Abstract
Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.
Highlights
Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic
This provides a similar separation to capillary electrophoresis (CE), which is driven by size and charge; based on the ion pair system used, Reversed phase ion pair chromatography (RP-IP) retains some selectivity to variations in mRNA hydrophobicity due to sequence or chemical modifications[24]
When RP-IP HPLC integrity analysis was applied to mRNA extracted from an mRNA-LNP, a late eluting-peak (LP) was detected by HPLC (Fig. 1b) that was not observed by CE (Fig. 1c)
Summary
Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. The effective delivery of mRNA-based vaccines and therapies is enabled by the use of lipid nanoparticles (LNPs), which protect nucleic acid degradation by exo- and endonucleases[7,8] and facilitate cellular uptake and expression[9,10] Used in both the Pfizer/BioNTech and Moderna COVID-19 mRNA vaccines, this delivery system is effective as it leverages LNP surface properties[11,12,13,14], the ability of LNPs to facilitate endosomal escape through ionization of the amino lipid[15,16], and tissue-specific mRNA delivery based on particle size[17]. These data can inform manufacturing protocols to limit the formation of lipid-mRNA adducts and ensure the high quality of nucleic acid-based products
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