Top Industrialization Challenges of Gene Therapy Manufacturing
Guest Authors:
Dr. Mark Szczypka
Global Director, Process Development Services
Pall Corporation
and
Clive Glover
Director, Cell & Gene Therapy
Pall Corporation
UPDATED 06/15/2026
This article now has been updated to answer some of the previous challenges highlighted by the Pall Corporation as issues in biomanufacturing processes of gene based therapies. This article has the following structure:
- An introduction to the current state of affairs of DNA production at scale for gene therapy with the introduction of
- non-cellular production means
- enzymatic DNA production at scale of therapeutic gene therapies
- the original article on what are gene therapies
- original article on manufacturing issues that were predominant at time of publication of original article
As genetic medicine continues to advance, the constraints of traditional plasmid DNA (pDNA) manufacturing have become increasingly difficult to ignore. Conventional bacterial fermentation—the industry’s standard for pDNA production—is inherently slow, labor‑intensive, and complex. It depends on cell banking, extended culture periods, and multi‑step purification workflows that frequently co‑purify unwanted bacterial elements, including genomic DNA, endotoxins, and antibiotic‑resistance markers.
The COVID‑19 pandemic made these limitations impossible to overlook. The unprecedented global demand for mRNA vaccines exposed the fragility of pDNA supply chains and highlighted the need for faster, cleaner, and more scalable DNA manufacturing technologies. As next‑generation genetic therapies move into clinical development, the pressure to improve pDNA production—without compromising quality, safety, or regulatory compliance—continues to intensify. The core challenge is not merely expanding capacity, but doing so while reducing cost and ensuring consistent product integrity.
dbDNA® (doggybone® DNA) offers a transformative alternative through fully enzymatic, cell‑free DNA synthesis. Unlike plasmid DNA, dbDNA is generated via rolling circle amplification (RCA) from a circular template, producing a linear, double‑stranded, covalently closed construct. This enzymatic workflow uses a defined set of amplification and processing enzymes and eliminates ligation‑based purification steps. The result is a streamlined, scalable process with smaller reaction volumes, reduced impurities, and minimal risk of generating non‑target DNA species.
What is doggybone DNA (dbDNA)?
Unlike pDNA, which can carry bacterial sequences and antibiotic resistance genes, cell-free DNA does not contain unwanted genetic elements. An enzymatic production process avoids fermentation and thermal cycling but maintains complex sequence integrity.
The structure of doggybone DNA (dbDNA). dbDNA is a linear, double-stranded DNA with covalently closed ends. Unlike pDNA, which can carry bacterial sequences and antibiotic resistance genes, cell-free DNA does not contain unwanted genetic elements. An enzymatic production process avoids fermentation and thermal cycling but maintains complex sequence integrity.
dbDNA is linear, covalently closed-ended dsDNA that is generated in a cell-free
environment, meaning no bioincubators such as bacterial cultures are needed. The process begins with rolling circle amplification (RCA), which
creates long concatemers of double-stranded DNA. A protelomerase enzyme then
cleaves and covalently seals the ends of the DNA into linear constructs, while
restriction enzymes and exonucleases remove the unwanted backbone sequences.
The final dbDNA product can then be purified by chromatography and filtration,
resulting in a highly pure DNA molecule free from bacterial sequences, antibiotic
resistance genes and endotoxins.
A Converstion with TouchLight’s Chief Scientific Officer and how dbDNA is making a difference in gene therapy manufacturing
Source: https://touchlight.com/
Cell and gene therapy has long relied on plasmid DNA (pDNA) to power clinical innovation. But cell-free DNA is already in use, already treating patients, and rapidly becoming established as a scalable, consistent, and cost-efficient alternative. As manufacturing demands grow, developers are reassessing which DNA format best supports speed, reliability, and future-proofing.
In this Q&A, Touchlight’s Jill Makin, Ph.D., Chief Scientific Officer, discusses the advantages of cell-free DNA, and the considerations developers should make when selecting a platform.
What is Touchlight’s approach to DNA production?
It’s a completely different, cell-free, approach. We use recombinant enzymes to amplify DNA at scales ranging from milligram to gram quantities, for both research-use-only and GMP applications. One of the key advantages to this approach is that we use the same materials and the same reaction components from small to large scale, which gives predictable scalability and yields, reliable comparability across scales, and consistent assurance of compliance. Enzymatic processes are also extremely rapid. And when it comes to cost, pDNA production is driven by CapEx and labour, whereas the cost associated with cell-free DNA is primarily driven by materials. The advantage of the difference in cost levers is that, with construct yield predictability on scale up, and with the volume discounts that apply to material supply on scale up, large scale production of cell-free DNA can be simple to predict and highly cost effective.
