Understanding GMP Grade Vaccinia Capping Enzymes Guanylyltransferase and 2'-O-Methyltransferase

Roche’s mRNA Vaccinia Capping System comprises Guanylyltransferase GMP Grade AOF and 2'-O-Methyltransferase GMP Grade AOF. Expressed in an E. coli host, these recombinant vaccinia virus enzymes generate the 5' cap structure—a critical modification for mRNA stability, nuclear export, and translation. Previously supplied exclusively to a leading mRNA therapeutics manufacturer, these critical enzymes are now available to support your mRNA manufacturing goals.

Both Guanylyltransferase and 2'-O-Methyltransferase are characterized by the following features:

• GMP Grade
• Antibiotic-free
• Animal-origin-free (AOF)
• Recombinant, expressed in E. coli
• Extended impurities tested
• Comprehensive regulatory document package
• Scalable solution suitable for R&D, clinical, and commercial manufacturing

The 5' cap is a crucial structural feature of eukaryotic messenger RNA (mRNA). It plays a vital role in translation initiation, mRNA stability, splicing, nuclear export, and immune evasion. When manufacturing synthetic mRNA, an appropriate Cap structure needs to be introduced. The Vaccinia Capping System works sequentially, as described below, to form the Cap 1 structure (m⁷GpppNm):

1. Guanylyltransferase (GuaT): Catalyzes the transfer of a Guanosine Monophosphate (GMP) group from a donor GTP to the 5'-diphosphate terminus of RNA, forming the cap-0 structure (m⁷GpppN).

2. 2'-O-Methyltransferase (MetT): Modifies the Cap-0 structure by catalyzing the addition of a methyl group to the 2'-OH position of the first nucleotide's ribose, resulting in the desired Cap-1 structure (m⁷GpppNm). Cap-1 is critical for immune evasion and efficient translation¹.

Yes, the enzymes are tested for the absence of several contaminating activities including:

• Unspecific endonucleases 
• Nicking activity
• RNase activity
• Proteases

Current total shelf life for 2'-O-Methyltransferase GMP Grade AOF is 24 months from the Date of Manufacturing (DoM), when stored at -15°C to -25°C. 

The maximal total life for Guanylyltransferase GMP Grade AOF is 36 months from the Date of Manufacturing (DoM), when stored at -15°C to -25°C.

Our capping enzymes are capable of highly efficient capping (>= 95%). Our capping enzymes are capable of highly efficient capping (>= 95%). For more detailed information on performance, refer to this webinar.

The recommended incubation temperature for our Vaccinia Capping System is 37°C. Optimal performance reaching up to 99.9% capping was achieved at 37°C for one hour. In addition, testing by an external partner found that the minimal amount of each enzyme (GuaT and MetT) required to achieve >95% capping efficiency was 0.25 ul per 10 mg of mRNA. Refer to our webinar: evaluating mRNA capping systems.

The enzymes are highly effective in capping both non-self-amplifying mRNA (NSA) (eg., a 2kb construct) and self-amplifying mRNA (SA) (eg., a 9kb construct).

Yes, the enzymes perform well even if modified nucleotides such as N1-methylpseudo-UTP are used in the IVT reaction.

The enzymes can be utilized to cap mRNA using two primary methods:

1. Two-Pot Method: IVT is performed, followed by an intermediate purification step to remove IVT components, then the purified mRNA is capped, followed by an optional final purification.
2. Modified One-Pot Method: IVT is performed, followed by a dilution step, and then the capping compatible buffer and enzymes are added directly to the reaction vessel. This method saves time by skipping the intermediate purification step.

No, the capping enzymes themselves do not significantly contribute to dsRNA formation. The contribution to dsRNA is typically IVT process-driven² and not enzymatic capping step-driven.

Enzyme removal is typically achieved during standard mRNA purification methods such as lithium chloride precipitation, oligo-dT chromatography, or ion-exchange chromatography.

There are no specific requirements to the DNA Template sequence following e.g. the T7 Promotor, in order for the vaccinia capping enzymes to introduce a cap-1 structure.

Yes, the launched 5 mL packs can be offered for sampling purposes: submit sample request here. Other sampling options may be available, contact us for more information.

Please contact us, your Roche CustomBiotech representative will be in touch with you.

For further processing into medical devices, IVD and pharmaceutical products only

Roche’s enzymes achieved high efficiencies (>99%) in testing. When compared to other competing capping systems, our solution showed as good as, or better capping efficiency.

Enzymatic (post-transcriptional) capping generally allows for higher IVT yields because the capping process does not compete with transcription for nucleotides. This typically leads to a higher overall recovery of capped mRNA compared to co-transcriptional methods.

A comprehensive set of certificates and statements is provided to accelerate regulatory filings. Supporting documents available include, and are not limited to: 

• Certificate of Analysis (CoA)

• Material Safety Data Sheet (MSDS)

• Certificate of Origin (CoO)

• Certificate of Stability (CoS)

• Animal-Origin-Free statement

• Allergen and additive statement

• Elemental impurities statement

• Viral safety statement

Switching raw material vendors often raises concerns about regulatory hurdles and re-qualification timelines. Roche proactively addresses these challenges to ensure a seamless integration into your existing workflows. We simplify the switching process by providing the documentation, data, and samples you need to validate technical equivalence quickly and confidently.