Identifying Key Factors Affecting mRNA-Lipid Nanoparticles Drug Product Formulation Stability

TL;DR

This study identifies key factors affecting the long-term stability of mRNA-lipid nanoparticle formulations, emphasizing the importance of cold storage and optimized components.

Contribution

The study systematically evaluates LNP stability under various storage conditions and identifies specific degradation mechanisms.

Findings

Storage at 5 °C and 25 °C caused significant degradation and loss of transfection efficiency in LNP formulations.

Oxidative and hydrolytic lipid degradation were identified as primary failure modes in specific LNP formulations.

Increasing Tris buffer concentration accelerated mRNA 5′-cap hydrolysis, highlighting the need for low-ionic-strength buffers.

Abstract

Background: The long-term stability of mRNA-lipid nanoparticles (LNPs), essential for mRNA vaccines and gene therapies, relies on managing physicochemical properties to preserve their integrity and effectiveness through optimized formulation components. This study systematically evaluated LNP formulations with varied compositions, e.g., Dlin-MC3-DMA and ALC-0315 as ionizable lipids, and DMG-PEG2k or ALC-0159 as polyethylene glycol (PEG)-lipids, stored at −80 °C, −20 °C, 5 °C, and 25 °C in Tris buffer (pH 7.4) for 12 months. Methods: Sixteen quality attributes were analyzed, including particle size, mRNA encapsulation, lipid oxidation, and transfection efficiency over different formulations and storage temperatures to mechanistically evaluate the long-term stabilities. Results: Formulations stored at −80 °C and −20 °C retained acceptable stability, while storage at 5 °C caused aggregation, reduced in vivo expression, and mRNA degradation. Storage at 25 °C led to complete loss of transfection within six months. Mechanistic studies identified oxidative and hydrolytic lipid degradation (e.g., DSPC) in ALC-0315 formulations and MC3 N-oxidation with subvisible particulates in MC3-containing LNPs as primary failure modes. Increasing Tris buffer concentration accelerated 5′-cap hydrolysis, emphasizing the importance of a low-ionic-strength buffer for LNP formulations. Conclusions: Findings re-emphasize the necessity of deep-cold storage (≤−20 °C) and optimized formulation components to preserve mRNA–LNP integrity, offering insights for designing next-generation LNPs with improved shelf-life.

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1. Introduction

mRNA–LNPs vaulted to the clinic with two pandemic-era vaccines (Comirnaty^®^ and Spikevax^®^) and a recently launched respiratory syncytial virus (RSV) LNP vaccine product (mRESVIA^®^). Despite success in vaccine application, mRNA–LNPs’ broader therapeutic use beyond vaccine, including protein replacement therapy [1] and genetic engineering [2,3], is still constrained by several factors, of which one is its unusually complex stability profile [4,5,6]. At the molecular level, the mRNA payload is inherently fragile and can be degraded through ribose-mediated backbone cleavage, endo- and exonucleases, nucleobase oxidation, and 5′-cap hydrolysis even at refrigerated temperatures [7]. Encapsulation by LNPs mitigates but does not eliminate these pathways of mRNA degradation. In fact, this mRNA encapsulation introduces new liabilities because ionizable and helper lipids used in the formulation of LNPs can autoxidize into aldehydes or peroxides that, in turn, form covalent mRNA-lipid adducts, leading to potency loss as well as raising safety concerns [8]. Structurally speaking, LNPs are self-assembled particles held together by weak non-covalent forces; thus, environmental stresses such as freeze–thaw, agitation, or shifts in pH and ionic strength can promote fusion, aggregation, or premature mRNA leakage, each of which compromises cargo integrity while reducing cellular uptake and transfection efficiency.

Formulation excipients play a critical role in helping to mitigate these risks. For instance, recently we have shown that tris buffers paired with sucrose outperform conventional Phosphate-Buffered Saline (PBS) or low-cryoprotectant systems, probably by damping pH excursions and ice-crystal stress during freezing, whereas sub-optimal cryoprotectant levels permit aggregation and hydrolysis [9]. At the same time, the manufacturing process (such as mixing, buffer exchange steps, etc.) adds another layer of complexity to the LNP stability. For example, microfluidic mixing at certain conditions and lipid types potentially produces solvent cavities and blebs whose prevalence affects long-term colloidal stability, though there may be improvement in endosomal escape and potency of LNP products at short-term storage, as reported in a few recent studies [10,11,12]. Other factors affecting LNP stability include storage temperature, light exposure, and agitation. Deep-frozen conditions (such as −80 °C or −60 °C) minimize chemical degradation. Yet, the conditions can trigger LNP fusion or aggregation upon thawing; −20 °C is logistically attractive but may allow slow oxidation of lipids; 5 °C simplifies distribution while accelerating hydrolysis; finally, ambient temperature rapidly erodes both physicochemical characteristics and in vivo potency. Recognizing these interwoven failure modes, regulatory agencies now call for orthogonal stability-indicating assays. Among these assays, nano- and micron-sized particle concentration and morphology images, robust liquid chromatography (LC) or mass spectrometry-based lipid-degradant and reactive oxidized species profiling, LC or capillary electrophoresis of mRNA purity, and proper functional read-outs, to name a few, can supplement standard physicochemical stability analyses, such as size distribution and encapsulation efficiency [13].

