dealt with many processes, qualities, customers, and authorities, which has helped to build our unique knowledge base. Our second selling point is flexibility. Although we are growing, we operate like a small organization, which enables us to focus fully on our customers’ demands. Our third and perhaps most important selling point is the quality of our products. We provide customers with the highest GMP quality, which has been proven by many authorities and inspections. This triangle of quality, experience, and flexibility differentiates Richter-Helm from all other players. We are also as reliable and transparent as possible thanks to our leading, hands-on project management team.

DA: How important is it for Richter-Helm to be able to support customers on the regulatory side?

TK: Our two main markets are EMA and FDA regulated, as we can completely follow the European pharmacopeia and the USP. We have covered all critical markets with a full, successful GMP system, which is identical to all the GMP operations that we have, independent of the sites. This is an advantage, because we can quickly exchange and interchange operations as we all work following the same system.

DA: Is there a general profile of the kind of companies you’ve worked with in the past or a certain type of organization that you think is the best fit specifically with Richter-Helm rather than another company?

TK: We have tremendous experience with phase III process validation approaches. If a company has a project in transition from late-phase validation to market, that’s a key interval to reach out. Of course, we can run a phase I project up to commercial. While there aren’t many registered plasmid DNA products, we are proud to have already contributed to one of the first cell and gene therapies.

DA: Where would you like to see Richter-Helm positioned five or 10 years in the future?

KP: We will continue to follow the path we’re on. We’ve invested €85 million into our expansion project, and I anticipate nothing but continued success. The idea is to grow by adding customers and products and continue to expand into new facilities to meet ongoing customer demand.

Originally published on PharmasAlmanac.com on May 17, 2022.

Supporting AAV and Lentiviral Vector Development and Commercialization

Plasmids are essential for the development of viral vectors used to manufacture novel gene therapies and viral vaccines. Aldevron is supporting the innovation of drug developers in this space by providing standardized royalty-free, bulk AAV helper and lentiviral packaging plasmids for research and GMP production.

Importance of Plasmids

Plasmids are circular DNA molecules found mainly in bacteria, but also in yeast and plants, that replicate independently from the host’s chromosomal DNA and enable bacteria to transfer genetic information from one to another via the process of horizontal gene transfer. They may also serve other functions, such as imparting antibiotic resistance. 

Natural plasmids contain an origin of replication, which controls the host range and copy number of the plasmid and allows initiation of replication. They also generally have at least one other gene that facilitates bacterial survival. Engineered plasmids are designed to introduce foreign DNA into other cells — typically mammalian cells during biopharmaceutical manufacturing. This process is referred to as plasmid transfection.

Like natural plasmids, engineered plasmids (also referred to as vectors or constructs) with specifically inserted genes have an origin of replication and a selection marker (e.g., antibiotic resistance to allow for the selection of plasmid-containing bacteria) and cloning site(s). Expression plasmids also have promoter regions that determine which cell types the gene is expressed in and the amount of recombinant protein produced.

Once the desired vector has been created, it is transformed into bacterial cells that are then selectively grown on antibiotic plates. The type of plasmid — cloning, expression, gene knockdown, reporter, viral and genome engineering — dictates the end-use application, including the production of viral vectors for gene therapies and viral vaccines.

Q2_Aldevron_sidebar_1

Triple-Transfection Process

There are several methods for manufacturing viral vectors. One popular technique involves the use of an insect cell/baculovirus expression system. Another system, which relies on a set of engineered herpes simplex viruses (HSVs) and a mammalian cell line, also has advantages, but is patent-protected and must be licensed. A producer cell method involves integration of all necessary genes into the genome of the cell, which allows for very stable expression but is highly complex and is generally only selected when one needs to manufacture a large quantity of a specific viral vector.

The most common plasmid transfection approach used for adenovirus (AV) and adeno-associated virus (AAV) vector production is transfection in mammalian cell lines, typically HEK293 cells. Multiple different plasmids are required in this method. For AAV, the cis-plasmid contains the gene of interest flanked by inverted terminal repeats (ITRs), which allow the genome to infect cells and then express the gene of interest. The trans-plasmid (also known as the Rep-Cap plasmid) contains the Rep and Cap AAV genes, which are not sandwiched between the two ITRs. The third plasmid — called the helper plasmid — contains the E4, E2a and VA genes for AAV (or AV), with the cell itself providing the AV E1A gene. As a result, no co-infection with adenovirus is required.

