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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. 4 — a Binary reactions of single lipid components shows that only combinations including the ionizable lipid resulted in significant adduct formation with mRNA by reversed phase-ion pair high performance liquid chromatography (RP-IP HPLC). PEG, polyethylene glycol. b The RP-IP HPLC adduct profiles of mRNA extracted from the binary system (blue) and mRNA-lipid nanoparticle (mRNA-LNP, red) with the same ionizable lipid show the same qualitative peak profile. mRNA main peak (MP). c Seven different lots of the same ionizable lipid were prepared in binaries with mRNA, yielding a variety of peak profiles and abundances of adduct species in the overlaid RP-IP HPLC chromatograms. d Adduct formation in a binary reaction with a highly reactive lipid was evaluated by RP-IP HPLC at 1 day (black), 2 days (blue), and 7 days (red), showing increased tailing likely corresponding to the accumulation of multiple adducts per mRNA molecule. e Adduct formation in binary reactions was evaluated with mRNA molecules of different lengths by RP-IP HPLC at equivalent mRNA masses. An increase in late eluting-peak (LP) with length is consistent with a constant rate of modification on the single nucleotide level. f RP-IP HPLC chromatographs of 659 (red), 1106 (dark blue), and 2498-nucleotide (blue) mRNAs show an increase and left shift of each adduct peak with increasing mRNA length. g Adduct formation as a function of mRNA length was assessed by RP-IP HPLC for mRNA-LNP and corresponding binaries. A positive correlation was observed, with more adduct at longer mRNA lengths. Representative data from ≥ 3 repeat experiments are shown. AU, absorbance units.