Since the first emergency authorizations, many groups have tackled isolated aspects of this problem for mRNA–LNP drug products, but there is very limited information on a head-to-head, long-term storage comparison of multiple clinically relevant lipid types under rigorously controlled conditions. We, therefore, executed an evaluation of storage stability up to one year with formulations containing diverse LNP combinations, containing DLin-MC3-DMA or ALC-0315 as ionizable lipids, and DMG-PEG2K or ALC-0159 as PEG-lipids formulated in an optimized tris/sucrose buffer. LNPs manufactured by microfluidic mixing were stored at −80 °C, −20 °C, 5 °C, or 25 °C and were analyzed using a comprehensive panel of testing, including physicochemical attributes such as lipid oxidation, mRNA integrity, nano-sized and subvisible particle counts, and in vitro and in vivo transfections.

This study evaluates pre-administration drug product shelf-life stability and does not assess post-dosing intracellular persistence, RNA turnover pathways, or clinical safety outcomes. Overall, the results indicate that the lipid and mRNA degradation correlate to particle aggregation and loss of function, and further highlight the impact of buffer strength and cryoprotectant concentration affecting freeze–thaw, short-term and long-term stability. Further, by correlating the quality attributes of mRNA–LNP to loss of in vitro and in vivo transfections, this study intends to provide a better understanding on design and selection of suitable formulation composition and storage conditions capable of sustaining mRNA-LNP efficacy during shelf-life storage.

2. Materials and Methods

2.1. Materials

Lipids, including cholesterol (CAS# 57-88-5), DSPC (CAS# 816-94-4), ALC-0159, ALC-0315, and DMG-PEG2k (CAS# 160743-62-4), were procured from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). DLin-MC3-DMA (CAS# 1224606-06-7) was obtained from BioFine International Inc. (Blaine, WA, USA). CleanCap^®^ firefly luciferase (FLuc) mRNA (CAS# L-7602) was acquired from TriLink BioTechnologies (San Diego, CA, USA). Absolute ethanol was procured from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Quant-it^™^ RiboGreen RNA Assay Kit, PBS, sodium citrate, molecular biology grade water, and other reagents were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA), unless otherwise mentioned. The Luciferase Assay System was bought from Promega Corporation (Madison, WI, USA). Other general chemicals and reagents were procured from VWR International (Radnor, PA, USA).

2.2. Cell Culture

HEK-293 cells (CRL-1573) procured from ATCC (Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, CAS# 2025-01-30). The media was supplemented with 10% fetal bovine serum (heat-inactivated) and 1% antibiotics, to maintain the cells in the presence of 5% CO_2_ at 37 °C.

2.3. Manufacturing of RNA-LNPs Using NanoAssemblr® Ignite

2.3.1. Cryoprotectant Concentration Screening Following Freeze–Thaw

LNPs encapsulating Fluc mRNA (FLuc mRNA–LNPs) were manufactured using the NanoAssemblr^®^ Ignite^™^ system through a microfluidic mixing approach (Precision NanoSystems Inc., Vancouver, BC, Canada). A freeze–thaw study was conducted to screen different concentrations of sucrose as a cryoprotectant in the LNP formulation. FLuc mRNA–LNPs composed of four lipids, namely DLin-MC3-DMA, Cholesterol, DSPC, and DMG-PEG2k (molar ratio 50/37.5/10/2.5) were prepared. Lipid solution in ethanol at a total lipid concentration of 50 mM, and FLuc mRNA at the target concentration in 10 mM sodium citrate buffer, pH 5.0, were used. LNPs were manufactured by mixing the lipid solution and FLuc mRNA solution at an optimized flow rate ratio of 1:3, while the total flow rate used was 12 mL/min. Obtained LNPs were diluted with sucrose (0–20% w/v) containing PBS buffer, followed by buffer exchange via ultracentrifugation (100 kDa molecular weight cut off, MWCO) (Millipore, Burlington, MA). LNP formulations were subjected to sterile filtration, filled in Schott 2R glass vials, and exposed to one overnight freeze–thaw cycle (−80°). Section 2.4 illustrates the analytical characterization performed on the formulated FLuc mRNA–LNPs.

2.3.2. Lipid Combinations and Formulation Buffer Screening Following the Freeze–Thaw Cycles

After identifying an optimized sucrose content, various concentrations of Tris buffer were screened and compared with 1× PBS to narrow down the formulation buffers that could protect the drug product quality following two freeze–thaw cycles (1st cycle overnight, and 2nd cycle 2 h incubation at –80 °C before thawing the samples for 30 min at room temperature). NanoAssemblr^®^ Ignite^™^ system (Precision NanoSystems Inc., Vancouver, BC, Canada) was used to formulate FLuc mRNA–LNPs containing various lipid combinations (Table 1). All five formulations (F1, F2, F3, F4, and F5) comprise an ionizable lipid/DSPC/cholesterol/PEG–lipid combination at a molar percent ratio of 50/10/37.5/2.5. These formulations were prepared using similar mixing conditions as described in Section 2.3.1. After microfluidic mixing, LNPs were diluted with either of the two buffers: 1× PBS or Tris (5, 10, 20, 30 mM) buffer, both containing 10% w/v sucrose, at pH 7.4. Diluted LNPs were subjected to ultracentrifugation for buffer exchange, and then sterile filtered to fill into 2R vials. FLuc mRNA–LNPs were characterized using different analytical techniques as mentioned in Section 2.4.