The cis-plasmid is unique for each recombinant AAV vector to be produced, as it contains the specific transgene of interest. The trans-plasmid containing the Rep and Cap genes is specific to the serotype of AAV being used. Different serotypes elicit a distinct immune response and also can have different tissue tropisms. The helper plasmid, however, is always the same regardless of the transgene and serotype.

In some cases, the trans-plasmid and helper plasmid have been combined into one larger plasmid, allowing for a two-plasmid system. This approach has some advantages, most notably a higher transfection rate, since only two plasmids need to enter the cell rather than three. There are potential cost savings to this method as well, but the combined plasmid is very large and more complex to produce. This approach tends to be limited to projects that involve only certain serotypes.

Bulk AAV Helper Plasmid

As a leading contract manufacturer of plasmids, Aldevron has extensive experience producing the three types of plasmids at research to commercial scale. Because the helper plasmid is the same for all triple-transfection projects, it is produced on a regular basis. Part of Aldevron’s growth strategy is to advance the field by becoming a full-service company and adding value for clients through various product offerings. Production of bulk helper plasmid as a standard, off-the-shelf product for research, clinical trial material production and commercial manufacturing is an ideal offering for Aldevron.

Our first focus was on the AAV therapy space. Two types of helper plasmids are used in AAV vector production: AAV AdΔF6 and AAV pXX6-80. There are various forms of each available that have various optimizations to perform better during transformation and viral vector production. 

Aldevron collaborated with Asklepios Biopharmaceutical, Inc. (AskBio), a gene therapy company, to make its helper plasmid available as an off-the-shelf product. AskBio’s pXX6-80 plasmid has been used in recombinant AAV (rAAV) production since 19981 and has been safely used in viral vectors administered to humans. In addition, Aldevron has produced all of AskBio’s clinical GMP material and is familiar with the production of this helper plasmid. Furthermore, both companies are dedicated to advancing gene therapy with enabling technologies that get treatments to patients as quickly as possible.

Our pALD-X80 standardized helper plasmid relies on kanamycin resistance rather than ampicillin resistance, which is commonly found in older helper plasmids. Many people have allergic reactions to ampicillin, and histamine responses can occur even with ampicillin reduced to very low levels. As a result, regulatory agencies are demanding that viral vector manufacturers avoid ampicillin resistance.

Another crucial element of this product offering is the agreement that no royalty fees would be passed on to Aldevron’s customers; they only pay for the pALD-X80 product itself. Given the intellectual property minefield in the gene therapy space, this approach helps dramatically simplify the use of the off-the-shelf material. Aldevron also makes small quantities of pALD-X80 available for free to academic and pharmaceutical industry researchers investigating its use for viral vector production.

Standardized Lentiviral Packaging Plasmids 

In the production of lentiviral vectors, the number and types of plasmids used differ, but the overall manufacturing process is similar. The process is based on a less-rugged, enveloped HIV virus, and four plasmids are involved: a plasmid containing the gene(s) of interest and three packaging plasmids. In this case, however, there are no serotypes, so the same three packaging plasmids are used for every project.

Aldevron has obtained rights to manufacture the set of plasmids developed by Oxford Genetics through a recent collaboration. These plasmids (pALD-Rev, pALD-VSV-G, pALD-GagPol and pALD-LentiEGFP) have been optimized to produce high-titer, high-infectivity lentivirus. Optimization includes minimization of vector backbones, reduced homology with HIV/VSV and inter-cassette homology. In addition, lentiviral vectors produced with pALD Lenti packaging plasmids transfect cells at a higher rate than commercially available kits, with infectious titers in the range of 5 x 107/mL. 

Once a client provides Aldevron with the gene of interest, it is cloned into the lentivirus expression plasmid (pALD-LentiEGFP) for custom production, replacing the EGFP sequence. The three packaging plasmids are available off the shelf. Aldevron’s production scale and experience with custom manufacturing of lentiviral expression plasmids, combined with the in-stock packaging plasmids, provide clients with a complete set of products and services for their programs. 

As with the AAV helper plasmid, the pALD lentivirus products are available royalty-free, and limited quantities of research material are provided free of charge. The products are currently available for research applications, with material suitable for the production of clinical trial materials ready for purchase in the summer of 2019. 

As a leading contract manufacturer of plasmids, Aldevron has extensive experience producing the three types of plasmids at research to commercial scale. 

Many Benefits for Vector Manufacturers

AAV and LV manufacturing have traditionally required unique custom batches of helper and packaging plasmids, respectively. It can take months for the production of these custom clinical and commercial plasmids. In addition, manufacturing capacity is currently limited, and wait times before projects can be initiated can be three months or longer.