How does Touchlight’s approach address the E. coli challenge?
With E. coli, we constantly battle evolutionary mechanisms. Engineering improved strains helps, but another way to avoid the issue entirely is not to use cells. By producing DNA outside of cells, with recombinant enzymes, we eliminate the need for bacterial antibiotic resistance genes and bacterial origins of replication, which are essential for cultivation in E. coli. We also avoid the difficulty of maintaining genetically unstable, or toxic sequences, during cell expansion, resulting in the emergence of large deletion mutations in target sequences, which is a common problem facing advanced therapy development. The industry, including plasmid manufacturing, is clearly moving toward solutions that avoid these inherent biological risks.
How has the industry’s perception of enzymatic DNA changed since you first entered the CDMO market?
At Touchlight’s emergence into the CDMO market, six or seven years ago, our biggest challenge was incumbency. Most clients came to us because they had a specific problem with their plasmid, usually genetic instability that caused scale-up issues or quality drift. Sometimes it was simply CDMO lead times. A notable early example: a cell therapy program needed mRNA and had secured an mRNA manufacturing slot, but was let down by plasmid supply. We stepped in and supplied the DNA template because the plasmid manufacturer couldn’t meet the timelines owing to a technical challenge. In recent years, we have seen a shift. One client undertook an extensive period of evaluation (against plasmid) and optimisation before selecting cell free DNA for their entire technology platform. Today, the dynamic has shifted. We are increasingly the first choice, especially for viral vectors, where predictable scalability and commercial cost-of-goods (COG) modelling favour enzymatic DNA. Developers also want to futureproof against safety or regulatory risks associated with plasmids. Gene editing is different—there’s no incumbency. The field has evolved as our technology matured, and, outside of cells, we can make DNA architecture and structures that are extremely difficult or impossible to produce in E. coli, but which may be useful in this setting.
Why are developers increasingly moving away from plasmid-based approaches and choosing enzymatic DNA instead?
Cost is often a significant driver. Recent cost-modelling exercises show clear advantages for DoggyboneTM DNA (dbDNATil over plasmids, because less DNA is required and less transfection reagent is needed. Additionally, you get more copies per gram with enzymatic DNA where plasmids include sequences you don’t need. Developers are also futureproofing against safety concerns. A recent Nature Medicine publication showed detection of pDNA backbone sequences from manufacturing constructs in a patient receiving AAV gene therapy. The combination of high patient doses and modern ultra-sensitive sequencing means these findings will only become more common. In fact, regulators have long scrutinized plasmid backbones, urging developers to try to avoid residual plasmid backbone sequences in therapies. As enzymatic DNA technologies mature and scale, the regulatory argument for plasmids weakens.
dbDNA is linear, covalently closed-ended dsDNA that is generated in a cell-free
environment. The process begins with rolling circle amplification (RCA), which
creates long concatemers of double-stranded DNA. A protelomerase enzyme then
cleaves and covalently seals the ends of the DNA into linear constructs, while
restriction enzymes and exonucleases remove the unwanted backbone sequences.
The final dbDNA product can then be purified by chromatography and filtration,
resulting in a highly pure DNA molecule free from bacterial sequences, antibiotic
resistance genes and endotoxins. Source: https:\\www.neb.com
Advantages of dbDNA Production over Traditional Plasmid Production
dbDNA enables multi-gram DNA manufacture in weeks, rather than months.
CRISPR‑based gene editing systems that rely on homology‑directed repair (HDR) remain foundational to ex vivo cell therapy. However, emerging technologies—including recombination, transposition, and homology‑independent targeted integration (HITI)—are expanding the landscape of non‑HDR gene insertion. These approaches often require payloads with highly specific structural and functional attributes. Touchlight meets this need with a portfolio of customizable circular DNA designs featuring user‑defined double‑stranded and single‑stranded regions, engineered to maximize compatibility across diverse gene editing modalities.