2.3.3. Long-Term Storage Stability

FLuc mRNA–LNPs were tested for long-term storage stability at different temperatures (−80 °C, −20 °C, 5 °C, and 25 °C) by incorporating the optimized formulation buffers and sucrose content from the aforementioned studies. FLuc mRNA–LNPs containing various lipid combinations were manufactured using microfluidic mixing as described in Section 2.3.1. Resultant LNPs were diluted with either of the two buffers: 20 or 30 mM Tris buffer containing 10% w/v sucrose, pH 7.4. Buffer exchange was performed, and LNPs were filtered by using a 0.2 µm syringe filter, followed by filling into Schott 2R glass vials (SCHOTT Pharma USA, Inc., Lebanon, PA, USA). Analytical characterization for all LNP formulations was performed as mentioned in Section 2.4.

2.4. Analytical Characterization of mRNA–LNPs

Drug product (DP) quality was evaluated by assessing the Quality attributes (QAs) of Fluc mRNA–LNPs as stated in Table 2.

2.4.1. Visual Inspection and pH

Visual appearance of all the prepared formulations was assessed against a black background for their clarity, color, and visible particulates. pH value of the FLuc mRNA-LNP drug product was measured using a pH meter (Mettler Toledo, Columbus, OH, USA). Visual inspection and pH measurement of LNP formulations during long-term storage stability were performed on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C).

2.4.2. Particle Size, Polydispersity Index, and Zeta Potential

Size and surface charge measurements of LNPs were measured using Malvern Zetasizer Ultra Red. Samples were diluted 10 times with sterile filtered 0.1× PBS pH 7.4 and transferred into a folded capillary cell (DTS1070). Measurements were performed in triplicate at 25 °C (refractive index of 1.45 and viscosity of 0.88 cP) using a 173° backscatter angle of detection. Particle size, polydispersity index, and zeta potential of LNP formulations on long-term storage stability were measured on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C). For quality control of dynamic light scattering (DLS) size measurement by Zetasizer instrument, the 100 nm polystyrene nanoparticles, calibrated by NIST traceable standards, were used (Malvern Panalytical, Chipping Norton, NSW, Australia, LTX3100A). For Zeta potential quality control, the zeta potential transfer standard with −40 ± 5.8 mV was used prior to each measurement (Malvern Panalytical, ZTS1240).

2.4.3. Particle Concentration

The nanoparticle tracking analysis (NTA) technique was employed to measure the LNP particle concentration using ZetaView^®^ (Particle Metrix Inc., Mebane, NC, USA). Prior to the measurements, samples were diluted with 0.1× PBS buffer and injected into the cell to avoid any air-bubbles. Sample dilutions were adjusted to maintain between 100 and 500 particles per frame (11 positions, 85 sensitivity) for accurate results. Particle concentration for LNP formulations on long-term storage stability was assessed on day 0 and the following one year at different storage conditions (−80 °C, −20 °C, 5 °C, and 25 °C).

2.4.4. Encapsulation Efficiency and mRNA Concentration

Quant-IT^™^ RiboGreen RNA Assay Kit (Invitrogen^™^) was used to evaluate the encapsulation efficiency percentage (EE%) and total/free mRNA concentration present in FLuc mRNA–LNP formulations. Standard curves of FLuc mRNA (50–2000 ng/mL) were generated in 10 mM Tris/1 mM EDTA (TE) buffer and 2% w/v Triton x-100/TE (TR) buffers. To determine the free mRNA concentration, LNPs were diluted 10× in the TE buffer, and for total mRNA concentration evaluation, LNPs were diluted 25× in the TE buffer followed by a 1:1 dilution with the TR buffer. The plates were incubated at 37 °C for 10 min to allow LNP lysis for mRNA quantification. RiboGreen assay was then performed by adding the reagent to each standard/sample, and the fluorescence (Ex485/Em528) was measured using a plate reader (Synergy Neo2, Biotek, Winooski, VT, USA). The encapsulation efficiency in LNPs was calculated using the following formula:

$$[eqn]$$

Encapsulation efficiency and mRNA concentration analysis for FLuc mRNA–LNP formulations on long-term storage stability were determined on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C).

2.4.5. Osmolality

The osmolality of LNPs was measured using the VAPRO^®^ Vapor Pressure Osmometer (ELITechGroup Inc., Logan, UT, USA) by following the manufacturer’s instructions. Briefly, a sample disk was placed in the sample holder, and 10 µL of the LNP solution was added onto the disk. The Open/Close key was pressed to have the sample placed in the measurement position. This allows automatic measurement of the sample osmolality. At least 3 measurements of each LNP sample were performed to consider an average value with an appropriate standard deviation. Osmolality of FLuc mRNA–LNP formulations subjected to long-term storage stability was measured on day 0 and the following one year at different storage conditions (−80 °C, −20 °C, 5 °C, and 25 °C).