Aldevron’s off-the-shelf AAV and LV plasmids are, on the other hand, immediately and universally available with acceptable plasmid elements (i.e., kanamycin resistance). As a result, the timeline for production of AAV and LV vectors can be significantly reduced, accelerating project timelines and lowering cost. Cost is also reduced because these products do not carry any pass-through fees after purchase.

As importantly, because Aldevron provides a reliable supply of consistent, high-quality plasmids and has demonstrated performance producing lentiviral and AAV vectors used in clinical trials, we facilitate supply chain risk reduction for our biopharma partners that are developing novel, life-changing therapies.

Customers also benefit from access to three quality grades of standardized plasmids for use in research applications, the production of clinical trial materials and commercial GMP manufacturing. Clients have the option of choosing the quality grade that fits their needs and stage of development with the assurance that the royalty-free product will be available when it is needed.

Straightforward Manufacturing Process

The bulk AAV and LV vectors from Aldevron are produced in Escherichia coli bacteria. First, the bacteria are subjected to a process that makes them competent (able to survive transient poration to allow the plasmids to enter), so they will be more accepting of the plasmid DNA. The bacteria are then transformed with the plasmid DNA. Exposure to antibiotic kills the bacteria that do not contain the plasmid. 

The remaining bacteria are grown and then lysed to release the plasmids. This step is challenging at large scale with large volumes, and Aldevron’s process development team has invested significant time and effort to develop large-scale processes that are highly efficient. The lysate is subjected to multiple filtration, chromatographic and buffer-exchange modalities before final formulation in the appropriate buffer to the desired concentration.

This process can be performed at very small to large scales. Currently, Aldevron can grow bacteria to a volume of 300 liters in a single-use fermentor, which can contain as much as 100 g of DNA. Recently, Aldevron has expanded their fermentation capacity to 1,000 L and will produce its first 1,000-L batch in the fall of 2019.

Product characterization includes measurement of the supercoiling density and sequencing, among other analyses. The level of impurities (e.g., endotoxins, residual host-cell proteins, DNA and RNA, dimerized plasmids) is also monitored. The basic attributes are dictated by the fact that these plasmids are used as raw materials for the production of viral vectors and are not intended for therapeutic applications that involve direct injection into humans.

Aldevron offers a series of integrated science platforms for the provision of nucleic acid, protein and antibody services. 

Q2_Aldevron_sidebar_2

Multiple Grades Fit for Purpose

The off-the-shelf plasmids from Aldevron are offered in three grades: research, GMP-Source™ and GMP.

Research-grade material is manufactured in a large, separate, open laboratory, in which multiple projects are performed simultaneously. The goal is to produce material rapidly at low cost and with high quality to enable effective research programs. Although the quality is high, it is not sufficient for use in humans because there is a small probability of contamination, even if at levels lower than can be detected. In addition, non-qualified assays are performed in a QC lab for these research materials.

GMP material is produced in a new facility designed specifically for plasmid manufacturing — the largest plasmid manufacturing facility in the world. Multiple independent air-handling systems enable production of multiple lots simultaneously, with no two lots sharing the same HVAC. Extensive environmental monitoring and rigorous cleaning and changeover procedures help ensure the quality of the products. This material is produced in compliance with current Good Manufacturing Practices and is of sufficient quality to be injected directly into humans.

GMP-Source™ material is manufactured in the same facility as our GMP products in separate suites that also have their own air-handling systems. The same raw materials are used for both GMP and GMP-Source materials, and all of the same qualified assays are used for both types of products and performed in a dedicated lab. The same quality system, which supports corrective actions and trending, is also used for both GMP and GMP-Source products. GMP-Source suites are not ISO classified, there is no environmental monitoring and the GMP-Source quality assurance review is limited to critical procedures. This quality level provides plasmids appropriate for use in viral vector production at a reduced cost and with a faster timeline without sacrificing any safety or efficacy in the final viral vector product. By offering this interim quality grade, we are helping viral vector manufacturers implement phase-appropriate GMP-compliant solutions.

Q2_Aldevron_sidebar_3

Advancing the Field

At Aldevron, we are driven to make a meaningful difference. We want to make real contributions to the lives of others by providing the basis for breakthroughs that improve human health and promote positive change throughout the world.

As a CDMO, Aldevron offers a series of integrated science platforms for the provision of nucleic acid, protein and antibody services. By supplying our customers what they need, when they need it, with expert support at every stage of development and production, we help them open up their laboratories for ground breaking science and breakthrough discoveries.

