In this technical note, the comprehensive characterization of impurities from the ionizable lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-(dimethylamino)butanoate, commonly known as DLin-MC3-DMA (MC3), is presented. Deep structural elucidation, including the localization of different oxidation products and saturation of double bonds in MC3, was achieved using EAD.
Lipid nanoparticles (LNPs) that are comprised of ionizable lipids are used to deliver oligonucleotides to work as therapeutics or to stimulate the immune system, as in the initial mRNA-based COVID vaccines. A recent study reported that N-oxidation of ionizable lipids might lead to covalent modification of ribonucleotides and a loss of mRNA potency.1 To ensure product quality, detailed and sensitive characterization of the ionizable lipid and its related impurities is necessary. However, obtaining the level of detail needed is challenging with current liquid chromatography-mass spectrometry (LC-MS)-based methodologies. Collision-induced dissociation (CID) provides head group and acyl or alkyl chain sum composition information but does not provide structural details. Data from alternative fragmentation techniques can help localize double bond positions within acyl or alkyl chains but alternative methods suffer from inefficient fragmentation, especially for singly charged species. They require long duty cycles or collision cell modifications to allow for the introduction of ozone. Only EAD has efficiently provided complete characterization of different naturally occurring lipids in a single LC-MS run.
Here, the applicability of this novel fragmentation mode for the detailed characterization of lipids used for LNPs was tested using MC3 and its related impurities as a model. Within a single experiment, the exact locations of oxygen incorporation of 2 isomeric species and the double bond reduction of another related impurity were pinpointed using the unique fragment ions produced by EAD. This information can be used to determine drug efficacy and safety from formulated LNPs. Additionally, it can be used to aid rational design of new synthetic lipids.
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