2.4.6. Microchip Capillary Electrophoresis (MCE) for mRNA Purity Analysis

FLuc mRNA and FLuc mRNA–LNP samples were diluted with 2% Triton x-100 to a final concentration of 5 µg/mL for further analysis. First, 80 µL of 1× Sample Buffer present in the RNA Pico Sensitivity Assay Kit (Perkin Elmer, Shelton, CT, USA) was added per well in a 96-well plate. Samples were denatured by heating in a 70 °C heating block followed by snap cooling on ice for a few min. RNA Pico ladder, gel-dye solution, and RNA LabChip^®^ were prepared by following the manufacturer’s protocol. The RNA LabChip was thoroughly cleaned, and the gel-dye solution and RNA Pico marker were then added to the appropriate wells. The assay was run using the instrument, LabChip^®^ GXII Touch^™^ (Perkin Elmer, Shelton, CT, USA). Electropherograms for FLuc mRNA and FLuc mRNA–LNP samples were evaluated by calculating the main peak and fragmentation as a percentage of the total peak area. The purity and integrity of FLuc mRNA encapsulated within LNP formulations on long-term storage stability were determined on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, 25 °C).

2.4.7. Particle Morphology Using Cryogenic Transmission Electron Microscopy (Cryo-TEM)

Cryo-TEM was used to determine the effect of freeze–thaw and storage temperature on the LNP’s morphology. Cryo-TEM analysis was performed for LNP formulations stored at different temperature conditions (−80 °C, −20 °C, 5 °C, and 25 °C), specifically at day 0 and following one month as well as one year of storage. Briefly, 3 µL of LNP solution was applied onto the mesh copper grids (Electron Microscopy Sciences, Hatfield, PA, USA), blotted, and vitrified by plunging into liquid ethane. Following vitrification, the grids were transferred to the autoloader of the microscope under liquid nitrogen for capturing images using a Glacios^™^ Cryo-Transmission Electron Microscope (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured at multiple scales for each grid by targeting the area of interest at lower magnifications (940x–8500x) before acquisition of movies at nominal magnifications of 28,000× (0.5 nm/pixel), 73,000× (0.2 nm/pixel), and 150,000× (0.1 nm/pixel), while highest magnification images (150,000x) were acquired with an electron dose between 12 and 25 e^−^/Å^2^.

2.4.8. mRNA 5′-Capping Integrity

The 5′-capping integrity of FLuc mRNA encapsulated within LNP formulations subjected to long-term storage stability was determined following one year at different storage conditions (−80 °C, −20 °C, 5 °C, 25 °C). For that, first the LNPs were treated with 2% triton-X100 to release the mRNA, then the released FLuc mRNA was analyzed using enzymatic treatment of mRNA–LNPs with a subsequent size-exclusion ultra performance chromatography (SE-UPLC) analysis. For enzymatic digestion of 5′-capping a sequential digestion using 5′ polyphosphatase (LGC Biosearch Technologies, Middlesex, UK, cat # RP8092H) and terminator 5′ phosphate-dependent exonuclease (LGC Biosearch Technologies, cat # TER51020) were used as previously described [7]. Following this enzymatic digestion, the mRNA with 5′ cap remained intact while uncapped transcripts were readily digested. Next, the intact transcripts and degraded mRNA fragments were resolved by an SE-UPLC method, and the percentage of capped mRNA was calculated using the peak areas by the following equation:

$$[eqn]$$

2.4.9. Subvisible Particles by Micro-Flow™ Imaging (MFI)

MFI measurements were performed with an MFI 5100 instrument featuring a flow cell depth of 400 μm (ProteinSimple). LNP samples were diluted 20-fold in a 0.2 μm-filtered 0.1× PBS. Data were acquired with MFI View System Software (MVSS) version 2-R4 and processed with MFI View Analysis Suite (MVAS) software version 1.4.0. A baseline measurement was performed with the sample diluent to ensure that less than 100 particles/mL were present. The optimizing illumination step was performed on the sample diluent, and then the samples were loaded into the sample port. The sample purge volume was set at 0.20 mL, and the sample volume analyzed was 0.571 mL. The reported particle size (in μm) is the equivalent circular diameter (ECD) detected by MFI following the guidance of United States Pharmacopeia (USP) Chapter <788>.

2.4.10. Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis for Lipid Quantification

An ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA) with an Accucore^™^ C30 column (2.6 µm, 150 Å, 2.1 mm × 150 mm, ThermoFisher Scientific, San Jose, CA, USA) was used for reversed-phase liquid chromatography (RPLC) to separate lipid components and degradants in each FLuc mRNA–LNP formulation under long-term storage stability conditions (−80 °C, −20 °C, 5 °C, 25 °C). Mobile Phase A consisted of 0.1% formic acid and 10 mM ammonium formate in a 40:60 water/acetonitrile mixture. Mobile Phase B consisted of 0.1% formic acid and 10 mM ammonium formate in a 10:90 acetonitrile/isopropanol mixture. The linear LC gradient was set up as follows: 30% B from 0 min to 1 min, 43% B at 3 min, 65% B at 13 min, 85% B at 19 min, 100% B from 21 min to 26 min, and 30% B from 26.1 min to 26 min. LC separation was conducted at a flow rate of 260 µL/min at 40 °C. Each LNP sample was diluted 200× and 1000× in 100% ethanol and analyzed in triplicate with a 4 µL injection volume.