We are constantly looking to innovate and advance our technologies, products and services through partnerships with those who share our goal of advancing science in our field. We attach great importance to the close, collaborative relationships that allow us and our partners to concentrate on our core efforts. This means giving organizations of all sizes access to affordable, high-quality products and services to advance their work.

We are also committed to bringing groups together to accelerate the process of drug and treatment discovery. In some cases, that requires changing traditional mindsets. With respect to our off-the-shelf plasmids for AAV and LV production, that means recognizing that some aspects of gene therapy and viral vaccine manufacturing do not need to be customized. Helper AAV and packaging LV plasmids do not require unique specifications that add complexity and cost. 

The off-the-shelf products Aldevron produces have specifications that are appropriate for viral vector manufacturing and are consistent with those applied to the production of viral vectors that have been administered to patients. Gene therapy development is highly complex, but identifying components that can be safely standardized allows innovation efforts to target other aspects that cannot be standardized. As the gene therapy field continues to grow and mature, we fully expect more players in the industry to realize — and leverage — the benefits offered by our off-the-shelf plasmids. 

References

  1. Xiao, Xiao, Juan Li and Richard Jude Samulski. “Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus.” Journal of Virology. 72: 2224–2232 (1998).

Originally published on PharmasAlmanac.com on May 24, 2019.

Transforming the Economics of AAV Manufacture While Optimizing Quality with dbDNA

Streamlining the production of viral vectors, especially on a large scale, entails significant technical challenges, such as suboptimal titers and packaging efficiencies, alongside high costs. These concerns are particularly significant as the industry strives to establish the commercial viability of gene therapies. Touchlight’s enzymatically amplified dbDNA™ (doggybone DNA) overcomes these challenges by reducing the amount of DNA and transfection agent needed, resulting in higher yields of functional adeno-associated viral (AAV) vectors with fewer non-functional particles. This efficiency not only cuts production costs, it makes therapies more accessible. Ready-to-use research-grade dbDNA for AAV vector production expedites initial evaluations, with both research- and GMP-grade dbDNA available on accelerated timelines. Through multiple mechanisms, Touchlight’s innovation reduces the cost of goods sold (COGS) for AAV-based therapeutics and paves the way for rapid development from preclinical stages to commercial scale.

Technical and Economic Limitations of Plasmid DNA at Scale for AAV

The production of AAV vectors for use in gene therapies requires the introduction of DNA sequences encoding viral components and the therapeutic gene of interest (GOI) into producer cells — typically mammalian HEK293 cells or their derivatives. Currently, the dominant technique for creating viral vectors is the transient transfection of plasmid DNA (pDNA), using a transfection reagent to facilitate entry into the cells. However, this approach faces significant pragmatic hurdles, especially as production scales up. One major challenge is that the bacterial origins of pDNA, including the antibiotic-resistance genes and endotoxins intrinsic to Escherichia coli–based bacterial fermentation, present potential safety complications if administered to patients (the U.S. Food and Drug Administration “recommend that you not use antibiotics for bacterial selection” and “prevent the introduction of bacterial endotoxins into drug and biological products and their components”). Removing these impurities requires rigorous and complex purification processes. Additionally, the manufacturing process for the production of pDNA itself is fraught with inefficiencies and high costs. The lengthy timeframes required to produce GMP-compliant pDNA, which can stretch into several months, further complicate the scenario, impeding rapid development and approval of new therapeutics.

As the gene therapy market evolves beyond a focus on rare diseases to target conditions affecting larger patient populations, the COGS becomes an increasingly critical factor. The existing pDNA-based production methods, with their complex purification needs and long lead times, result in pDNA representing the largest share (15–50%) of the raw material costs per GMP batch of AAV, which is not economically viable at the scale required for widespread treatments. This shift in market demand necessitates a new production paradigm that can reliably supply large quantities of therapeutic vectors without a corresponding increase in costs.

Owing to these challenges, interest has grown in the establishment of alternatives to pDNA for viral vector production. Various DNA constructs and processes have been developed over the years, and while each has its own advantages and limitations, all potentially present benefits compared with pDNA produced via fermentation.

In particular, enzymatic amplification of DNA not only simplifies the DNA production process and reduces manufacturing footprints but also yields high-quality DNA that is free from bacterial sequences and antibiotic-resistance genes — attributes that significantly enhance the safety profile of therapeutic products.

dbDNA from Touchlight

Touchlight’s dbDNA is linear, closed double-stranded DNA (dsDNA) produced enzymatically via rolling circle amplification from a very small quantity of plasmid template. dbDNA contains no bacterial sequences and, since it is not derived from bacterial cells, contains very low endotoxin, mitigating common contamination concerns. By utilizing a sophisticated combination of amplification and processing enzymes, dbDNA is rapidly amplified with high fidelity and a remarkably smaller benchtop production footprint compared with pDNA.

