Challenges and Regulatory Requirements in Plasmid DNA Manufacturing: Ensuring Safety, Quality, and Compliance

Introduction

Plasmid DNA, a small, circular, double-stranded DNA found in bacteria and some other organisms, plays a crucial role in genetic engineering and molecular biology. A typical plasmid contains an origin of replication, an antibiotic resistance gene as selective marker during replication, and optimized genetic elements (promoter, cloning site & gene, etc.), which is transferred and replicates within the host cells (such as E. coli), as shown in Figure 1 [1]. Starting from the 1970s, plasmid DNA (pDNA) has been employed as useful vehicles to introduce and propagate foreign genes inside microbes for different application. 

Since the Covid-19 breakout, the demand for pDNA has massively increased as it’s the critical starting material in the advanced therapy medicinal products (ATMPs) supply chain. Plasmid DNA is stable and easy to manipulate and replace non-essential genetic material with therapeutic genes of interest, therefore they have been broadly used as crucial starting materials for vaccines (viral vector vaccines, mRNA vaccines), gene and cell therapies against several infectious-, acquired-, and genetic-diseases. The gene therapy market size obtained a value of USD 7.81 billion in 2023. The market is anticipated to grow at an average growth rate of 22.8% during the forecast period of 2024-2032 to reach a value of USD 49.60 billion by 2032 [2]. Recently, there are over 1,500 ongoing clinical trials in the fields of cell and gene therapy, driven by industry’s steadily increasing funding and investments [3].

           Figure 1. Design, selection, and replication of recombinant plasmid DNA within E. coli [1]. 

Plasmids DNA Manufacturing Challenges and New Platforms

pDNA Manufacturing Challenges

With the rapid advancement of cell and gene therapy-related technologies, such as CRISPR, CAR-T, RNA therapeutics, and nucleic acid-based vaccines, commercialization of ATMPs comes a tremendous demand for plasmid DNA [4, 5, 6]. Typically, the pDNA manufacturing process includes several key steps such as plasmid design and construction, fermentation, cell harvest and lysis, purification (chromatography & filtration), and formulation, as illustrated in Figure 2. Appropriate control of these key steps ensures the production of pure, stable, and functional plasmid DNA suitable for use in gene therapy, vaccine development, gene editing, and other biotechnological applications.

Figure 2. Key steps of plasmid DNA manufacturing process. 

Plasmid DNA is typically produced by fermentation methods using K-12 strain of E. coli in stainless steel or single use bioreactors. The fermentation process is inherently slow and expensive, with limited capacity, and is also prone to batch failure, while the downstream purification process is similarly expensive, slow, and difficult to scale. Some challenges that can arise during the manufacturing of plasmid DNA, including: 

• Low Yield. Achieving high yields of plasmid DNA can be challenging, particularly for large-scale production. The vector design, bacterial strain, plasmid stability, culture conditions (temperature, pH, aeration, and nutrient availability etc.), DNA extraction methods, and purification protocols are crucial factors to maximize yield without compromising product quality.
• Purity Issues. Purification of plasmid DNA at large scale requires robust and scalable downstream process or methods. Contaminants such as RNA, genomic DNA, proteins, endotoxins, and other impurities can affect the purity of plasmid DNA. To address the purity issues, it’s essential to optimize purification protocols that effectively remove contaminants while preserving the integrity, functionality, and yield of plasmid DNA.  Meanwhile, utilizing regular quality control testing methods such as UV spectrophotometry, gel electrophoresis and analytical techniques like HPLC to analyze the purity of plasmid DNA during downstream processing is useful to identify any potential contamination issues. 

