Product Overview

Sufficient length of poly (A) tail is required to maintain mRNA stability and translation efficiency, and therefore, poly (A) tail length is a key quality attribute of mRNA vaccines. The mRNA poly (A) Tail Length Test Sample Preprocessing kit allows efficient and reproducible processing of mRNA samples that detect poly (A) tail length for LC-MS analysis of the 3' end sequence. The kit contains preformulated 10x RNase T1 Mix, detergent, eluate and filler and is ready for sample preparation within 1 hour. The poly (A) tail is cleaved from mRNA using RNase T1 enzyme, and then the average length of released poly (A) tail is analyzed by size-exclusion chromatography and high-resolution ion-pair reversed-phase liquid chromatography (IP RP LC) can be used to evaluate the size heterogeneity of the poly (A) tail.

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

Product Advantages

Performance Data

Sample Pre-processing

Incubate 100pmol of mRNA at 95°C for 5min and then quickly cool down to 25°C; Add RNase T1 digestion mix and incubated for 15min at room temperature; After digestion, perform purification with oligo dT binding. The optimized purification and elution system can stably recover the mRNA 3'end poly (A) tail fragment, which is ready for LC-MS analysis. The whole process can be completed within 1 hour.

LC-MS analysis

The treated samples were subjected to LC-MS determination to analyze the poly (A) tail length distribution. The retention time of target component 30A was 3. 25min and 70A was 3. 58min in sample 1. The TIC profiles are shown in Figure 2, and the deconvolved molecular weight maps of sample 1 at different retention times are shown in Figure 3, 4. MS result matched the targets in sample 1, and the MS matching table is shown in Table 1-2.

Figure 2. The TIC profile was detected by LC-MS of sample 1. Sample 1 was analyzed by liquid mass to isolated two peaks.

Figure 3. The TIC profile was detected by LC-MS of sample 1. Different amounts of PolyA were identified after deconvolution result analysis of the peaks with 3. 25min retention time. Based on the molecular weight results, the PolyA distribution of sample 1 is 29A-34A, which matches the theoretical sequence.

Figure 4. The TIC profile was detected by LC-MS of sample 1 Sample 1 retention time 3. 58min deconvoluted the molecular weight map. Different amounts of PolyA were identified after deconvolution result analysis of the peaks with 3. 58min retention time. Based on the molecular weight results, the polyA distribution of sample 1 was 63A-81A corresponding to the theoretical sequence.

Table 2. Nucleotide sequence matching of sample 1 target component 70A and PolyA length distribution.