The versatility of dbDNA lies in its ability to carry small, large, and complex genes, with a capacity for sequences over 30 kilobases in length. This makes dbDNA a valuable tool for a considerable array of therapeutic applications. Its ability to carry sequences that far exceed the size limits of AAV and lentiviral vectors — approximately 4.7 kb and 8–10 kb, respectively — positions dbDNA as a universal tool for the production of viral vectors employed in cell and gene therapies. Beyond these, dbDNA may also be pivotal for advancing other genetic medicines, including mRNA therapies, gene-editing techniques, non-viral gene therapies, and DNA vaccines.

The production of dbDNA begins with a circular template, which undergoes rolling circular amplification initiated by the ɸ29 DNA polymerase. Protelomerase then catalyzes covalent closures that convert the amplified DNA into a linear form, and other enzymes mediate the degradation of unneeded backbone sequences. Enzymatic DNA production removes the need for clearance of host cell proteins, genomic DNA, and all the endotoxins normally associated with a plasmid biological replication system, as those impurities are not there to begin with. The purification process needed for dbDNA is simpler and more straightforward, as it only needs to remove excess enzymes and low-molecular-weight DNA particles using chromatography and stringent filtration. dbDNA then undergoes a final fill/finish step, preparing it for use in various applications.

This scalable platform (currently up to 5-g batch size with further scale-up in the pipeline) can produce significant quantities of dbDNA at a fraction of the volume required for traditional fermentation, exemplified by the equivalence of material produced in less than 5-liter (L) scale to that of 200-L to 400-L fermentation batches. Such efficiency not only circumvents the waste generation and contamination risks of bacterial systems (e.g., excess bacteria, bacterial DNA, proteins, and endotoxin) but also maintains excellent fidelity. This high fidelity is particularly evident in complex sequences prone to errors in other systems, such as inverted terminal repeats (ITRs), which are essential elements for viral vector production.

Having manufactured over 1,400 client constructs as of Q1 2024, Touchlight has positioned dbDNA for applications across diverse modalities ranging in size and complexity. The company offers a spectrum of dbDNA grades — Catalogue, Discovery, Research, Smart-GMP, and GMP — tailored for varying stages of development, from early research to clinical and commercial manufacturing, demonstrating the versatility and applicability of this technology.

Operational Efficiencies and Improved Safety in AAV Production

The utilization of dbDNA in the manufacturing of AAV vectors achieves enhanced titer levels and improved ratios of full to empty capsids, especially after protocol optimisation.

The efficiency of dbDNA in transfection stems from its linear, minimal configuration. A lesser quantity of dbDNA is required — up to 40% less on average — for effective transcription, resulting in heightened cellular uptake and productivity and circumventing the overproduction of viral particles.

Beyond these operational efficiencies, the absence of bacterial sequences and antibiotic-resistance markers in dbDNA improves the resulting safety profile. Such safety considerations, coupled with the streamlined scalability of dbDNA production and the swift turnaround time for the generation of GMP-grade material, enhance the overall attractiveness of dbDNA. The capability to produce dbDNAs cost-effectively for various AAV serotypes — including AAV2, AAV9, AAV6, and AAV8 — has been verified, with data on AAV5 coming in 2024.

Better Fidelity for Complex Viral Sequences

Touchlight’s technology employs the high-fidelity proofreading DNA polymerase ɸ29 in rolling circle amplification, resulting in production of DNA constructs with a substitution error rate in the range of 1 in 10–6 to 10–5 and insertion/deletion mutations at a rate of less than 1 in 10–9. The error rate in a dbDNA molecule is indistinguishable from that of the plasmid template from which it is derived. This high fidelity is particularly advantageous for the production of dbDNA for AAV, where the constructs containing the GOIs also incorporate complex sequence structures, such as ITRs, which are prone to substantial errors in pDNA production, including multibase truncations and rearrangements. Such sequences remain stable during the dbDNA enzymatic process, avoiding the unintended alterations that can occur during bacterial fermentation.

Next-generation sequencing methods (including Illumina, Nanopore, and others) are routinely applied to characterize the performance of the dbDNA process, and these data have been provided to regulatory agencies as part of IND/IMPD dossiers.

