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46 Pharmaceutical Technology ® The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com AnAly tics understand RNA stability and purity, as high‑tempera‑ ture IP reversed‑phase chromatography enables highly efficient peaks and RNA is relatively stable in the basic conditions of AEC mobile phases (30,31). Nevertheless, it remains challenging to determine peak purity due to the low ionization efficiency of large nucleic acids and low chromatographic resolution of large RNA struc‑ tures. Although chromatographic techniques, such as AEC and IP reversed‑phase, can identify some syn‑ thesis impurities, analyte and impurity adsorption to metal surfaces within the chromatography system can lead to a reduction in chromatographic efficiency and sensitivity (32). Hybrid surface technologies, such as ethylene‑bridged siloxane polymers, that reduce these effects are a welcome addition to improving chromato‑ graphic performance. Capillary gel electrophoresis is an alternative tech‑ nique that can be utilized to separate large oligonu‑ cleotides and understand DS purity. The application of diluted solutions of aqueous hydroxyethylcellulose sep‑ aration polymer can enable the resolution of large RNA fragments differing by as little as 700 nucleotides (33). Analysis of drug product mRNA, like all nucleic acids, is negatively charged and therefore incompatible with the lipid membrane bilayer of the cell. To deliver the mRNA therapeutic to its target, this incompatibility must be overcome. This is achieved by encapsulating the mRNA thera‑ peutic into a nanoparticle, which is more amenable to endocytosis or cell delivery. In addition, the lipid nanoparticle protects the mRNA from degradation post dosage. Delivery nanoparticles are composed of cationic lipids or polymers and may also contain peptides for cellular membrane targeting (4). The two registered mRNA vaccines targeting SARS‑CoV‑2 are + Retention time (min) SARS CoV-2 Spike protein mRNA eGFP mRNA Retention time (min) Absorbance (mV) Absorbance (mV) 5 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.2 10 5 2 8 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.2 FIGURE 4. After purification, mRNA is highly pure and free from DNA and nucleotide contaminants, as can be seen using IP reversed-phase conditions (5 mM tributylammonium acetate, 10–80% acetonitrile). Adapted and reproduced with permission from reference 25 under a Creative Commons Attribution 4.0 International License: https://creativecommons.org/licenses/by/4.0/