(W0930-09-49) Rapid Analysis and Characterization of Lipid Nanoparticle Components for mRNA Delivery

Purpose: The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems. Incorporating the mRNA into LNP protects the mRNA from enzymatic attack and enhances cell uptake and expression. A LNP is typically composed of four lipids. The majority of a LNP is comprised of an ionizable/cationic lipid that drives potency. Cholesterol is the next most prevalent species and it enhances LNP stability. The two other lipids include a polyethylene glycol-lipid (PEGylated lipid) and a phospholipid like 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). A simple, rapid, and routine LC-MS method was developed for the characterization and analysis of LNP components using an ACQUITY™ Premier CSH C18 Column and the BioAccord™ System.

Methods: Chromatographic separation was performed using ACQUITY UPLC™ System with ACQUITY Premier CSH C18 Column (2.1x100mm, 1.7µm), equipped with a binary solvent delivery system and autosampler. The mobile phase consists of (A) ACN-Water-1M aqueous ammonium formate (600:390:10, v/v/v) in 0.1% formic acid and (B) IPA-ACN-1M aqueous ammonium formate (900:90:10, v/v/v) in 0.1% formic acid with a column temperature of 55 °C and at a flow rate of 0.4 mL/min. The column effluent was monitored using a BioAccord LC-MS System operated in positive ionization mode using the following instrument settings: capillary 1.5 kV, cone voltage 30 V, scan rate 10 Hz and fragmentation cone voltage 120-200 V.

Results: Identification and purity are both critical quality attributes of lipid materials used for LNPs and almost equally important is the monitoring and quantification of the integrity of the raw material impurities and potential degradants. The lipid ingredients for LNPs were separated using a RP ACQUITY Premier CSH C18 Column to provide improved coverage and resolution. The standard mixture of the four lipids in methanol was analyzed using the BioAccord LC-MS System. The four LNP components were eluted in the order of DMG-PEG-2000, cholesterol, ionizable lipid MC3 and DSPC. DMG-PEG-2000 forms an ammonium adduct under the LC-MS conditions with multiple charge states (+2, +3 and +4). A serial dilution of each lipid in a concentration range of 0.1-500 pg/µL was prepared and analyzed at six concentration levels to evaluate the detection limit of the BioAccord System. The lower limit of detection was determined to be 5 pg/µL (25 pg on column) for ionizable lipid MC3, DSPC and DMG-PEG-2000 and 250 pg/µL (1.25 ng on column) for cholesterol. Lipid nanoparticle ratio were prepared to mimic the actual product and analyzed. The binary comparison functionality in UNIFI™ Software was used to directly compare the analysis results of an unknown lipid sample with those of a reference sample. This is a useful feature, with the LC-MS profile of the raw lipid material or product of interest being generated, to allow differentiation from similar products, to identify uncommon impurities, or to compare the quality of lipids from multiple production batches. Bovine liver extract purchased from Avanti Polar Lipids was spiked with synthetic ionizable lipid MC3 and DMG-PEG-2000. The binary comparison plot identified only those spiked two peaks in the unknown sample compared to control. In summary, a simple, rapid, and routine reversed-phase LC-MS method was developed for the analysis of LNP composition.

Conclusion: Lipid nanoparticle component analysis using a simple, rapid, and routine reversed-phase LC-MS method.

References: 1. Vaccines 2021, 9(1), 65; https://doi.org/10.3390/vaccines9010065


(A) Extracted Ion chromatograms and (B) corresponding spectra of the four lipid components


Component summary plot showing (A) the identified lipid nanoparticles of cholesterol, cationic lipid MC3, DSPC and 14 different DMG-PEG-2000 (B) Example extracted ion chromatogram of DSPC (C) Low energy exact mass of DSPC and (D) High energy fragment ion spectrum of DSPC. The blue icon in panel D indicates matched predicted in silico and experimental fragment ions.


(A) Chromatogram binary comparison of liver lipid extract (reference) compared to a sample with additional lipids spiked into the sample (unknown). New peaks detected identified with arrows. (B) Difference plot of reference and unknown chromatograms. (C) Combined spectra binary comparison of ionizable MC3 lipid (RT 6.6 min). (D) Spectra difference plot between the reference and unknown from figure C.