Figure 1 presents NGS data for pDNA and dbDNA encoding two wildtype or untruncated ITR constructs and two truncated ITR constructs. The left bar of each graph displays the percentage of reads of the pDNA from which the dbDNA was made that matched the original sequence, while the right bar contains the reads from those dbDNAs. Error rates were low and nearly identical for both the untruncated and purposely truncated ITRs. These data reinforce the reliability of dbDNA for reproducing intricate genetic sequences from pDNA with high fidelity, ensuring that the ITRs and therapeutic gene sequences will remain intact and function as intended.

NGS sequencing data — amplification of untruncated and truncated ITRs for dbDNA.Figure 1. NGS sequencing data — amplification of untruncated and truncated ITRs for dbDNA.

Increased Full/Empty Capsid Ratios with dbDNA

The transient transfection process for viral vectors usually employs three plasmids: one carrying the GOI, another serving as the adenoviral helper plasmid, and a third containing the Rep and Cap genes. dbDNA can be utilized in each of these roles effectively.

Figure 2 shows the titers obtained for an AAV9 vector produced using dbDNA and pDNA by two different customers in suspension-adapted 293 cells. Viral particles (VP) per mL and viral genomes (VG) per mL titers were determined using enzyme-linked immunosorbent assay (ELISA) and enhanced green fluorescent protein (eGFP)-targeted dPCR.

Improved percentage of full capsids using dbDNA.Figure 2: Improved percentage of full capsids using dbDNA.

Using the initial optimization conditions in Table 1, customers compared AAV9 expressing eGFP dbDNA, matching pDNA versus their own plasmid and using their optimized protocol for plasmid. The viral genomes (VG) per mL titers were determined using digital polymerase chain reaction (dPCR). Percent (%) full was determined by VG per mL divided by viral particles (VP) per mL (VP titers were determined using AAV9-specific ELISA, not shown). Suspension-adapted 293 cells were used. AAVs were harvested at 72 hours and clarified.

Recommended starting parameters for dbDNA AAV production.Table 1. Recommended starting parameters for dbDNA AAV production.

The advancement of affordable AAV vector–based therapies is significantly hindered by the challenge of achieving high titers of fully encapsidated vectors. Regulators mandate minimizing the percentage of empty or partial capsids due to their negative impact on therapeutic efficacy and safety. Hence, improving the production of full capsids is vital not only for enhancing overall yields but also for simplifying the purification process.

When dbDNA is employed in the place of pDNA using conventional protocols, the resulting AAV titers are similar; however, the total viral particle count is significantly lower. Touchlight’s bespoke dbDNA starting ratio not only matches these titers but also substantially decreases the proportion of empty viral particles.

Comparable AAV Yields with dbDNA Using Unoptimized Protocols

Transitioning from pDNA to dbDNA can be simple and benefits from optimization, as would be expected. One customer wanted to jump in quickly and decided an important knowledge point was how the production of AAV9 varied with different transfection reagents, as their clients may have specific transfection reagent requirements. Figure 3 displays this very early testing of dbDNA versus sequence-matched pDNA versus the customer’s own pDNA system and standard operating protocols. Using Transporter™ 5, PEIpro™ and FectoVIR™, the results show that dbDNA produced similar or increased VG titers.

AAV9 yields are comparable between dbDNA and pDNA.Figure 3. AAV9 yields are comparable between dbDNA and pDNA.

VP per mL and VG per mL titers were determined using AAV9-specific ELISA and dPCR. % full was determined by VG/VP per mL. Suspension-adapted 293 cells were used with either Transporter™ 5, PEIpro™, or FectoVIR™ as the transfection reagents. AAVs were harvested at 72 hours and clarified.

dbDNA consistently produced a lower VP/mL titer while maintaining a similar VG/mL titer to the matching pDNA using the standard protocol after minimal optimization of the dbDNA process. In addition, the customer’s own control pDNA system yielded only somewhat better VG/mL but drastically more VP/mL, resulting in much lower percentage (%) full outcome. Notably, these dbDNA results were achieved by using 40% less DNA. Such efficiency in early-phase production underscores dbDNA’s potential to streamline AAV vector manufacturing and reduce COGS.

An example of why optimization is important is that utilizing 0.6 µg of dbDNA in contrast to 0.8 µg results in equivalent viral genome titers, yet the former generates roughly half as many empty particles, thereby increasing the full-to-empty capsid ratio. This enhancement in packaging efficiency translates to increased yields, reduced waste, and a streamlined downstream purification process.