Mass spectrometry analysis was performed using an Orbitrap Exploris^™^ 480 mass spectrometer (ThermoFisher Scientific, Bremen, Germany) in positive electrospray ionization (ESI) mode. Instrument parameters included the following: capillary voltage of 3.5 kV, ion transfer tube temperature of 300 °C, sheath gas at 40, auxiliary gas at 10, sweep gas at 1. MS1 acquisitions were conducted at a resolution of 120,000, mass range 300–1200 m/z, normalized automatic gain control (AGC) target of 300%, and maximum injection time of 50 ms. MS2 acquisitions were performed at a resolution of 30,000, normalized AGC target of 100%, maximum injection time of 50 ms, isolation window of m/z = 1, and stepped higher-energy collisional dissociation (HCD) with collision energy of 15%, 30%, and 50%, using top10 data-dependent acquisition (DDA). Compound identification was performed using Compound Discoverer (version 3.3, ThermoFisher Scientific, San Jose, CA, USA) and relative quantification of LNP component and degradants was determined using TraceFinder (version 4.1, ThermoFisher Scientific, San Jose, CA, USA).

2.4.11. LC-MS Analysis for Nucleosides

Cold isopropanol precipitation was used to isolate mRNA from the FLuc mRNA–LNP formulation [8]. Specifically, 900 µL of 60 mM ammonium acetate isopropanol solution was added to a 100 µL aliquot of the sample. After a brief vortex, the mixture was centrifuged at 14,000 g for 15 min at 4 °C, and the supernatant was discarded. The pellet was then washed twice with 1000 µL of cold isopropanol, vacuum dried, and rehydrated in 100 µL of ultrapure water for quantification by UV Nanodrop 2000 spectrophotometer (260 nm, ThermoFisher Scientific, San Jose, CA, USA). Subsequently, the isolated mRNA (approximately 2 µg) was enzymatically digested to nucleosides for the LC-MS analysis. In brief, the mRNA samples were incubated with 10 units of nuclease P1 in 25 mM Tris-HCl (pH 7.2), 50 mM NaCl, 1 mM ZnCl_2_ (New England Biolabs, Ipswich, MA, USA), 2 units of phosphodiesterase I (Sigma-Aldrich, Inc., St. Louis, MO, USA), and 2 units of quick calf intestinal alkaline phosphatase in 25 mM Tris-HCl (pH 7.5), 1 mM MgCl_2_ and 0.1 mM ZnCl_2_ (New England Biolabs, Ipswich, MA, USA). The digestion reaction was carried out at 37 °C for 1 h.

Afterward, nucleoside analysis was conducted using the UPLC I-Class system (Waters Corporation, Milford, MA, USA) with an Atlantis Premier BEH Z-HILIC Column (1.7 µm, 2.1 mm × 150 mm, Waters Corporation, Milford, MA, USA) at a column temperature of 50 °C. This setup was used to quantify the four nucleosides in each mRNA–LNP formulation under long-term storage stability conditions (−80 °C, −20 °C, 5 °C, and 25 °C). The mobile phases were 200 mM ammonium acetate aqueous solution (Mobile Phase A) and acetonitrile (Mobile Phase B). Separation was achieved at a flow rate of 0.5 mL/min with a step gradient: 100% B from 0 to 1 min, 50% B at 11 min, 50% B from 11 to 12 min, and 100% B at 15 min. Each mRNA sample was analyzed in triplicate with a 5 µL injection volume. Mass spectrometry scans were acquired using an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific, Bremen, Germany) in both positive and negative ESI modes. The instrument parameters were as follows: capillary voltage of 3.2 kV in positive mode and 2.5 kV in negative mode. The ion transfer tube temperature was set to 260 °C. The sheath gas was set at 8, the auxiliary gas at 5, and the sweep gas at 1. Both MS1 and MS2 acquisitions were conducted at a resolution of 60,000, with a mass range of 60–760 m/z. The AGC target was 100%, and the maximum injection time was 100 ms. MS2 acquisitions were performed with an isolation window of 1.5 m/z. Stepped HCD collision energies of 20%, 35%, and 50% were used with top10 DDA. Compound identification and quantification were performed using FreeStyle (Version 1.3, ThermoFisher Scientific, San Jose, CA, USA).

2.5. In Vitro Transfection Efficiency

HEK-293 cells at a density of 10,000 cells per well were treated with various FLuc mRNA–LNP formulations at 10 ng mRNA per well, and Lipofectamine^™^ MessengerMAX^™^ Transfection Reagent (Thermo Fisher Scientific, Fair Lawn, NJ, USA) was used as a positive control. Following 24 h of treatment, ONE-Glo^™^ Luciferase Assay reagent was added to each treatment and control wells to incubate at room temperature for a few min, then the supernatant was measured for luminescence using a microplate reader (Tecan, Männedorf, Switzerland). The in vitro transfection efficiency of LNP formulations on long-term storage stability was determined on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C).

