mRNA-based therapies and vaccines have emerged as a revolutionary approach in the field of medicine, offering a versatile and powerful tool for addressing a wide range of diseases. The success of mRNA vaccines, such as those developed for COVID-19, has highlighted the potential of this technology and the need for efficient large-scale production techniques. Scaling up mRNA production involves overcoming several challenges, including optimizing synthesis, purification, and ensuring product quality and consistency. This article provides a comprehensive overview of the techniques and strategies used in large-scale mRNA production.

Overview of mRNA Production Process

The production of mRNA involves several key steps, including in vitro transcription (IVT), purification, and post-transcriptional modifications. IVT is the process by which mRNA is synthesized from a DNA template using an RNA polymerase enzyme. After transcription, the mRNA must be purified to remove contaminants, such as enzymes, residual DNA, and aberrant mRNA species. Post-transcriptional modifications, such as the addition of a 5' cap and polyadenylation, are then performed to enhance the stability and translation efficiency of the mRNA.

In Vitro Transcription (IVT) Techniques

Traditional IVT Methods

Traditional IVT methods involve the use of a DNA template, RNA polymerase, and nucleotides to synthesize mRNA in a reaction mixture. These methods are well-established and widely used in laboratory settings. However, they may not be easily scalable for large-scale production. One of the challenges with traditional IVT is the removal of the DNA template after transcription, which is typically achieved through DNase digestion. This step can be time-consuming and may introduce additional purification challenges.

Figure 1. Schematic illustration of in vitro transcription (IVT) of mRNA. mRNA is synthesized in vitro by using a linear DNA template and RNA polymerase (T7). The IVT mRNA is composed of five domains: 5'-cap, 5'- and 3'-UTR, an ORF encoding the protein of interest, and a poly(A) tail. (Ma et al., 2023)

Solid-Phase IVT

Solid-phase IVT is an innovative approach that addresses some of the limitations of traditional IVT methods. In this technique, the DNA template is immobilized on a solid support, such as magnetic beads, allowing for direct scalability from small to large volumes. The immobilized template enables efficient transcription and simplifies the purification process, as the mRNA can be easily separated from the reaction mixture by washing the beads. This method offers significant advantages in terms of scalability and ease of automation. For example, using automated magnetic bead handlers, it is possible to process up to 24 wells (total 48mL IVT) in parallel, producing up to 150mg of purified mRNA. Alternatively, scaling up in a reactor can yield up to 3g of purified mRNA in a single 1L IVT reaction.

Figure 2. RNA solid-phase synthesis cycle. The most common approaches rely on temporary protection with the 4,4'-dimethoxytrityl (DMT) group and coupling using phosphoramidite chemistry. Abbreviations: 2-cyanoethyl (CE). (Flemmich et al., 2024)

Purification Techniques

Chromatography Methods

Chromatography is a mainstream purification process widely accepted in the pharmaceutical industry due to its selectivity, versatility, scalability, and cost-effectiveness. Several chromatography techniques have been explored for mRNA purification, including size exclusion chromatography (SEC), ion exchange chromatography (IEC), and affinity chromatography.

• Size Exclusion Chromatography (SEC): SEC separates molecules based on their size and is considered the simplest form of chromatography for purifying oligonucleotides. It can effectively remove unreacted nucleotides, enzymes, short transcripts, and high-molecular-weight DNA templates from the desired IVT mRNA products. However, SEC has limitations in removing impurities of similar size, such as dsDNA.
• Ion Exchange Chromatography (IEC): IEC exploits the charge difference between the target mRNA species and different impurities. This technique is scalable, cost-effective, and allows the separation of longer RNA transcripts. Weak anion exchange chromatography has been successfully implemented to separate mRNA from IVT impurities. However, IEC must be performed under denaturing conditions, which adds complexity to the process.
• Affinity Chromatography: Affinity chromatography using oligo-dT is a widely used method for capturing mRNA by binding to its poly(A) tail. This technique produces high-purity products suitable for cGMP but has limitations in binding capacity and cost-effectiveness

Non-Chromatography Methods

Non-chromatography methods, such as tangential flow filtration (TFF), have emerged as fast and efficient alternatives for large-scale mRNA purification. TFF involves filtering and concentrating solutions containing biological molecules by flowing the liquid tangentially to the filter surface. This method can be combined with mRNA precipitation to replace traditional precipitation methods. TFF has been successfully applied in the production process of approved COVID-19 mRNA vaccines.

Process Development and Optimization

Scalable Platform Processes

Developing scalable platform processes is crucial for large-scale mRNA production. A scalable downstream platform process typically includes several unit operations, such as ultrafiltration/diafiltration (UF/DF), chromatography, and bulk drug substance filtration and fill. For example, a platform process based on a 300mL IVT reaction has been developed and demonstrated to be scalable in increments of 300mL. This process yields approximately 80% purified mRNA bulk drug substance relative to the non-scalable lithium chloride purification process. The purification operations eliminate process residuals and product contaminants, including dsRNA.

Process Optimization Strategies

Optimizing the mRNA production process involves several strategies, including improving reaction conditions, selecting appropriate purification methods, and implementing efficient downstream processing. For example, optimizing the IVT reaction conditions, such as temperature, pH, and reagent concentrations, can enhance mRNA yield and quality. Additionally, selecting the right chromatography resins and membranes for purification can improve process efficiency and scalability. Implementing in-process analytical tools, such as size exclusion chromatography (SEC) for assessing mRNA purity, can also aid in process optimization.

Challenges and Future Directions

Supply Chain and Raw Material Constraints

One of the significant challenges in large-scale mRNA production is the supply chain and raw material constraints. The availability of high-quality raw materials, such as nucleotides and enzymes, is crucial for consistent mRNA production. Addressing these challenges requires risk-based strategies, such as those recommended by the FDA Q9 Quality Risk Management Guidance. Implementing additional controls, such as release testing of incoming materials and vendor-based testing for RNase, can help mitigate these risks.

Creative Enzymes stands out as a trusted partner, offering a robust supply of GMP-grade enzymes backed by stringent quality control and regulatory support. With our expertise and reliability, we help you lay the foundation for compliant, high-yield mRNA production.

Cold Storage and Delivery Requirements

Another challenge is the cold storage and delivery requirements of mRNA products. mRNA is sensitive to degradation, and maintaining its stability during storage and transportation is essential. Developing more stable formulations and delivery systems can help address these challenges.

Innovations in mRNA Production

Future innovations in mRNA production may focus on improving scalability, reducing costs, and enhancing product quality. For example, the development of more efficient solid-phase IVT methods and novel purification techniques can further streamline the production process. Additionally, advancements in mRNA delivery systems, such as lipid nanoparticles, can improve the stability and efficacy of mRNA-based therapies.

Recommendation Products

Scaling up mRNA production introduces new levels of complexity, requiring robust processes, high-efficiency workflows, and enzymes that can perform reliably under intensified conditions. The specialized enzymes offered by Creative Enzymes are engineered to meet the demands of large-scale manufacturing—from high-throughput plasmid linearization to efficient in vitro transcription, capping, and polyadenylation. By delivering consistent performance and technical expertise, we support the scalable production of mRNA therapeutics and vaccines, helping to meet global health demands at industrial scale. Contact us today for more information and personalized assistance.