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PharmTech.com The Real Message Behind Commercial mRNA Products April eBook 2023 Pharmaceutical Technology ® 45 AnAly tics as uridine and pseudouridine, that may exhibit low res‑ olution in other chromatographic conditions. Increased polarity in pseudouridine containing RNA fragments induce stronger retention to the HILIC column. A method to identify the nucleoside stoichiometric composition of an mRNA used capillary zone electro‑ phoresis (CZE) coupled with tandem MS (28). The mRNA was first digested into nucleosides and separated using a bare fused‑silica capillary and 10% acetic acid solution, pH 2.2. This was followed by tandem MS detection to quantify modified and unmodified nucleosides, which can be an orthogonal approach to RNA mass mapping using LC–MS approaches. Purity analysis of mRNA is challenging as the longer an oligonucleotide becomes, the harder it is to resolve molecules of similar length chromatographically (29). In addition to IP reversed‑phase, anion exchange has been used to determine mRNA purity using a poly‑ meric positively charged column and 10 mM sodium hydroxide to elute mRNA at pH 12 without on‑column degradation (30). Figure 4 displays a chromatogram obtained after mRNA produced by IVT was purified using a silica membrane. It shows the mRNA in a highly pure form—free from small IVT contaminants, such as free ribonucleotide triphosphates. IP reversed‑phase and AEC are optimal chromatographic modes to Retention Time (min) RNase T1 Relative intensity % 2 0 10 20 30 40 50 60 70 80 90 100 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Retention Time (min) Relative intensity % 2 0 10 20 30 40 50 60 70 80 90 100 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Retention Time (min) Relative intensity % 2 0 10 20 30 40 50 60 70 80 90 100 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 FIGURE 3. Partial RNase digests with increasing concentration of immobilized RNase T1 to digest FLuc mRNA. Lower concentrations create larger mRNA fragments, as indicated by late retention in the top total ion chromatogram. Longer mRNA fragmentsare more unique and therefore more facile to map. Adapted and reproduced with permission from reference 25 under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/