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PharmTech.com The Real Message Behind Commercial mRNA Products April eBook 2023 Pharmaceutical Technology ® 47 AnAly tics formulated within positively charged lipid nanoparti‑ cles to enable cell delivery (1), and as of December 2021, 32 clinical trials were registered for lipid nanoparti‑ cle‑mRNA vaccines against cancer and infectious dis‑ eases (34). Within the drug product formulation, there may also be excipients, such as acetic acid, sodium ac‑ etate, potassium phosphate, sodium phosphate, or su‑ crose, for further stability (13,35). Critical quality attributes for DP formulations include the amount of the encapsulated vs. un‑encapsulated mRNA in formulation, composition, or ratio of lipid species within the nanoparticles and the size distribu‑ tion of the nanoparticles (36,37). Characterization and quantitation of lipid nanoparticle formulations em‑ ploy capillary electrophoresis (CE) or reversed‑phase chromatography coupled with UV, refractive index, multi‑angle light scattering (MALS), evaporative light scattering (ELS), and charged aerosol detectors (CAD). MALS detectors measure the light that is scattered by a sample passing through the detector whilst in solu‑ tion, whilst ELS detectors nebulize the column efflux into aerosol droplets. These droplets are dried, leaving the dried analyte to pass a detector that measures the amount of scattered light. Charged aerosol detectors are similar to ELS detectors; however, they charge the dried particles and detect them using an ion trap. These types of detector are useful for the analysis of molecules that do not absorb light, such as lipids; therefore, the benefit of using CAD lies in its ability to characterize both lipid and nucleic acid peaks within the same separation and over a larger DP concentration range (13). There is also future potential to use supercritical fluid chromatogra‑ phy (SFC) for lipid analysis, as has been demonstrated for lipidomic studies derived from biological tissues (38). Identification of lipid species of mRNA‑loaded nanopar‑ ticles using LC–CAD has been demonstrated using a C18 column and reverse phase chromatography, which facilitated the characterization and quantification of the ratio of lipids present within the nanoparticle (39). SEC‑MALS and field‑flow fractionation (FFF) have both been used to characterize loaded nanoparticle size het‑ erogeneity (37). FFF enables the separation of analytes without a stationary phase, using a perpendicular field (for example, electric or thermal) to the mobile phase flow, fractionating the solution mixture according to differing analyte mobilities under the field. In addition, FFF‑MALS is able to determine nanoparticle morphol‑ ogy, concentration, stability, and aggregation, which are also CQAs of interest (40). Drug encapsulation analysis often begins with the separation of un‑encapsulated mRNA from the nanopar‑ ticles and then comparative quantification of the free mRNA by IP reversed‑phase or AEC chromatography vs. the encapsulated fraction. IP reversed‑phase chro‑ matography has been shown to resolve lipid nanopar‑ ticles from smaller ribonucleotides using a C18‑phenyl column and dibutylammonium acetate as the IP re‑ versed‑phase ion‑pairing agent (41). This approach could also be utilized for lipid nanoparticle‑mRNA formulations and may also provide an indication of stability. Stability‑indicating methods are crucial for DP characterization to ensure medicine quality over long periods of time. IP reversed‑phase chromatog‑ raphy has been employed to analyze the stability of mRNA up to 1000 nucleotides in length using 0.1 M triethylammonium acetate within the mobile phase (and acetonitrile as organic solvent) and a polymeric reverse‑phase stationary phase (31). Under a range of stress‑inducing conditions, degradation products were quantified and relative stability was evaluated. Conclusions The diversity of the analytical methods used to evalu‑ ate mRNA CQAs shows that chromatography plays a leading role in the purification and characterization of mRNA therapeutics. It is poignant to consider that mRNA therapeutic technologies are continually being advanced and developed. With future advancements in the structure and chemistry of mRNA therapeutics, we will continue to require separation techniques to resolve analytes and critical impurities. In addition, effective delivery of mRNA therapeutics to target tissues remains a challenge, which will also lead to further technologi‑ cal advancement of delivery vehicles, thereby adding to the complexity in developing accurate analytical meth‑ ods. What is exciting is the range of detection methods available for hyphenation in separations of complex mixtures, such as formulated drug product. Chromato‑ graphic stationary phases are also being developed to address more complex and challenging separations. With regard to mRNA and other large nucleic acids, it is clear that low resolution of similarly sized oligonucle‑ otides needs to be addressed—possibly by using novel stationary phases and chromatographic conditions. The wide interest in developing analytical methods for mRNA therapeutics is demonstrable from the wealth of literature, and chromatography will continue to remain crucial for purification and analysis of DS and DP. Key Points • The mRNA therapeutic market is expected to reach a value of $101 billion by 2026. • Chromatography is a common approach for critical quality attribute characterization and quantification. • Mass spectrometry-based detection is difficult due to large mRNA size, thus enzyme degradation is employed in sample preparation. • Advances in stationary phases and chromatographic conditions could improve resolution challenges.