2.6. In Vivo Transfection Efficiency and IVIS® Imaging

Female CD1 mice (8–10 weeks old) were bought from Charles Rivers (Wilmington, MA, USA) and maintained under pathogen-free conditions. All the animal protocols were performed as approved by the Institutional Animal Care and Use Committee (IACUC). The in vivo transfection efficiency was analyzed for LNP formulations subjected to long-term storage stability, specifically on day 0 and the following 1, 3, 6, and 12 months when stored at different storage temperatures (−80 °C, −20 °C, 5 °C, 25 °C). CD1 mice were intravenously injected with various FLuc mRNA–LNP formulations at a dose of 5 µg/kg (n = 4 per group). Following 24 h of injection, the mice were injected with 150 mg/kg of D-luciferin (I.P., 15 mg/mL; Perkin Elmer, Waltham, MA, USA) and exposed to 1–2% isoflurane to perform imaging of whole animals using IVIS^®^ Spectrum (Perkin Elmer, Waltham, MA, USA). Mice injected with PBS were used as a negative control. Next, the mice were euthanized by cervical dislocation, the liver and spleen were harvested and the luminescence signals of the tissue samples were quantified ex vivo using IVIS^®^ Spectrum (Perkin Elmer, Waltham, MA, USA). Bioluminescence signal within the mouse liver (total liver flux) was determined for all the tested LNP formulations following intravenous injection.

2.7. Statistical Analysis

GraphPad Prism software (version: 10.1.0) was used to perform statistical analysis and all the data were presented as mean ± SD/SEM for any in vitro/in vivo experiments. Statistically significant differences were considered for a p-value of less than 0.05.

3. Results

3.1. Stabilizing LNPs Against Freeze–Thaw Stress

Sucrose screening revealed that ≥10% w/v was sufficient to protect FLuc mRNA–LNPs during a single freeze–thaw cycle, preserving particle size (≤100 nm), encapsulation efficiency (≥90%) and mRNA purity (≥70%) at levels indistinguishable from a control sample (unexposed to freeze–thaw sample) (Figure S1a–c). Additionally, cryo-TEM confirmed that 10% sucrose prevented the extensive aggregation seen without cryoprotectant and maintained spherical morphology after thawing (Figure S1d). On this basis, 10% sucrose was selected for subsequent buffer and lipid-composition screens.

Using formulations F1–F5 (Table 1), we compared Dulbecco’s Phosphate-Buffered Saline (DPBS) with Tris buffers (5–30 mM, pH 7.4) containing 10% sucrose for protecting LNP qualities against freeze–thaw. While maintaining charge and mRNA purity (Figure S2a–d), PBS and 5 mM Tris failed to control size or PDI after two freeze–thaw cycles, whereas 20–30 mM Tris kept F1–F4 at ≤100 nm with PDI ≤ 0.3; F5 remained heterogeneous in particle distribution (PDI > 0.3) regardless of buffer (Figure 1a–d). Tris–sucrose also yielded physiologically acceptable osmolality (≈300–350 mmol kg^−1^) and consistently higher encapsulation efficiency than PBS-containing formulations (Figure 1e–f and Figure S2e–f). In vitro, F1–F4 maintained acceptable transfection (luciferase expression of ~10^6^ a.u.) across Tris concentrations, while F5 lagged by ~3 orders of magnitude (Figure 1g–h). Collectively, these data identified 20 to 30 mM Tris + 10% sucrose as the most suitable composition for LNP formulations. These 20 and 30 mM Tris buffers were further selected to formulate and evaluate the long-term stability of F1–F4 formulations.

3.2. Long-Term Storage of LNP Drug Product: Effect of Storage Temperature

After establishing 10% w/v sucrose in 20–30 mM Tris (pH 7.4) as a robust freeze–thaw stable LNP buffer composition for four selected lipid combinations (F1–F4), we subsequently manufactured and evaluated whether these formulations are stable during long-term storage conditions.

The following sections detail the results of the comprehensive panel of quality attributes tested for the manufactured LNPs, including physicochemical properties, lipid and mRNA integrity, and transfection efficiency at in vitro and in vivo, over 12 months at different storage temperatures (−80 °C, −20 °C, 5 °C, and 25 °C).

3.2.1. pH, Appearance, Osmolality, and Particle Morphology

Throughout 12 months of storage, the pH of all four formulations (F1–F4) stayed between 7.0 and 7.5 with no meaningful drift, confirming that 20 mM and 30 mM Tris + 10% sucrose effectively buffer the system irrespective of temperature or lipid composition (Figure S3). Formulations remained clear and no visible particulates were detected at −80 °C, −20 °C, 5 °C, or 25 °C (a representative image of the LNPs’ visual appearance is shown as Figure S4). Also, osmolality was consistently 340–360 mmol kg^−1^ across conditions over 12 months (Figure S5).

Cryo-TEM revealed that all formulations retained spherical morphology after one year at −80 °C (Figure 2 and Figure S6). At −20 °C, only F4 preserved its morphology, while the rest of the formulations showed early aggregation, large internal blebs, and size growth, detectable within one month and pronounced by 12 months (Figure 2 and Figure S6). Storage at 5 °C maintained morphology for the first month, but at 12 months, all samples displayed reduced particle counts (based on the visual observation of the samples in the scanned regions under cryo-TEM) and distorted shapes. Here, F1 showed moderate aggregation, while F4 formed oversized (>200 nm) vesicles lacking internal structure, indicative of advanced instability; F2 and F3 showed few distinct ultrastructural alterations (Figure 2 and Figure S6). At 25 °C, all formulations showed early signs of particle growth under cryo-TEM at one month of storage, though the other physical characteristics, such as pH and osmolality, did not indicate any significant changes during this time.

