Getting IVT Right: Improving Capping Efficiency

October 30, 2024 by Clare Whitewoods

Uncapped, capped, co-capping and mRNA

Part 2 of a two-part discussion on using IVT with mRNA therapeutics

As regulatory bodies grapple with how to handle the emerging class of mRNA vaccines and therapeutics, they are placing increasing scrutiny on standards for quality, manufacture, and purity. Of particular concern to those agencies are byproducts generated during in vitro transcription (IVT), which can cause undesirable effects in the host and affect the stability and function of the mRNA itself.

In our previous post, by Kyle Studey, we addressed the challenges associated with double-stranded RNA (dsRNA) byproducts. This time, we’re looking at mRNA capping and the impact of uncapped species, along with the strategies used to reduce them.

A quick refresher on the 5’ cap
The typical structure of an mRNA consists of an open reading frame (ORF) that encodes the protein of interest, flanked on either end by the 5’ and 3’ untranslated regions (UTRs). The 3’ end contains the polyadenylated (poly(A)) tail and the 5’ end contains a cap structure.

The cap’s functions are numerous and include activating and stabilizing the transcript, preventing immunogenicity, and it is also involved in the splicing and transport of the transcripts. Given the prominent role of the 5’ cap, it is hardly surprising that improperly capped mRNA is problematic when generated during the IVT process.

Juggling effectiveness and simplicity
During the manufacture of synthetic RNA, the cap needs to be manually added to the end of the 5’ chain. There are two options to achieve this: enzymatic post-transcriptional capping or using a co-transcriptional cap analog.

In post-transcriptional capping, the uncapped IVT product is subjected to a separate enzymatic reaction to add the 5’ cap to the full-length transcript. Although somewhat effective, post-translational capping adds a separate step after the IVT reaction, and the post-transcriptional capping reaction is relatively inefficient for some transcripts.

Co-transcriptional capping is ideal in theory because it eliminates the additional reaction step, as the 5’ cap is added to the transcript during the IVT process. However, not all mRNA obtained from the IVT reaction is capped, therefore capping efficiency, or the percentage of mRNA transcript that is successfully capped during transcription, is crucial.

Pursuing simplicity isn’t always simple
The ideal scenario from a manufacturing perspective is to make use of the simplicity of co-capping while ensuring that the capping efficiency remains high, to avoid generating significant uncapped byproducts. A common approach to raising capping efficiency when using co-capping methodologies is to use a high molar excess of the cap analog to bias the capping reaction towards cap initiation, as the cap analog competes with nucleotides as the initiator component.

While this does significantly improve capping efficiency, this approach also comes with significant disadvantages. Using a large excess of capping analog is extremely costly and the competition between cap analogs and nucleotides results in reduced total mRNA.

Another approach is to simply remove the uncapped species. This can be achieved using an enzymatic reaction, but adding this to the manufacturing process removes the initial benefit of co-capping.

A new approach to simplicity
Thankfully, a new generation of RNA polymerases is tackling these issues head-on. Codex® HiCap RNA Polymerase, an engineered enzyme that improves cap incorporation, enables the simplicity of co-capping while eliminating the need for excess capping reagent. HiCap has been shown to achieve >95% capping efficiency with up to 62% less capping reagent, delivering high-yield mRNA synthesis that today’s mRNA-based vaccines and therapeutics demand.

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To learn more about how Codex HiCap RNA can help optimize IVT processes for speed, cost, and safety, take a look at our datasheet.

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