mRNA therapeutics have delivered on the promise of simple and fast vaccine production. Since then, the field has been rapidly evolving as it has tremendous potential to combat a range of incurable diseases. As the demand for mRNA-based therapeutics continues to grow, it is essential to explore more sustainable production technologies that reduce environmental impact while ensuring the quality and efficiency of the products. By implementing environmentally friendly strategies, researchers can contribute to a greener future without compromising on results. In this blog post, we will delve into the concept of more sustainable mRNA production and highlight more environmentally responsible methods for mRNA synthesis by solid-phase in vitro transcription (IVT) and purification.
Steps in more sustainable mRNA production
The mRNA production process is a multi-step procedure that starts with preparing a DNA template, followed by IVT of mRNA drug substance, purification, and encapsulation to formulate the final drug product. Currently, plasmid is most often used as the DNA template. Plasmids are produced in E. coli host, with the use of antibiotics which adds to the environmental impact. Thorough purification of the plasmid is required also to remove host DNA before it can be used in the production of an mRNA drug substance. For IVT, the plasmid template needs to be linearized. Removal of remaining circular DNA and linearization reaction components is needed before the next step to reduce unwanted IVT reaction products. The purified plasmid is mixed with IVT reaction components to produce mRNA. After IVT, the template DNA is an undesired contaminant that needs to be removed together with unused reaction components. This is done by DNase I-based digestion and multiple purification steps that are usually column-based. They utilize solid matrices to selectively bind and separate mRNA from the reaction mixture. These steps are long and complex, require specialized equipment and know-how, and utilize a lot of buffers. At the same time, each purification step adds to the loss of the mRNA drug substance. More sustainable mRNA production involves adopting practices that reduce waste generation, lower energy consumption, and optimize resource utilization.
Harnessing the power of solid-phase mRNA production
Solid-phase mRNA drug substance production utilizes solid support, such as magnetic beads during IVT and mRNA purification. Solid-phase IVT is a technology, where the DNA template is immobilized on streptavidin-coupled magnetic beads during mRNA synthesis. The process starts with the linearization of the plasmid DNA template followed by biotinylation. After that, the DNA template is mixed with magnetic beads and after a short incubation, placed on a magnet. This serves as a purification step – only biotinylated fragments of DNA bind to the bead surface. Then IVT components are added and after the reaction, the DNA template is placed on a magnet and removed from the mRNA. The template can be reused for the next round of mRNA synthesis by adding more IVT reagents. At the same time, the resulting mRNA does not need a DNase digestion step, which greatly decreases the complexity of subsequent purification allowing one-step purification by Carboxylic Acid magnetic beads. The beads are mixed with the mRNA and accompanying purification buffer resulting in mRNA binding to the bead surface. This is followed by simple wash and elute steps, resulting in the final purified mRNA drug substance (Figure 1).
Figure 1. mRNA synthesis by solid-phase IVT and purification using Dynabeads magnetic beads
The solid-phase technology for IVT and purification offers several advantages that contribute to a more sustainable mRNA production:
- Reduced resource and material consumption
- Enhanced efficiency
- Reduced waste
- Energy efficiency
- Scalability
- Automation
Reduced resource and material consumption
Plasmid: The immobilization of the plasmid DNA template on solid support such as magnetic beads allows the reuse of the same template up to six times reducing the amount of the plasmid needed.
DNase I: solid-phase IVT has a built-in DNA template removal via magnetic separation, which eliminates the DNase digestion step.
Antibiotics: The reuse of the plasmid template six times reduces by the same amount the need for antibiotics during plasmid production. For example, to produce 50g of mRNA (Figure 2), 32L of E. coli culture is needed, while solid-phase IVT would require only 6L. Importantly, this could be reduced even further if the PCR template was used for mRNA production instead of plasmid. Here, to produce 50g of mRNA, only 2 mL of E. coli culture is needed.
Figure 2. Dynabeads magnetic beads IVT workflow compared to conventional in-solution IVT workflow
Purification buffers: Solid-phase IVT eliminates the purification of the plasmid template after linearization. Also, the elimination of DNase digestion simplifies the matrix complexity (no additional protein or DNA fragments) for the next purification steps. Multiple column-based purification steps with their buffers are replaced by one-step generic capture on magnetic beads surface, greatly reducing the volumes of buffers needed. Also, magnetic bead-based purification allows for the control of the final elution volume yet eliminates the use of tangential flow filtration buffers.
Equipment: Magnetic bead-based technology is very simple and requires the same type of tube/reactor vessel for the IVT reaction and purification. Reusable magnet replaces HPLC systems and expensive columns.
Magnetic beads: Both the solid-phase IVT bead and purification bead can be reused up to six times decreasing their contribution to the cost of the mRNA drug substance production process.
