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With messenger RNA (mRNA) emerging as a transformative platform for vaccine and therapeutic research, the evolution of biopharmaceutical manufacturing has been rapid. After the success of COVID-19 mRNA vaccines, it is now also worth looking at ways to maximize mRNA production for diseases. It's the purification step that is of crucial importance to mRNA production and, therefore, directly to the efficiency, stability and efficacy of the end drug product. The TFF has been a valuable technology for mRNA purification because of its high recovery and the fact that it allows the RNA integrity to be maintained during the process.
mRNA Drugs
mRNA-based therapies are already fast becoming a bedrock of contemporary medicine. mRNA drugs work by signalling cells to manufacture therapeutic proteins, a promising new treatment option for genetic disorders, cancers and viruses. The most visible mRNA use case to date has been in the form of mRNA vaccines. These vaccines work by inserting a synthetic mRNA sequence into the body that codes for an antigen (or a viral protein) associated with a pathogen (for example, the spike protein of the virus SARS-CoV-2). The mRNA tells the host cells to make this protein, so there's an immune reaction and immunity without live pathogens. This approach offers several advantages:
- Speed of Development: Unlike traditional vaccine development, which requires the growth of viruses or bacterial cultures, mRNA vaccines can be designed and manufactured in a matter of weeks. This rapid response is a significant advantage in the context of emerging infectious diseases.
- Scalability: mRNA vaccines can be produced at a large scale using cell-free systems, which eliminates the need for complex biological culture systems, thus enabling faster and more efficient production.
- Flexibility: mRNA vaccines can be easily adapted to target different pathogens by simply changing the mRNA sequence, allowing for quick adaptations to evolving viruses or new infectious agents.
As the pipeline for mRNA-based treatments expands, so too does the need for efficient and scalable manufacturing processes that can produce high-purity mRNA drug substances. The success of these drugs relies heavily on the purity and integrity of the mRNA, which is essential for the accurate and reliable expression of therapeutic proteins within the body.
Diagram of the SARS-CoV-2 virus mRNA vaccine.
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mRNA Purification
The purification of mRNA is a fundamental step in the production of mRNA-based therapeutics, directly influencing both the quality and efficacy of the final drug product. As mRNA therapeutics evolve into a prominent modality for treating various diseases, including genetic disorders, cancers, and viral infections, the demand for efficient and scalable purification technologies has become paramount. However, this process is not straightforward. It requires careful handling to ensure that the final mRNA product maintains its biological activity, stability, and safety for clinical use.
Impurity Removal for Drug Safety and Efficacy
During in vitro transcription (IVT), mRNA synthesis often results in impurities such as residual nucleoside triphosphates (NTPs), enzymes, and by-products. These contaminants can degrade mRNA stability, hinder protein translation, and trigger immune responses. Purification is critical for removing these impurities to ensure mRNA stability, biological activity, and safety.
Ensuring mRNA Integrity
mRNA is inherently unstable and prone to degradation. The purification process must minimize degradation risks, preserve mRNA integrity, and remove harmful agents. Techniques like Tangential Flow Filtration (TFF) can help recover intact mRNA by minimizing shear stress and filtering out impurities without damaging the RNA.
Controlling Critical Quality Attributes (CQAs)
Key CQAs for mRNA drugs include integrity, purity, size distribution, concentration, and endotoxin levels. The purification process must meet Good Manufacturing Practices (GMP) standards and maintain these attributes to ensure safety and efficacy, which are crucial for regulatory approval.
Post-Purification Considerations for Formulation and Delivery
After purification, mRNA is typically encapsulated in lipid nanoparticles (LNPs) for delivery. The purity of the mRNA is crucial for ensuring LNP stability and efficient delivery. Residual impurities can negatively impact these processes.
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Tangential Flow Filtration
Tangential Flow Filtration (TFF) is a membrane-based filtration technology commonly used in the biopharmaceutical industry for the separation and purification of biomolecules, including mRNA. TFF is based on the principle of cross-flow filtration, where the feed solution flows tangentially across the membrane surface. This setup allows the permeation of smaller molecules through the membrane, while larger molecules—such as mRNA—are retained and concentrated. In contrast to traditional dead-end filtration methods, where the entire feed is forced through the membrane, TFF employs a cross-flow mechanism that helps to minimize membrane fouling. This results in increased filtration efficiency and higher permeate flux, which is critical when processing large volumes of material, as is often required in mRNA drug production.
How Does Tangential Flow Filtration Work?