Beyond CRISPR: Enabling the Next Wave of Gene Insertion Technologies While tools such as base editing and prime editing have revolutionized the correction of point mutations and small genetic lesions, the next major challenge is the efficient integration of larger DNA sequences. This capability is essential for therapeutic strategies involving full gene replacement, addition of heterologous genes, or installation of complex synthetic circuits. Although advances in synthetic biology and the growing array of gene insertion platforms provide powerful solutions, one factor consistently determines success: the architecture of the DNA payload. The structure, composition, and stability of the template directly shape integration efficiency, cytotoxicity, and ultimately the therapeutic performance.
from: https://touchlight.com/ Touchlight offers a suite of novel circular DNA architectures developed to enhance gene therapy technologies. These include mbDNA™, single-stranded circles (sscDNA), hybrid single-stranded circles (hsscDNA), and double-stranded circles (dscDNA). Each format is designed with user-defined sequences and structural flexibility to support a range of genetic engineering applications.
Our platform offerings are compatible with various editing technologies, including homology-directed repair (HDR), homology-independent targeted integration (HITI), recombination, and transposition. These technologies are of interest because of their ability to overcome CRISPR HDR’s reliance on dividing cells.
Original Article
What Is Gene Therapy? How Does It Save and Improve the Quality of Life?
What Is Gene Therapy?
Gene therapy is a new and exciting technique, defined as the use of genetic material to cure or alleviate disease. It is considered revolutionary, yet still in its infancy, with many new therapies currently undergoing clinical trials.
Gene therapy has the potential to transform the treatment for diseases, significantly changing how doctors manage and treat patients.
Two Types of Gene Therapy
There are two main types of gene therapy.
The first corrects a specific disease causing genetic mutation. These are targeted towards inherited genetic disorders such as hemophilia or Duchenne muscular dystrophy. The second gives new functions to cells allowing them to fight disease.
A good example of these therapies are chimeric antigen receptor T cell (CAR-T) therapies. Both Novartis’ Kymriah♦ and Gilead’s Yescarta♦ are examples of CAR-T therapies, that have demonstrated exceptional cancer remission rates where other forms of treatment have failed.
Cancer is the by far the largest category of disease with 65% of gene therapy clinical trials being investigated, followed by 11.1% for inherited monogenetic disease, 7% for infectious disease, and 6.9% for cardiovascular disease1.
How Does Genetic Material Get Delivered to Host Cell(s)?
Genetic material gets delivered to a host cell via a delivery system known as a vector. Vectors deliver genetic material via one of the two methods. By directly injecting genetic material into the patient (in vivo), and where selected cells collected from the patient, undergo modification outside (ex vivo) before introducing them back into the patient.
The most commonly used type of vector is a virus. While there are other methods of delivering genetic material into a cell, viruses have now been developed that demonstrate a good balance between efficacy and safety.
Commercially Successful Gene Therapies
Developing a commercially successful gene therapy is challenging. It requires balancing several different considerations. Having a clinical effective therapy is essential, but this alone is not sufficient to ensure product success. In addition to this, reimbursement, quality and regulatory considerations, and manufacturing also must be considered.
To date, a total 11 gene therapies have received marketing approval. However, behind this there is a strong clinical pipeline with >1000 clinical trials underway, and 92 drugs in Phase 32.
Furthermore, there has been significant investment with >$50B being invested in the area in the past 3 years3.
This investment, coupled with the accelerating understanding of disease at the genetic level, holds immense potential. Academic, commercial manufacturers, and industry suppliers are actively seeking new approaches that deliver these therapies as quick as possible to a waiting population.
Author Details:
Clive Glover
Director, Cell & Gene Therapy
Pall Corporation
Top Industrialization Challenges of Gene Therapy Manufacturing
Manufacturing and scale-up of industrialized processes to manufacture gene therapy products are accompanied by many challenges that must be overcome to succeed in the marketplace. Commercialization of gene therapies for patient use is time consuming and requires substantial financial investment and dedicated resources.
Despite the unique range of challenges associated with gene therapy development, the quest to bring these therapies to market is worthwhile because the therapeutic potential of the treatments is revolutionary and the commercial opportunity is considerable. The process to industrialization is complex, but the benefits of successful development of robust processes are huge. The industry is rapidly expanding and is implementing novel approaches to overcome existing challenges, using innovative methods for medicinal application and developing new drugs to treat rare diseases.
