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. 2021 Nov 22;12:6777. doi: 10.1038/s41467-021-26926-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Isolated main peak (MP) and late eluting-peak (LP) alongside control mRNA-lipid nanoparticle (mRNA-LNP) and an assay positive control were tested for in vitro protein expression in BJ fibroblasts after 48 h. RNA was extracted from 2 hEPO-LNP formulations by isopropanol precipitation and purified by RP-IP to generate MP and LP prior to transfection. The assay control is a pure hEPO mRNA standard, the control sample is mRNA extracted from each formulation prior to RP-IP separation, and the MP and LP are isolated fractions. Results from 2 replicate transfection wells is plotted with the mean. b Five different mRNA-LNP samples were prepared using different ionizable lipids and incubated at 5 °C to generate varying levels of adduct and degradation. RNA was extracted from the mRNA-LNP by isopropanol precipitation and evaluated by reversed phase-ion pair (RP-IP), capillary electrophoresis (CE), and in vitro protein expression in HeLa cells as measured by mean fluorescence intensity. Relative expression as a percentage of the neat mRNA expression is plotted versus relative integrity as a percentage of the neat mRNA integrity, determined by relative area using both CE and RP-IP HPLC. c Loss in mRNA purity to adduct formation in 2 vaccine formulations is plotted over 3 months at refrigerated conditions. Poor process control led to high LP in Vaccine A, but adduct was well-controlled in Vaccine B. Representative data are shown for 3 repeat experiments for Fig. 6a and multiple repeat experiments for Fig. 6b and c.