In a TFF system, the feed solution is pumped along the surface of a membrane, which serves to separate molecules based on size. Molecules smaller than the pore size of the membrane pass through the filtration barrier and are collected as the filtrate, while larger molecules, such as mRNA, are retained in the retentate. TFF operates in two primary modes: concentration and diafiltration. In concentration mode, the volume of the retentate is reduced while the concentration of retained molecules increases. In diafiltration mode, the retentate is diluted with fresh buffer to remove impurities and replace the surrounding solution with a buffer suited for subsequent steps in the production process. For mRNA purification, TFF is particularly advantageous because it enables selective removal of contaminants like unreacted NTPs while retaining the intact mRNA.
Cross-Flow Filtration vs Tangential Flow Filtration
While both cross-flow filtration and tangential flow filtration describe similar principles, they are sometimes used interchangeably. However, there are subtle differences:
- Cross-flow filtration refers to the overall mechanism where the feed is filtered across the membrane surface, and it can be used in both dead-end and tangential flow configurations.
- Tangential flow filtration, on the other hand, specifically refers to a process where the feed is continuously pumped tangentially across the membrane, allowing for better flow dynamics and reduced fouling.
In mRNA purification, the benefits of tangential flow filtration over dead-end filtration are evident, as it offers higher recovery rates and reduced membrane fouling, leading to less frequent cleaning and lower operating costs.
Advantages of Tangential Flow Filtration
High Recovery and Purity
TFF allows for the selective retention of mRNA while effectively removing contaminants. By fine-tuning operational parameters such as membrane pore size, transmembrane pressure (TMP), and feed flux, recovery rates exceeding 70% can be achieved, with minimal degradation of the mRNA. This is crucial for ensuring that the purified mRNA retains its therapeutic potential.
Scalability
TFF is highly scalable, making it suitable for both research-scale and commercial-scale mRNA production. The ability to handle large volumes efficiently is essential for industrial-scale manufacturing, where high throughput and cost-effectiveness are key considerations.
Reduced Fouling
The tangential flow mechanism minimizes membrane fouling, a common issue in filtration processes. This results in lower operational costs as it reduces the need for frequent cleaning or replacement of the filtration system. The reduction in fouling also translates into more stable operation and longer filtration runs, improving the overall process efficiency.
Preservation of mRNA Integrity
The gentle nature of TFF, combined with the ability to operate under optimized conditions (e.g., low transmembrane pressure and moderate flow rates), ensures that mRNA integrity is preserved during filtration. This is crucial for maintaining the stability and efficacy of the mRNA drug substance.
Flexibility in Buffer Exchange
The diafiltration mode of TFF allows for easy exchange of buffers, ensuring that mRNA is transferred into a buffer composition that is optimal for subsequent processes like chromatography or lipid nanoparticle (LNP) encapsulation.
Prospects for Tangential Flow Filtration in mRNA Purification
As mRNA technology continues to advance, the demand for highly efficient, scalable, and cost-effective purification methods will only increase. The future of TFF in mRNA drug production will likely be shaped by several key trends:
Enhanced Membrane Materials
The development of more selective and durable membranes is expected to improve the efficiency of TFF in mRNA purification. Membranes with smaller pore sizes, improved fouling resistance, and higher throughput will help to further increase the purity and recovery of mRNA drug substances.
Integration with Automation
The integration of automated systems for process monitoring and control will streamline TFF operations. Automation can help ensure that critical parameters, such as transmembrane pressure and feed flux, are continuously optimized, leading to more reliable and consistent production of high-quality mRNA.
Improved Process Modeling and Simulation
With the increasing complexity of mRNA manufacturing, advanced modeling and simulation tools will play a key role in optimizing TFF operations. By integrating experimental data with mathematical models, manufacturers will be able to predict and control the effects of fouling, process scaling, and other variables, ensuring the most efficient use of resources.
Customized Process Design for Specific mRNA Sequences
The purification process may be tailored to specific mRNA sequences or formulations, allowing for personalized or targeted therapeutics. Research into how different mRNA sequences interact with filtration membranes could lead to the development of more customized purification strategies that improve yield and quality.
Tangential Flow Filtration (TFF) stands as a cornerstone of mRNA drug purification, offering unparalleled advantages in terms of efficiency, scalability, and preservation of RNA integrity. The optimization of TFF processes will continue to play a pivotal role in the future of mRNA therapeutics, particularly as the demand for personalized and highly effective treatments grows. As mRNA-based drugs become increasingly prevalent, the continued advancement of TFF technologies will be crucial in meeting the challenges of large-scale manufacturing, ensuring the production of high-quality, stable, and potent mRNA drug substances.
* Only for research. Not suitable for any diagnostic or therapeutic use.