Figure 4 illustrates the potential of dbDNA to improve AAV9 vector packaging efficiency. Here, AAV9 viral titers were measured by mass photometry. The same AAVs produced in the experiments for Figure 3 were run through an anion-exchange column purification and used to generate the mass photometry results.

Packaging efficiency is improved with dbDNA, as confirmed by mass photometry.Figure 4. Packaging efficiency is improved with dbDNA, as confirmed by mass photometry.

These initial results display a greater than 100% increase in the proportion of full AAV capsids without any deliberate efforts to optimize encapsidation, hinting at the possibility of further refining dbDNA packaging efficiency, an area that continues to be explored with the intent of maximizing the therapeutic potential of AAV vectors.

These results represent only a starting point. Touchlight customers who are initially transitioning from pDNA to dbDNA have benefited from adopting Touchlight’s foundational protocol and then optimizing the process for the unique product. Design-of-experiment (DoE) studies with the support of Touchlight’s experienced Application Specialist team are crucial to fine-tune the process for each unique application, maximizing both yield and quality of the final AAV product.

Cost-Effectiveness of dbDNA for AAV Manufacturing

The reduction of the COGS for AAV manufacturing enabled by dbDNA marks a significant step forward in gene therapy, facilitating the development of treatments for widespread diseases and broadening patient access. Addressing the economic challenges in viral vector production — specifically DNA, transfection reagent and process duration — is pivotal. The switch from pDNA to dbDNA offers a straightforward strategy to improve the economics of transient transfection.

On the upstream side, dbDNA significantly reduces the DNA requirement by over 40% and transfection reagent needs by as much as 50%. For example, in a 200-L batch, where traditional AAV production might consume around 410 mg of pDNA and 820 mL of PEI, dbDNA operations could be conducted with under 245 mg of dbDNA and just 410 mL of PEI — allowing the production of nearly three dbDNA batches with the same resources used for two pDNA batches.

Figure 5 illustrates these substantial savings when dbDNA is applied to AAV production. Moreover, downstream efficiencies are realized through on average twofold or greater increase in packaging efficiency, which simplifies operations and reduces loss during purification. The elimination of bacterial contamination assays further cuts both costs and time.

Real savings by using dbDNA for AAV production.Figure 5. Real savings by using dbDNA for AAV production.

Producing more viral vector per batch translates into more potential doses from each batch. Coupled with the enhanced percentage of full capsids in the dbDNA-produced AAV product, this may reduce the dosages needed to be administered to achieve the therapeutic effect, multiplying the number of doses possible from a single batch and further decreasing the cost per dose. This dual impact of quantity and quality in dbDNA AAV production ushers in a new era of cost-efficiency in gene therapy manufacturing.

Regulatory Status of dbDNA for AAV Manufacturing

The regulatory landscape for dbDNA in AAV vector manufacturing is favorable, thanks in part to its enzyme-based production process, which offers a superior safety profile. The lack of bacterial origins means that standard assays for antibiotic resistance genes, endotoxins, and mycoplasma, typically mandated by regulatory bodies, are technically unnecessary for dbDNA. In the context of heightened regulatory scrutiny regarding antibiotic resistance and guidance aimed at mitigating the risk of transferring antibiotic-resistance markers to pathogenic bacteria, the ability to avoid the use of the resistance genes positions dbDNA favorably in the AAV-mediated gene therapy market going forward, and a shift in regulatory standards is anticipated as the understanding of dbDNA’s distinct production process becomes more widespread.

Touchlight’s dbDNA has garnered regulatory recognition, securing a Drug Master File (DMF) with the FDA in 2022. Furthermore, it has achieved the Innovative Licensing and Access Pathway (ILAP) designation from the UK’s Medicines and Healthcare products Regulatory Agency (MHRA), signaling its potential to streamline the regulatory process.

The use of dbDNA is being actively adopted in the clinic, with a phase II clinical trial in France currently investigating an AAV-based therapy produced with dbDNA. Beyond the AAV sector, dbDNA has received FDA approval for Investigational New Drug (IND) applications for its use in mRNA-based cell therapies and therapeutic vaccines, underscoring its versatility and acceptance as a key component in the manufacturing of advanced therapies.

Enabling the Switch from pDNA to dbDNA for AAV Manufacturing

Transitioning from pDNA to dbDNA for AAV production represents a paradigm shift, leveraging dbDNA’s linear and minimal design that includes only the necessary genetic elements. For the triple transient transfection essential in AAV vector production, dbDNA versions of the GOI, Rep-Cap, and helper sequences are up to 40% smaller than their pDNA counterparts, owing to the absence of bacterial genetic elements.

