Fig. 1: Identification of LP in formulated mRNA-LNP by RP-IP HPLC.

a Reversed phase-ion pair high performance liquid chromatography (RP-IP HPLC) provides high resolution mRNA length-based separations to assess content and quality of mRNA products, as shown by the separation of 6 mRNAs of different lengths (659, 785, 914, 1106, 2498, and 2993 nucleotides) in a single lipid nanoparticle (LNP) formulation across retention times of 9.5–15 min. Peaks prior to 9.5 min are degradants and the peaks at 22 min form part of the column wash. b The RP-IP HPLC analysis of pure mRNA (blue) yields a single MP (retention time 15 min), with shorter degradation products and impurities eluting prior (retention time, 10–14.5 min), whereas mRNA extracted from an mRNA-LNP formulation (black) yields an additional late-eluting peak region (LP; retention time 19–21 min). The UV spectrum at each peak apex obtained from an on-line 3D UV detector shows an identical profile with maximum absorbance of 260 nm (inset). c CE analysis of extracted mRNA shows a single peak, with no additional late-eluting species. d LP species in mRNA extracted from an mRNA-LNP formulation can be further resolved with adjusted gradient conditions and show a polydisperse fingerprint of species. e An mRNA-LNP formulation was stored for 3 months at 4 different temperatures (40 °C, red; 25 °C, blue; 5 °C, green; −20 °C, orange), and sampled at 1, 2, and 3 months for analysis by RP-IP HPLC; each data point is a single incubation condition run in a single RP-IP HPLC assay. Representative data are shown. AU, absorption units.