Per FDA guidelines 2007, the purity of plasmid DNA must meet the following specifications [13]:

• Scalability Limitations.  Transitioning from small-scale laboratory production to large-scale manufacturing can pose scalability challenges. For example, conditions that works well at small scale may not directly translate to large scales due to difference in mixing, aeration and nutrient distribution. Maintaining adequate oxygen levels in large bioreactors is challenging, which can affect bacterial growth and plasmid yield. Another concern is efficiently lysing large volumes of bacterial cells without degrading the plasmid DNA is difficult. Also, scaling up purification processes, such as filtration, chromatography (typically ion exchange (IEX) and hydrophobic interaction chromatography (HIC)), and ultrafiltration, can be very challenging. High-volume purification requires robust and reproducible methods to ensure high purity and yield. To address the scalability limitations, process design (bioreactors, downstream purification units) and optimization, equipment scalability, quality control, regulatory compliance (such as GMP) and cost considerations must be carefully considered to ensure consistent production at higher volumes.
• Quality Control Complexities. Rigorous quality control measures are essential to assess the identity, purity, stability, and functionality of plasmid DNA throughout the multi-step manufacturing process, including fermentation, cell lysis, purification, and formulation. Analytical techniques such as gel electrophoresis, UV spectrophotometry, and sequencing are employed to ensure final product identity, stability, purity, safety, and efficacy. 

New Platforms 

Instead of employing K12-based E. coli production strains, recently a startup company called Novel Biotechnology Inc has developed a new robust bacterial platform which does not use E. coli as a host, Novel’s NBx Platform™, uses a highly productive BSL-1 bacteria which has been highly mutated and optimized to produce plasmid DNA [7]. In Novel Biotech’s published white papers, the bench/pilot run results indicated that the pDNA yield was up to 10 times more than E. coli, with half of the turnaround time. Additionally, the endotoxin level was 100-400 times lower and host cell protein contaminants were up to 40% lower as compared to E. coli, which simplifies the downstream operation. This new platform is quite promising to overcome the low yield and scalability limitations during pDNA manufacturing. Novel Biotech has its platform commercially and currently working with multiple strategic partners to test and license its platform.

As an alternative using bacterial to produce plasmid DNA, Touchlight has developed a solution to synthesize DNA utilizing an in vitro dual enzyme process [8]. In the process, an amplification enzyme and a processing enzyme are used to quickly produce high-quality product within a relatively small footprint. The synthetic DNA vector is called as “doggybone” or dbDNA™, which is classified by regulatory authorities as a chemical, rather than a biological product. The company’s technology and application has been employed for expeditious DNA production to meet urgently global demand to some extent. So far, the technology has already been employed by multiple mRNA companies to develop Covid-19 vaccines and other therapies.

Regulatory Requirements

The regulatory requirements for plasmid DNA manufacturing vary depending on the intended use and jurisdiction in which it is produced and distributed. In general, plasmid DNA used for pre-clinical study and clinical phase I/II, is less stringent than being employed for clinical phase III studies and commercial application. Figure 3 outlines the phase-dependent plasmid production [9].

Figure 1. Phase-dependent plasmid DNA production. [9]

Regulatory Agencies

Plasmid DNA production is governed by several regulatory agencies, each ensuring safety, efficacy and quality through stringent guidelines and requirements. This paper shall mainly focus on the guidelines and requirements by the U.S.Food and Drug Administration (FDA) and European Medicines Agency (EMA). 

EMA guidelines on plasmid DNA

Since becoming effective in 2001, the initial requirements for plasmid DNA described in EMA guidance reflected its primary application in gene therapy as a gene transfer vector/DNA vaccine. During that time, the plasmid DNA was usually used as an active substance administered either in a simple salt solution or a more complex one with a carrier or adjuvant [10]. Until early 2019, EMA outlined the most relevant existing regulatory requirements of plasmid (linearized template) as a raw starting material in the draft guideline for quality, nonclinical and clinical requirements for investigational advanced therapy medicinal products (ATMPs) in clinical trials [11]. The EMA claimed that where cell lines were used, an appropriately characterised Master Cell Bank (MCB) and Working Cell Bank (WCB) should be established, whenever possible. While a MCB should be established prior to the initiation of phase I trials, the WCB may not always be established early on.

