Listicle: Five tips on scaling up IVT for mRNA production

In this listicle, Thermo Fisher Scientific outlines five measures to help you ensure consistency in your workflow and produce reproducible, high-quality batches of IVT mRNA at scale.

The success of messenger RNA (mRNA) vaccines in combatting the global SARS-CoV-2 crisis brings a new era of vaccine development. Applications of in vitro transcribed (IVT) RNA in mRNA therapeutics have exciting implications for modern medicine, as the ability to easily change mRNA sequences provides a flexible platform to create robust therapeutics against a wide variety of chronic and infectious diseases.

IVT is a complex reaction, requiring a mixture of RNA polymerase and supplementary components (e.g., RNase inhibitor, inorganic pyrophosphatase), nucleoside triphosphates (NTPs), and a DNA template, to make each mRNA. There is not a single solution for optimal RNA yield, as it depends on a series of reaction variables and is sequence dependent.

Consider these five measures to help you ensure consistency in your workflow and produce reproducible, high-quality batches of IVT mRNA at scale.

1. Recognise that raw materials matter

You can help ensure a seamless transition to production scale by using raw materials suitable for scaling up. When beginning to scale up, it’s a good idea to move to materials suitable for GMP manufacturing early, meaning process development is completed with the same raw materials and formulations, allowing a quick transition to GMP manufacturing. The early use of GMP-quality materials minimises the need for the costly and time-consuming testing, auditing, and documentation needed for changing materials.

Figure 1: Scaling up IVT reactions for mRNA production

Designed with scale, quality, consistency, and regulatory needs in mind, Thermo Scientific TheraPure GMP* standard enzymes and nucleotides (Figure 2) are readily available for IVT reactions, providing an excellent starting point to enable seamless transition from early-stage mRNA production to scale-up for commercial manufacturing using GMP-quality materials.

* “TheraPure GMP” refers to the quality level of the raw, ancillary, or starting materials to be used for further manufacturing. TheraPure GMP products are manufactured in facilities with ISO 9001–certified quality management systems that operate in accordance with relevant good manufacturing practice (GMP) principles, as outlined in ICH Q7 or equivalent guidance documents or standards.

Figure 2: TheraPure GMP products for mRNA production 

2. Optimise IVT reaction parameters for yield and efficiency

There is much attention and effort on developing conditions to maximise the yield and efficiency of IVT reactions used to produce mRNA. For example, increasing IVT reaction time (Figure 3) and optimising temperature can help to maximise yield and product quality. Temperatures both lower and higher than 37 degrees celsius have been shown to improve mRNA quality. Regardless of the optimal reaction temperature, it is important to maintain a constant temperature throughout the IVT reaction volume, to minimise generating truncated transcripts.

Figure 3: Yield of RNA of varying lengths at different reaction times

Furthermore, different buffer conditions and nucleotide concentrations can impact the IVT reaction (Figure 4) and, subsequently, mRNA yield and purity. Also keep in mind that optimisation is not linear and can be process- and product-dependent. Starting with single components rather than premixed solutions will allow you to test a range of conditions for optimisation. It’s also worth noting that RNA polymerases from different suppliers often require additional optimisation, which may also increase your time investment and costs.

Figure 4: Determining buffer components impacting IVT. In the context of this analysis, LogWorth values larger than 2 are significant

3. Secure a sufficient supply of DNA, or consider going synthetic

The DNA template is a critical raw material for mRNA production. Due to increased demand for plasmid manufacturing for mRNA and gene therapy applications, supply may be limited, especially for GMP-grade plasmid DNA. Before you begin to scale up, you must ensure a sufficient and steady supply of your DNA template for your desired output. At Thermo Fisher Scientific, Patheon Pharma Services offers CGMP-grade plasmid DNA for all stages of development and commercialisation, with capacity and flexibility to scale with you.

Alternatively, you could choose from synthetic options for generating plasmid DNA, such as rolling-circle DNA amplification approaches or Invitrogen GeneArt plasmid construction and purification services, to mitigate against supply chain issues.

4. Consider a modular approach for flexible scale, or automation where possible

The ability to adapt a manufacturing production process can provide flexibility, allowing you to scale up or down according to your need without extra expense or time commitment. Rather than working towards a large-capacity process, it is worth considering a modular approach, using the same technology suitable for all required volumes, from microliter to liter scale. This can cut manufacturing costs and minimise your footprint.

Manufacturing is a cost-sensitive process. Automatable workflows can help reduce strain on staffing, which could prove to be a bottleneck once you begin to scale. Although automation can require an initial outlay to acquire capital equipment, it’s worth considering as it could reduce overall costs as well as potentially reduce turnaround times and increase productivity.

For synthesis and purification of mRNA, Thermo Fisher offers the Invitrogen Dynabeads platform (Figure 5) to enable a modular manufacturing approach with flexible manual or automated workflows.

Figure 5: Invitrogen Dynabeads Streptavidin for in vitro transcription and Dynabeads Carboxylic Acid for RNA purification

5. Speak to your dedicated technical team

Working with a supplier who is experienced in not just IVT but also in IVT process development and scale-up can help accelerate your project significantly. As many companies are new to mRNA production, the value and availability of local, experienced technical support and partnership is more important than ever. Working with a supplier like Thermo Fisher, you’ll gain access to a team of technical professionals who can proficiently support you throughout your process development and scale-up process.


The main challenge for efficient and effective mRNA process development is a lack of standard methodologies and practices. Thermo Fisher is committed to offering standard IVT products backed by quality, experience, and technical support for consistent, robust, and reproducible mRNA production. This means you can scale mRNA production as required while maintaining consistent product quality, and reap rewards with rapid acceleration in the development of future mRNA therapeutics.

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Intended use of the products mentioned varies. For specific intended use statements, please refer to the product label or contact Thermo Fisher Scientific.

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