Boost Your mRNA QC Workflow with Hzymes’ 1.5-Hour Sample Prep Kit

Product Overview

In recent years, mRNA technology has taken center stage in biotechnology and pharmaceutical innovation, driving breakthroughs in vaccines, therapeutics, and advanced research. Its rapid rise has been fuelled by the immense potential to create personalized treatments and address previously untreatable diseases. However, the journey from in vitro transcription to a functional mRNA therapeutic involves multiple steps of fine-tuning and quality control — one of the most crucial being capping efficiency detection.

Table 2. Characterization and release testing for mRNA Drug Substance

Figure 1. mRNA drug substance characterization and release testing in USP mRNA Vaccine Quality Analysis Methods-Draft Guidelines.

Key Features

Easy to operate and User-friendly

Breakthrough one-step workflow from probe combination to RNase H digestion, the whole process only takes 1.5 hours.

Reliable results

100% mRNA cutting efficiency and high recovery rate of 5 ʹ end sequence. Combined with LC-MS analysis, it can achieve accurate analysis of the cap structure at the 5 ʹend of mRNA.

High stability of RNase H Mix

Tolerant to 37°C thermal acceleration for 45 days, tolerant to open tubes for 10 times with no effect on the enzyme digestion activity.

Ready-to-use Positive Control Template

The kit contains a positive control mRNA which is capped by co-transcriptional capping.

Product Performance

Compatible with multiple modes of administration

1. Probe design and synthesis

This kit does not contain any specific probes but is needed. The user needs to design a 14-30 nt probe complementary to the 5 ʹ end sequence of the target mRNA with 4-6 DNA bases at the 5 ʹ end or middle and the 2 ʹ -O-methyl modified RNA base, and the 3 ʹ end should carry Biotin TEG tag as shown in Figure 2.

Figure 2. Schematic diagram of the probe design.

2. Sample processing procedure

Figure 3. Hzyme’s Fast Sample treatment process within 1.5h.

3. LC-MS analysis

EGFP mRNA Capped by enzymatic method was processed by Sample Preparation Kit for mRNA Capping Efficiency Detection (Cat No. HBP005001) and analysed by LCMS to check the capping profile of EGFP control mRNA. The 2.65 min peak is the target fragment and 3.29 min peak is the probe. The large 2.65min peak area of TIC indicates that the kit has a high cleavage efficiency and recovery efficiency for sample treatment (Cat No. HBP005001).

Figure 4. HPLC-MS TIC profile of EGFP mRNA Capped by enzymatic method.

Figure 5. Target peak deconvolved molecular weight map (left) and probe peak deconvolved molecular weight map (right) of EGFP mRNA Capped by enzymatic method.

Table 1. Target deconvolution profile.

The capping rate of the EGFP mRNA capped by enzymatic method is 97.60%. Peak deconvolution of the 2.65min target sequence(left) and the 3.69min probe (right). The data indicated that the target sequence is with the correct molecular weight therefore is suitable for downstream data analysis.

EGFP mRNA positive control within the kit Capped by co-transcriptional capping was processed by Sample Preparation Kit for mRNA Capping Efficiency Detection (Cat No. HBP005001) and analysed by LCMS to check the capping profile of EGFP control mRNA. The 2.71 min peak is the target fragment and 3.37 min peak is the probe.

Figure 6. HPLC-MS TIC profile of EGFP mRNA Capped by.

Figure 7. Target peak deconvolved molecular weight map (left) and probe peak deconvolved molecular weight map (right) of EGFP mRNA Capped by co-transcriptional capping.

Table 2. Target deconvolution profile.

The capping rate of the EGFP mRNA Capped by co-transcriptional capping is 99.46%.

③ Cap 0: The methyltransferase activity of the capping enzyme adds a methyl group to the G-Cap.

④ Cap 1: Methyltransferase adds a second methyl group to Cap 0 to cap the m RNA 5'end.