3.2.2. Particle Size, Surface Charge, and Encapsulation Efficiency

Figure 3, Figures S7 and S8 summarize the physicochemical characterization of four tested mRNA–LNP formulations. Particle size distribution (measured by DLS) showed no meaningful difference over 12 months (Figure 3). MC3-based formulations (F1 and F3) remained ~20 nm larger than their ALC-0315 counterparts (F2 and F4) across all temperatures, and both Tris concentrations with no variation were detected over this time period. Polydispersity indices remained low overall across the study. DMG-PEG2k formulations (F1 and F2) consistently showed the narrowest distributions (PDI < 0.10), whereas ALC-0159 formulations (F3 and F4) exhibited modest fluctuations with occasional values higher than 0.18, consistent with Figure S7.

Zeta potential values varied from −10 mV to +10 mV and were largely unaffected by temperature or buffer concentration (Figure S8). ALC-0315 systems (F2 and F4) trended slightly more negative than MC3 analogs (F1 and F3), but differences were <5 mV and did not change with storage.

Encapsulation efficiency (EE) was maintained above 95% for MC3 LNPs and 80–90% for ALC-0315 LNPs throughout the study, independent of temperature or buffer, except for F3 (MC3 with ALC-0159) at −80 °C, which declined ~40% by month 12 (Figure 3). Notably, this EE% drop occurred without parallel changes in size or PDI, indicating possible selective mRNA leakage rather than gross particle destabilization. More evaluation is needed to determine the observed discrepancy between EE% and the size and PDI data observations for the F3 formulation.

3.2.3. Subvisible Particle Concentration

One of the key observations of this study was the changes observed in subvisible particle counts measured by MFI. These results showed that after 12 months, all samples contained less than 600 particles mL^−1^ of ≥10 µm except F3 at −20 °C (≈1200 particles mL^−1^, Figure 4). The highest incidence of LNP aggregates followed the order −20 °C > 5 °C > 25 °C > −80 °C.

Across formulations, 20 mM Tris generated fewer subvisible particles than 30 mM Tris. Together, the data highlights F3 as the most aggregation-prone formulation and reaffirm −80 °C with 20 mM Tris as the optimal storage condition for maintaining both nanoparticle integrity and minimal subvisible particulate burden.

3.2.4. Lipid Composition and Oxidative Degradants (LC-MS)

Lipid content and integrity of LNPs were evaluated by LC-MS post-one year storage at different temperatures. This LC-MS profiling at month 12 showed that the lipid composition of every formulation was essentially unchanged at −80 °C and −20 °C (≤10% deviation from baseline; Figure S11) when 20 mM or 30 mM tris/sucrose buffer was used. At 5 °C and 20 mM tris/sucrose, total lipid loss reached 10–25%, and at 25 °C as much as 40%, possibly driven by degradation of DSPC and PEG-lipids. Notably, DSPC content fell to ~70% of baseline in F1 and 60% in F3, while DMG-PEG2k in F1 decreased by ~20%. When the tris concentration increased to 30 mM, a significantly higher amount of lipid loss was observed for F1 and F3 (MC3-based formulations), even at −20 °C, while F2 and F4 (ALC-0315-based formulations) were surprisingly unaffected. Additionally, we observed that the lipid content of F2 and F4 was not affected at 20 mM tris buffer over the storage time by 12 months.

At the same time, oxidative markers showed an increase in parallel to the lipid loss. Using 20 mM tris, the potentially mRNA reactive DSPC-C_18_H_34_O increased 2–6 fold at 5 °C and >10-fold at 25 °C (Figure 5). Increasing the tris concentration to 30 mM caused a severe increase in this reactive DSPC derivative at 5 °C and 25 °C by approximately 10–20-fold higher compared to the frozen samples at −80 °C and −20 °C. MC3 N-oxide, a predominant MC3 reactive impurity in MC3-containing F1 and F3, climbed up sixfold at 25 °C in 20 mM tris and up to threefold when 30 mM tris was used. On the contrary, the oxidized ALC-0315 in F2 and F4 showed either no significant changes at any temperature and tris concentration or rose only by <0.5-fold, indicating the greater stability of the ALC-0315 head group against oxidation. Collectively, these results confirm that frozen storage preserves the lipid integrity, while DSPC hydrolyzation can be the earliest indicator of chemical degradation at elevated temperatures. Lastly, as indicated by DSPC and MC3/ALC-0315 lipid data, the buffer concentration exhibits a mixed effect on each of the oxidized species of lipids and overall, we have observed less susceptibility of ALC-0315-based formulation lipid loss and oxidation compared to MC3-based LNPs.

3.2.5. mRNA Integrity: Purity, 5′-Cap Retention, and Nucleoside Conservation

Next, we tested the mRNA integrity at one year of storage at different temperatures. MCE for mRNA purity showed that frozen storage preserved mRNA integrity, where all formulations retained ~80% purity after 12 months at −80 °C or −20 °C (Figure 6). At 5 °C, purity began to decline after month three and fell to 30–40% by month 12, regardless of buffer strength. At 25 °C, purity dropped by 40–60% within three months and continued to degrade, confirming rapid hydrolytic/oxidative decay at ambient temperature.

LC-MS quantification of enzymatically digested nucleosides partially corroborated these findings. Adenosine, cytidine, guanosine, and uridine levels were unchanged at −80 °C, −20 °C, and 5 °C but fell slightly at 25 °C (Figure S12), indicating mRNA backbone (ribose-phosphodiester structure) fragmentation rather than selective base loss for the liquid formulations.