Manufacturing sufficient quantities of high quality product, is an area that requires substantial developmental effort. Challenges surrounding reimbursement for treatment, and the pressures associated with shorter time to approval, both increase burden placed on manufactures to rapidly develop suitable processes that are cost-effective. Cost of goods (COGs) need to be kept below critical threshold levels to drive sufficient profit margins, even though process development timelines are aggressive and short. There are a multitude of critical decisions and considerations to overcome.
This blog explores some of these fundamental manufacturing challenges in more detail.
Scalable Manufacturing Platform
Technologies used to manufacture gene therapy biologics are advancing at very rapid pace. Not having a platform that is suitable nor scalable is a significant challenge many manufacturers face. It is a necessity throughout clinical development stages to be able to optimize the manufacturing process. However, any change in the manufacturing process that increases product yield or enhances quality is accompanied by the risk of changing the product. It is therefore essential that close attention is paid to tracking variation throughout the development process at every stage.
A substantial amount of early stage development is still being performed using outdated, non-commercially viable platforms and transferring processes to new platforms is required. To achieve manufacturing platform advancement, the product needs to be very well characterized during development so that investigators can generate data sets which demonstrate comparability between products used in clinical studies and those generated with the final manufacturing process.
Cost of Goods
COGs associated with manufacturing any drug product impacts the overall price of the therapy and heavily influences the profit margin realized by gene therapy manufactures. High production cost is a challenge that affects profitability. This is reflected in the high costs associated with newly approved gene therapy drugs such as Yescarta♦, Kymriah♦ and Luxturna♦ which are currently priced in the 100 thousands dollar range per dose. The challenge becomes a critical concern when the product in development cannot be sold at a price high enough to achieve a commercially-viable profit margin. If acceptable margins cannot be reached, developers may choose to terminate production making the drug unavailable to patients. However, due to the remarkable value and life changing nature of the treatments the entire industry is committed to the pursuit of cost effective methods for manufacturing. There is a significant effort that has been mounted by all players to reach this end.
Currently, the main cost contributor to the overall COGs for gene therapy products is high quality clinical grade plasmid DNA containing the therapeutic gene of interest. This reagent is required for transient transfection of cells and it is imperative that the reagent is of high quality. It is an essential component of the process to assure an acceptable safety profile. Another example of an expensive gene therapy product is Zolgensma♦. This new drug was recently approved for the treatment of spinal muscular atrophy (SMA), which is a rare disease that causes severe muscle weakness for suffers. It affects their ability to breath, speak and move. Most babies born with a common form of SMA die by the time they reach two years of age. Currently there is no cure. Zolgensma represents the only treatment option now available to cure the 10,000 – 25,000 affected individuals in the US. However, the current challenge with this therapy is that it could costs $2.1 million per patient1.
Reimbursement
Market size is an important factor that can limit effective commercial return. If the market size is too small, profitability is limited due to the small number of doses required to treat the patient population. This decreases the profit margin realized by the drug developer and can lower motivation to commercialize the therapy. The most encouraging aspect of the gene therapy revolution is that the first round of gene therapy products has been developed for extremely rare diseases, with small patient populations indicating the commitment to treat previously untreatable diseases. Amazingly, these patients can be cured by a single drug application, however, this inherent property of the therapy can further limit commercial profitability. Patients are often not required to pay for these high-cost medicines themselves, and look to government programs and health care insurance providers to reimburse the manufacturer for treatments. Health insurance reimbursement plans for new products is challenging, particularly so for new category products like gene therapy. It is expected that the process of reimbursement will differ from country to country and it will also be guided by factors like economics, demographic data and politics. If the current cost of manufacturing stands then drugs such as Zolgensma could place a huge financial strain on health systems. In the US for example, it is surmised that treating common diseases such as hemophilia, which affects around 20,000 people in the US alone, could cause a financial crisis1. If we look to the future of modern medicine, commercialization of gene therapies will require not only significant advancement in manufacturing processes to reduce costs but also a practical reimbursement strategy that will allow for drug developers to continue to forge into the new frontiers of medicine.
References:
1. Business Insider. http://www.businessinsider.com/gene-therapy-treats-disease-but-prices-could-strain-us-health-system-2019-2
♦Kymriah is a trademark of Novartis AG., Luxturna is a trademark of Spark Therapeutics, Inc., Yescarta is a trademark of Kite Pharma, Inc., Zolgensma is a trademark of AveXis Inc.
Author Details:
Dr. Mark Szczypka
Global Director, Process Development Services
Pall Corporation
2012pharmaceutical
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