Efficient mRNA Preparation: How to Overcome the Difficulties of Precise Temperature Control and Low Shear in Very Small Volumes During the IVT Stage?

As a new generation of therapeutic drugs, mRNA has shown great potential in the fields of tumor immunity and vaccine development. However, the most critical step in the mRNA production process - in vitro transcription (IVT) - puts forward strict requirements for reaction conditions: sensitive biomacromolecules coexist with complex enzyme systems,  requiring both mixing efficiency and shear force control. When reaction volumes are scaled down to 10–200 mL for R&D or small‑scale production, achieving precise temperature control and sterile mixing becomes a real challenge for process developers.

Overview of the mRNA Process Workflow

In recent years, mRNA technology has extended beyond vaccine development to tumor immunity, gene therapy, protein replacement therapy and other fields, and is regarded as the third‑generation drug platform after small molecule and antibody drugs. However, clinical translation of mRNA products heavily depends on robust, controllable manufacturing processes. The complete production process generally comprises the following steps:

1. Sequence design and plasmid preparation: Starting from the target antigen or protein sequence, a plasmid DNA (pDNA) containing the mRNA coding sequence is designed and constructed. After amplification and linearization, the pDNA serves as the template for *in vitro* transcription.

2. In vitro transcription (IVT): The linearized pDNA template, T7 RNA polymerase, nucleotide triphosphates (NTPs), capping reagents, etc., are mixed to synthesize mRNA under specific conditions.  This is the core step determining mRNA yield and quality.

3. Purification and quality control: Impurities such as enzymes, unreacted materials, truncated products, and double‑stranded RNA (dsRNA) are removed by chromatography, tangential flow filtration (TFF), etc., ensuring mRNA purity and safety.

4. LNP encapsulation and formulation: Purified mRNA is encapsulated into lipid nanoparticles (LNPs) to form the final stable mRNA‑LNP drug product.

Pain Points and Challenges of IVT Stage: Cost, Temperature Control and Mixing

In the whole process of mRNA production, in vitro transcription (IVT) is the most critical, but also the most challenging. This enzymatic reaction system contains sensitive biological components such as mRNA molecules, T7 RNA polymerase, and NTPs, demanding very tight reaction conditions.

1. Cost Pressures: The polymerases (mainly T7 RNA polymerase) and related enzymes used in IVT are expensive. Enzyme costs account for over 90% of the total material cost in IVT [1]. The raw material cost (enzymes + substrates) for a single dose (100 μg) of mRNA is about 20 yuan at lab scale and 6–7 yuan at industrial scale. For the production of 1 billion doses of mRNA vaccine, the enzyme demand alone is valued at 6–7 billion yuan [1,2]. Therefore, precise control of enzyme usage during the R&D phase is a core strategy to reduce costs and improve efficiency—and small‑volume reactions is the direct means to control enzyme dosage.

2. Shear Force Sensitivity: Long mRNA strands are prone to breakage under shear forces generated by vigorous stirring, and enzyme activity can also be impaired by mechanical stress.

3. Contamination Risk: Even trace amounts of RNase contamination in the IVT system can lead to complete mRNA degradation, requiring extremely high cleanliness of reaction vessels.

4. Temperature Control Precision: IVT requires tight temperature control. The optimal reaction temperature for conventional T7 RNA polymerase is 37 °C; deviations reduce yield or cause failure. The formation of by‑products such as dsRNA is also highly temperature‑sensitive, and controlling other conditions at 37 °C effectively minimizes dsRNA formation. For GC‑rich or long RNA templates, high temperatures (37–52 °C) help resolve complex secondary structures, but conventional polymerases have very low activity at 50 °C. Therefore, dedicated thermostable polymerases and more accurate temperature control systems are needed to ensure reaction efficiency and product quality.

5. Homogeneous Mixing Requirements: Template, enzymes, substrates, etc., must be uniformly distributed in the reaction system to ensure reaction stability and batch‑to‑batch consistency.

Unique Challenges at Small Volumes

When the reaction volume is  scaled down to 10-200mL, temperature probes and stirring paddles of conventional bioreactors are difficult to operate effectively, while the ordinary water baths cannot provide low-shear mixing and sterile enclosed environment. How to achieve uniform mixing, precise temperature control, and sterility in an extremely small reaction system?

Figure 1: CytoLinX^® RW Single-use Rocking System

Figure 2: Schematic diagram of a typical Cellbag component

To address above challenges, the BioLink CytoLinX^® RW Single-use Rocking System introduces an ingenious design: the "Bag-in-Bag" configuration. Through the structural innovation of double-layer single-use bioreactor bags, this design perfectly solves the problems of precise temperature control and low-shear mixing in extremely small volumes:

• Precise Temperature Control to Eliminate Local Temperature Differences:

The large liquid volume in the outer bag serves as a thermal buffer, ensuring exceptionally uniform heating of the reaction system within the inner bag. Coupled with the advanced PID feedback control technology of the CytoLinX^® RW Single-use Rocking System, this configuration achieves temperature control accuracy within ±0.2°C, effectively overcoming the susceptibility of small-volume reactions to ambient temperature fluctuations.

• Low-Shear Mixing to Protect Sensitive Molecules:

Mixing within the inner bag is indirectly driven by the wave-like motion of the outer bag, requiring no mechanical impellers or gas sparging at any stage. This non-invasive, low-shear environment is exceptionally benign to the integrity of long-chain mRNA and the activity of T7 RNA polymerase.

• Completely Enclosed to Eliminate RNase Contamination:

Both the inner and outer bags are made of single-use, RNase/DNase-free materials that have been pre-sterilized by irradiation. The reaction system never comes into contact with any reusable equipment components throughout the entire process. BioLink can even provide 100% RNase/DNase testing and release, eliminating contamination risks at the source.

• Flexible Adaptability from R&D to Production:

The "bag-in-bag" design is optimized specifically for ultra-small volumes of 10 mL to 200 mL in development, while the CytoLinX^® RW Single-use Rocking System also supports a wide volume range from 0.3 L to 100 L, enabling seamless scale-up from process development to commercial manufacturing.

A Domestic Solution That Directly Addresses mRNA Process Pain Points

As mRNA technology expands further into vaccines, tumor immunotherapy, gene therapy, and other fields, the market demand for high-quality, large-scale mRNA production is becoming increasingly urgent. The IVT step, as the core of mRNA manufacturing, presents three major hurdles for process developers: high enzyme costs, stringent temperature control requirements, and a sensitive reaction mixture. During early-stage R&D, process exploration at ultra-small volumes of 10–200 mL is particularly critical, often determining the success or failure of subsequent scale-up.

BioLink's CytoLinX^® Single-use Rocking System, featuring an innovative "bag-in-bag" design, provides an ideal platform for this stage: the outer bag delivers precise temperature control, the inner bag ensures low-shear mixing, and the fully enclosed system provides aseptic assurance—turning small-volume IVT reactions into a process that is truly "small yet precise, stable and accurate."

If you have any further questions about the application of the "bag-in-bag" design of the CytoLinX® WB Cell Culture Bag in IVT, please feel free to reach out to us to discuss at any time.

Reference

1.McKinsey & Company. The mRNA vaccine supply chain – Building resilience at speed[R]. 2021.

2.mRNA疫苗商业化带来哪些投资机会？酶是价值链最大的一块 https://www.163.com/dy/article/GL2V1SM10552BZ8U.html