Historical transient transfection methods tailored around pDNA yield variable results when dbDNA is introduced without adjustments. Recognizing this, Touchlight has conducted extensive research to establish a robust starting protocol for dbDNA-based AAV transfection. This protocol considers key changes, such as a reduction in the total quantity of DNA required and adjustment of the DNA to transfection reagent ratio, emphasizing molar rather than mass ratios for precision.

Touchlight has streamlined access to dbDNA, whether by synthesizing from a Genbank sequence or using an existing customer plasmid containing the GOI. Both are simple and can be quickly made into dbDNA for the chosen modality.

Touchlight also offers off-the-shelf Catalogue dbDNAs specifically developed for AAV production and available for initial evaluation and testing, including Rep-Caps 2 and 9, an AdHelper, and a luciferase and GFP combined reporter transgene. These products are shipped next day. They can be purchased individually or in a kit that also contains the plasmids from which each dbDNA was derived, which allows for direct comparison studies.

Technical support from Touchlight is robust, with direct access to Application specialists with extensive experience in viral vector scale-up. We are poised to assist in designing and refining processes through DoE strategies.

Finally, Touchlight’s manufacturing capabilities for dbDNA span from laboratory to commercial scales, with a notable reduction in lead times compared with pDNA. Discovery- (1 mg) or R&D-grade material (5 mg to 2.5 g) can be delivered in 4–6 weeks, and SmartGMP or GMP material within 5–6 weeks of receiving the GOI sequence — significantly faster than traditional pDNA timelines (on the order of months), thereby accelerating the pace from concept to clinic.

Investing in Additional Capacity and Further Innovation for AAV Applications

Touchlight has taken steps to ensure supply chain robustness, establishing strategic partnerships with GMP suppliers, dual supply of critical raw materials, and immediate access to off-the-shelf consumables, eliminating lead times for customization. The infrastructure is further bolstered by dual production facilities equipped with independent backup generators, ensuring resilience and the option for third-party dbDNA manufacturing to meet escalating demands.

Pioneering achievements in synthetic DNA technology have cemented Touchlight’s leadership, with an impressive portfolio of 100 granted patents, groundbreaking GMP-grade material production, substantial scaling capabilities, FDA DMF accreditation, and tangible human study advancements — yet these are just the early milestones.

In 2023, Touchlight tripled capacity for dbDNA at its production facility to 8 kg per year. In 2024, a grant-funded lab focused solely on optimizing dbDNA AAV vector production opened. In addition to exploring in detail the use of dbDNA in transient transfection reactions, the lab will reinvestigate the architectures of the Rep-Cap, helper, and GOI constructs with the goal of determining the optimum designs that will maximize AAV production. The expectation is that dbDNA can provide even greater productivity and cost benefits for advanced therapies than have already been demonstrated, and Touchlight is committed to realising those gains for therapy developers and their patients.

Transforming AAV Economics

Touchlight’s enzymatically synthesized dbDNA presents transformative potential to improve process economics and quality of AAV vectors for use in gene therapies. By replacing pDNA manufacturing with a simplified DNA production process and eliminating bacterial sequences and antibiotic-resistance genes, dbDNA improves the safety profile of therapeutic products. With lower DNA and transfection agent requirements, dbDNA facilitates higher yields of functional AAV vectors, resulting in cost reductions and accelerating the transition from preclinical stages to commercial scale. In particular, dbDNA production demonstrates superior fidelity in replicating intricate genetic structures like ITRs, which are crucial for the integrity of gene therapies.

Additionally, the use of dbDNA has been shown to significantly enhance the ratio of full to empty AAV capsids, increasing the efficiency of AAV vector production. This increased packaging efficiency is not only promising for the quality of AAV vectors but also for streamlining the purification processes, further contributing to cost savings. The economic benefits of employing dbDNA are substantial, with the potential to markedly decrease the COGS in AAV manufacturing. These benefits are matched by a favorable regulatory outlook for dbDNA, as evidenced by its recognition by agencies like the FDA and MHRA, which may lead to a shift in regulatory standards and further adoption of this technology.

Touchlight’s dedication to innovation in AAV applications is evident in their strategic investments and capacity expansion, as well as their ongoing research aimed at refining AAV vector production. Their approach not only paves the way for more affordable and accessible gene therapies but also underscores the company’s commitment to supporting therapy developers in bringing advanced treatments to patients more efficiently.

Originally published on PharmasAlmanac.com on July 2, 2024