Over the past several years, mRNA therapeutics have become increasingly common. Beginning with the initial approval of Modern’s COVID vaccine, the demand for mRNA medicines to treat a variety of conditions is expanding. To keep up with this demand, drug developers must increase the scale of their mRNA therapeutic production. 

In vitro transcription (IVT) is the answer for scaling-up mRNA therapeutics. IVT synthesizes mRNA from a DNA template in vitro in the laboratory to make mRNA and can synthesize any RNA sequence. These characteristics can make IVT great for scale-up, yet this process is not as straightforward as it seems. 

When performing IVT scale-up, you must use caution throughout every step of the protocol. Increasing the amount of mRNA you produce can be challenging, and scale-up involves more than simply adding additional raw materials. If any impurities or transcription issues occur due to scale-up, then the mRNA could produce low yield or be ineffective or unsafe in a therapeutic. 

In this blog, we will explain some of the challenges of IVT scale-up and how to overcome them. 

Designing a high-quality template 

The first challenge of IVT scale-up is at the start: ensuring your input material is high-quality. Beginning with pure, high-quality raw materials will lay the foundation for building an effective mRNA therapeutic. One of the most critical raw materials for IVT is the DNA template. Small-scale IVT templates are often generated by PCR, which can serve to linearize the DNA and simultaneously add the poly(A) tail. 

For larger scale IVTs, plasmid or rolling circle-amplified DNA is generally preferred due to the large amount of DNA required. If using plasmids or rolling-circle amplified DNA, it must also encode the poly(A) tail and be fully linearized before use in IVT. Thus, it is important to consider how the mRNA will be used and therefore how much DNA template you need. Generally more scale-up requires more DNA, but excessive template DNA can lead to double-stranded RNA (dsRNA) formation, which can reduce the safety and efficacy of the therapeutic. 

Besides quantity, you need the DNA to be high quality, which involves a few factors. First, selecting an optimized DNA sequence design of your gene of choice will help you smoothly start your template preparations. In the lab, parameters will need to be adjusted for linearizing the plasmid or amplifying the region of interest with PCR, depending on the sequence. Finally, the DNA template must be purified and pass a quality check before proceeding to building IVT mRNA. 

Performing a balanced IVT reaction 

Once you have your linear, clean DNA template, you are ready to begin the IVT reaction. This presents another group of challenges to ensure a high yield, therapeutic-ready mRNA. 

For the IVT reaction, you need the correct ratio of template, enzymes, and nucleotides. This is not necessarily just increasing all components in the same ratio compared to small-scale IVT. You will need to consider the costs and benefits of each element. For example, increasing the amount of T7 RNA polymerase can improve yield, but too much polymerase can lead to dsRNA formation if the polymerase produces a runoff transcript. 

You also need to balance the length of time of the reaction. If your IVT reaction is too short, you may end up with truncated RNAs that reduce the desired therapeutic effect, but if the reaction is too long, you risk dsRNA formation that may cause immunogenicity. 

Adding the cap and tail 

Another consideration for your IVT reaction is capping and tailing your mRNA. The 5’ cap and 3’ tail improve your mRNA’s stability and ability to be translated, which are both necessary elements for successful scale-up. There are a number of ways to cap and tail an mRNA. Capping can be performed enzymatically following mRNA transcription and purification or included co-transcriptionally. Likewise for the tail, it can be encoded on the DNA template or added enzymatically after transcription.  

One benefit of co-transcriptional capping is that it allows the use of the numerous commercially available, chemically-modified synthetic cap analogs that may help increase the mRNA’s potency.  

Post-transcriptional capping and tailing involves the use of a specific cascade of enzymes. Producing the 5’ cap involves three enzymatic reactions to produce the cap0 structure as well as optional additional methylation to produce the cap1 structure common in higher eukaryotes and vaccine therapeutics. The 3’ poly(A) tail is produced via polyadenylation using poly(A) polymerase.  

Purifying your mRNA post-transcription 

Transcribed mRNA must also be cleaned, purified, and characterized to be ready for therapeutics. Using DNase, you should remove any remaining DNA template to ensure an mRNA product free of DNA contamination. You should also remove dsRNA, abortive or truncated transcripts, and other undesired elements like leftover enzymes or excess nucleotides. 

There are a number of existing methods to clean up IVT reactions including LiCl precipitation, silica membrane columns, and oligo(dT) chromatography. For large scale IVTs, oligo(dT) chromatography is often recommended because of its high throughput and strong single-step purification capabilities. Smaller scales may use precipitation or column-based methods to remove proteins and free nucleotides in addition to cellulose-based methods for dsRNA depletion. Removing these undesired parts to the transcript is critical for having effective, nonimmunogenic mRNA. 

Managing IVT scale-up challenges 

While IVT scale-up increases the ability to produce and deliver mRNA therapeutics, you must take steps to ensure you produce a successful mRNA. Eclipsebio’s eMERGE offers characterization analytics to keep your IVT scale-up on track. Using next-generation sequencing, the suite of assays in eMERGE offer nucleotide-level insights into your RNA, revealing where you need to focus to produce a strong mRNA therapeutic.  

Locate dsRNA impurities with eSENSE dsRNA, identify breakage hotspots with eSENSE Break, and confirm RNA identity and poly(A) tail length with nanopore sequencing. Through these targeted RNA insights, you can perform successful IVT scale-up and get your mRNA therapeutics to the clinic efficiently. 

References 

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