Later, in February 2021, the EMA released a guidance document named: “Questions and answers on the principles of GMP for the manufacturing of starting materials of biological origin used to transfer genetic material for the manufacturing of ATMPs” [12]. In this Q&A, the EMA provided clarification and basic guidance for both plasmid and emerging synthetic DNA as alternative starting materials for gene therapy. Furthermore, the EMA has stated the GMP requirements of plasmid as a starting material, the difference between the principles of GMP and full GMP, and how to select the GMP principles to be followed using a risk-based approach by ATMPs manufacturers. The most useful clarifications is listed as below [12]:

“A GMP certificate is not required for manufacturing and testing sites of starting materials for ATMPs. For certain starting materials of biological origin [according to the Directive 2001/83/EC] such as linear DNA used as template for ex vivo transcription into mRNA, plasmids to generate viral vectors and/or mRNA, and vectors) used to transfer genetic material for the manufacturing of ATMPs it is, however, mandatory that the principles of GMP are complied with.”

According to EMA, the ATMP manufacturers are responsible to verify that appropriate GMP requirements are implemented. Table 1 is presented in the 2021 EMA Q&A Guideline on the principles of GMP manufacturing to help clarify the EMA’s expectations around the level of GMP controls needed for different products using plasmid DNA [12]. 

FDA guidelines on plasmid DNA

The US FDA published a guidance document named, “Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications” in 1996. In 2007, the US FDA released a revised recommendation titled “Considerations for Plasmid DNA Vaccines for Infectious Disease Indications” with accumulated preclinical and clinical experiences [13]. However, both the guidance and the revised recommendation focus on plasmid DNA only as an active pharmaceutical ingredient (API). 

Only until 2020, the new released guidance titled “Chemistry, manufacturing, and control (CMC) Information for human gene therapy investigational new drug applications” addressed the requirements of plasmid DNA when used as starting material [14]. This guidance mentions the plasmid DNA should be made from qualified cell banks and the manufacturing procedures, reagents, and specifications should be listed in the investigational new drug (IND). Testing suggested for the plasmids to include sterility, endotoxin, purity (including percent of supercoiled form and residual cell DNA, RNA, and protein levels), and identity to ensure the identity, purity, potency, and safety.

Like the EMA, the FDA also recommends that the plasmid DNA be derived from qualified and traceable cell banks, establishment of a bacterial MCB, as it can provide a consistent starting material for the manufacture of plasmid DNA. However, MCBs may not be necessary for all manufacturing situations if the plasmid intermediate is appropriately qualified (eg, for early phase studies when the plasmid is used to make a vector for ex vivo modification of cells). 

As outlined in the FDA 2020 guidance, the genotype and source of the microbial cells must be fully described for the cell bank material itself, and information provided to include how the material was generated, how the bank is stored and maintained, as well as information regarding the cell bank qualification, therefore to adequately establish the safety, identity, purity, and stability of the microbial cell preparation employed in the manufacturing process. The testing requirements for bacterial cell banks used to produce plasmid DNA were also prescribed in the same FDA guidance. 

Overall, taking account of the various recommendations provided by the EMA and FDA, plasmid manufacturers should determine the level of controls they will establish for their plasmid manufacturing platform and the associated quality control testing strategy. Contract plasmid manufacturers must also assess the types of plasmid DNA they intend to produce, which include starting material plasmids for viral vectors, in addition to other plasmid DNA therapies and vaccines.

Good Manufacturing Practices (GMP) for pDNA Manufacturing

Good Manufacturing Practice (GMP) requirements for the large-scale pDNA manufacturing is critical to the production of high-quality, safe, and consistent products for use in research, diagnostics, and therapeutics. Key GMP requirements for pDNA manufacturing are recommended to be considered as below: 