Labor and footprint: Solid-phase magnetic bead-based mRNA production is shorter than the traditional process decreasing the labor needed. The reuse of the beads allows a decrease of the production footprint as the same quantity of mRNA drug substance can be obtained from five times smaller vessels. Additional footprint reduction comes from the elimination of sophisticated purification equipment.
Enhanced efficiency
Higher yield per template unit: The solid-phase IVT technology can result in approximately a 20% decrease in mRNA yields compared to the traditional in-solution method. At the same time, due to the six-times reuse of the plasmid template on the solid support, approximately five times more mRNA is produced per unit of the template.
Efficient purification: The generic capture of mRNA on the magnetic bead surface is an extremely efficient process leading to over 90% recovery rates in a single step. Column-based purification is usually much less efficient with an average 70% recovery rate. Combined with the need for multiple purification steps, the final yield of the purified mRNA drug substance is much lower compared to the solid-phase one.
Reduced waste
Antibiotics: The reuse of the plasmid template reduces the impact of the waste antibiotics from the plasmid production.
Less contamination: The solid-phase approach minimizes the risk of contaminants coming from host derived plasmid production. As only linearized and biotinylated plasmid DNA template binds to the streptavidin bead surface, no endotoxins or E. coli DNA is carried over to the IVT step. This can reduce the number of times a sample needs to run to achieve your desired yield and purity grade.
Direct waste reduction: The solid-phase technology simplifies the mRNA production and purification process by reducing multiple purification steps. This results in less liquid and plastic waste compared to traditional methods, reducing environmental impact.
Energy efficiency
Lower energy consumption: The streamlined process with reduced purification steps is faster and the elimination of temperature-sensitive DNase digestion and energy-dependent advanced purification equipment leads to lower energy consumption.
Scalability
Ease of scale-up: Scalability is crucial for producing large quantities of mRNA for therapeutic applications. Solid-phase IVT is directly scalable from uL (tube) to L (reactor) scale as the steps remain similar even at higher throughput volumes. The obtained mRNA yield is a linear function depending only on the amount of magnetic beads/template and IVT reagents. It is crucial especially in the purification steps as traditional column-based purification is not easily scalable. This scalability streamlines the mRNA production process reducing time and resources as the same technology can be used during the development and production phases.
Flexibility: Depending on the production scale needed, solid-phase mRNA production can be either scaled out using automated magnetic bead handlers with multiple parallels or scaled up in a reactor. For example, using KingFisher bead handlers, one can process up to 24 wells (total 48mL IVT) in parallel, producing up to 150mg of purified mRNA (without bead re-use). Or with scaling-up, one can produce 3g of purified mRNA in one glass reactor (1L IVT).
Automation
Resource efficiency: Magnetic beads are inherently automatable allowing screening up to 96 samples in 4h, from template immobilization to purified mRNA. Automation also increases precision and reproducibility reducing the likelihood of errors and the need for repeat processes. This facilitates process optimization and leads to a reduced number of resources consumed and reduces waste.
Advantages of more sustainable mRNA synthesis and purification
By adopting more sustainable practices in mRNA synthesis and purification, researchers can enjoy several benefits:
- Lower environmental impact: More sustainable methods reduce waste generation, lower energy consumption, and optimize resource utilization.
- Cost efficiency: Efficient mRNA synthesis and purification methods reduce production costs, leading to more accessible mRNA-based technologies, such as mRNA vaccines and gene therapies.
- High-quality mRNA: More sustainable methods ensure the production of pure mRNA, free from impurities and contaminants, resulting in reliable and reproducible research outcomes.
Achieving more sustainable mRNA synthesis and purification is crucial for the development of mRNA-based technologies, including vaccines and gene therapies. By harnessing environmentally responsible methods such as solid-phase IVT and magnetic bead-based purification, researchers can reduce waste, lower energy consumption, and optimize resource utilization. Embracing more sustainable practices not only contributes to a greener future but also helps ensure the production of high-quality mRNA for various applications in research and therapeutics. More sustainable mRNA production is especially critical in mRNA vaccine development, where the quality, yield, and purity of the mRNA molecule directly impact the safety and efficacy of the vaccine. By prioritizing sustainability in mRNA synthesis and purification, researchers can advance the field while reducing environmental impact and promoting the accessibility of mRNA-based technologies. For research use or further manufacturing. Not for diagnostic use or direct administration into humans or animals. Looking to produce more sustainable and high-quality mRNA? Learn about Dynabeads magnetic beads for vaccine development and production. Interested in learning more about mRNA synthesis by IVT? Watch our on-demand webinar here.
Authors
- Iwona Grad, Staff Scientist, Thermo Fisher Scientific, Oslo, Norway
- Ausra Kaleckiene, Staff Scientist, Thermo Fisher Scientific, Oslo, Norway
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