• Facility Design and Maintenance. Plasmid DNA manufacturing facilities must be designed, constructed, and maintained in a way to prevent contamination and facilitate clean and orderly operation environment, which includes proper ventilation, temperature and humidity control and segregation of manufacturing areas. The facility should also be qualified, such as critical HVAC for temperature and humidity controls for manufacturing areas. 
• Equipment Qualification and Calibration. Manufacturing equipment, such as fermenters, centrifuges, process vessels, purification systems (chromatography and filtration systems), and process analytical instruments (such as pH meter, fluorometry, or quantitative polymerase chain reaction (PCR), high-performance liquid chromatography (HPLC) etc.), must be qualified, calibrated, and maintained to ensure accurate and reproducible results.
• Personnel Training and Hygiene Practice. All personnel involved in plasmid DNA manufacturing must take appropriate training in GMP principles, procedures, and safety protocols. Personal hygiene practices, including gowning, handwashing, and use of personal protective equipment, are essential to prevent contamination.
• Documentation Control. Comprehensive documentation of all aspects of pDNA manufacturing, including standard operation procedures (SOPs), batch records, test results, deviations, and corrective actions, must be maintained in compliance with GMP requirements. Documentation control procedures ensure the accuracy, integrity, and traceability of manufacturing records.
• Quality Control Testing. Plasmid DNA batches must undergo rigorous and comprehensive testing and analysis to ensure identity, purity, potency and safety, which typically includes the followings at a minimum: (1). Identity verification to confirm that the plasmid DNA matches the intended sequence and construct; (2). Concentration measurement using validated quantitative methods such as spectrophotometry, fluorometry, or quantitative polymerase chain reaction (qPCR); (3). Purity profile assessment by quantifying impurities such as host cell DNA, RNA, proteins, endotoxins, and other contaminants; (4). Functional assays to validate the functionality of the plasmid DNA, including its ability to replicate, transcribe, and express the desired gene or genes in relevant host systems. 

Analytical methods must be validated for accuracy, precision, specificity, and robustness.

• Incoming Raw Material Control.  Raw materials used in plasmid DNA manufacturing, including DNA templates, enzymes, reagents, cells, and cell culture media, must be of appropriate quality (such as transmissible spongiform encephalopathy (TSE) and bovine spongiform encephalopathy (BSE) free) and sourced from audited and approved suppliers. Plasmid DNA manufacturers should establish criteria for supplier qualification and materials specifications, conduct risk assessment and on-site audits to assess the suppliers’ facilities, quality systems, manufacturing process, and adherence to relevant regulations and standards. Incoming material must be inspected, tested to prevent contamination, and ensure consistency. The raw materials must be traceable throughout the supply chain with necessary documentation, including certification of analysis, batch records, and regulatory documentation. 
• Manufacturing Process Validation. Plasmid DNA manufacturing processes, including fermentation, cell lysis and downstream purification (filtration, chromatography), must be validated to demonstrate their consistency, reproducibility, and capability to produce the desired quality and yield of plasmid DNA. Process validation typically includes qualification of critical process parameters and purification protocols validation.
• Equipment Cleaning and Sanitization. The process contact stainless steel equipment must be designed as per ASME BPE-2024 standards. Cleaning and sanitization protocols must be established and validated to prevent cross-contamination and ensure cleanliness of manufacturing equipment and facilities. Cleaning and sanitization are not required if single use equipment is used instead. 
• Plasmid DNA Stability Testing. Plasmid DNA products are required to undergo stability testing to evaluate their stability under various storage conditions and establish shelf-life specifications. Stability study is helpful to ensure product quality and efficacy throughout its intended shelf life.

Abiding by the above GMP requirements is vitally important for plasmid DNA manufacturers to maintain product quality, ensure patient safety, and comply with regulatory standards enforced by agencies such as the FDA and EMA.

Summary 

The production of plasmid DNA has evolved as an increasingly critical element in the supply chain for ATMPs. With the fast expansion of the traditional pDNA manufacturing capacity, and the development of new pDNA manufacturing platforms, the shortage problems for pDNA shall be gradually resolved. The contract plasmid manufacturers should establish their own process platforms and testing strategy to meet the GMP requirement, while abiding by the most updated guidelines released by the FDA and EMA to produce high quality pDNA to ensure the quality, efficacy and safety of their product.