Ion-pair RP-HPLC revealed near-quantitative 5′-capping retention (>95%) in 20 mM Tris after one year at both frozen temperatures. The 30 mM Tris buffer reduced 5′-cap levels by 5–15% at both frozen temperatures, suggesting mild cap hydrolysis at higher ionic strength (Table 3, Figures S13 and S14). Additionally, capping analysis was not possible for 5 °C and 25 °C samples because bulk mRNA was already degraded below the assay’s threshold (Figures S13 and S14). Together, these data reinforce the need for sub-zero storage, preferably −80 °C, in a low-ionic-strength buffer to protect both the chemical and structural integrity of encapsulated mRNA.

3.2.6. In Vitro Transfection Efficiency

The transfection efficiency (TE) results (Figure 6) indicated that, in general, the baseline TE% ranked F1 ≈ F2 ≈ F3 ≫ F4. The three higher performing formulations produced ~10^7^ mean luminescence intensity (MLI) in HEK-293 cells, whereas F4 reached only ~10^6^ MLI, consistent with its slightly lower encapsulation efficiency (80–90%, as indicated in Figure 3).

TE was largely preserved for all formulations stored at −80 °C and −20 °C, showing ≤0.2-log variation over 12 months. At 25 °C, TE% declined in parallel with mRNA purity, dropping one log by month three and becoming negligible by month 12. The 5 °C condition showed a biphasic pattern, where transfection remained comparable to the frozen controls through six months, despite measurable mRNA purity loss, but fell sharply between 6 and 12 months. This lag suggests that a critical threshold of intact mRNA must be crossed before in vitro functional activity is compromised. Overall, −80 °C and −20 °C storage fully preserves in vitro TE, while even mild refrigeration (5 °C) affords short-term (up to six months) protection for luciferase mRNA–LNPs.

3.2.7. In Vivo Transfection Efficiency and Biodistribution

We next evaluated the in vivo transfection trend of LNPs over one year of storage at different temperatures at 30 mM tris composition. Long-term storage temperature exerted a clear, formulation composition-independent hierarchy on in vivo potency (Figure 7). Intravenous dosing of the four LNPs into CD1 mice showed that −80 °C preserved whole-body, liver, and spleen luminescence similar to the day one levels for every formulation. At −20 °C, formulations F2–F4 largely maintained in vivo activity over 12 months, while F1 showed a modest ~15–20% reduction in liver signal relative to its corresponding −80 °C LNPs but not statistically significant (Figure 7c, One-way ANOVA, p-value = 0.73). Also, the spleen expression was unaffected for F1, suggesting slight instability specific to this lipid blend. LNPs stored at 5 °C stayed stable up to six months (Figure S16) but showed a uniform ~10-fold drop in liver and spleen signals by month 12 (Figure 7), mirroring the late decline in the in vitro TE%. Finally, 25 °C storage led to near-complete loss of detectable luminescence by month six for all formulations, confirming functional failure at this temperature. These results align with mRNA integrity data and reinforce the obvious requirement for frozen storage, preferably −80 °C, to ensure the retention of systemic transfection efficiency.

4. Discussion

The long-term stability profile of mRNA–LNPs is governed by an interplay between formulation excipients that reduce external stresses and the intrinsic reactivity of the constituent lipids and mRNA [14,15]. In the present work, we first established a freeze–thaw-tolerant suitable composition containing 10% w/v sucrose combined with 20–30 mM Tris pH 7.4, to prevent particle size increase, particle aggregation, maintain ≥ 90% encapsulation efficiency and preserve mRNA purity after a freeze–thaw cycle (Figure 1). Among the formulations, F5, which contained DSPE-PEG2K, failed at producing and maintaining an acceptable particle quality (Figure 1, evidenced by the high PDI at all buffer conditions) at pre- and post-freeze–thaw and at transfection efficiency. DSPE-PEG2K contains a longer lipid chain (C18) compared to DMG-PEG2K (C14), and consequently, the LNPs prepared by DSPE-PEG2K are shown to be more stable in the bloodstream and have longer serum half-life than DMG-PEG2K, mainly due to its slower shedding/cleavage rate from the LNP’s surface [16]. Also, it has been reported that LNPs containing DSPE-PEG2K can transfect cells both in vitro and in vivo [17]. However, in this one-year study, non-ideal particle size and physical quality, as well as the lack of transfection in vitro (Figure 1) was observed for this formulation. We speculate that a slight negative charge of DSPE-PEG2K lipid (as observed in zeta potential data, too) and its very slow shedding rate of PEG from the LNP surface may be the reasons behind the lack of potency of the LNPs containing DSPE-PEG2K. Nevertheless, the optimized composition was selected for a one-year evaluation of the other four prototypical LNPs (F1–F4) that differ in the ionizable and PEG-lipid chemistries (MC3 or ALC-0315 and DMG-PEG2k or ALC-0159).

In general, from the comprehensive panel of sixteen assays that we selected to thoroughly analyze the long-term stability of the drug product, several assays were not indicative of the stability issues of the samples. For instance, particle size and PDI measurements by DLS and EE% by RiboGreen assay results stayed consistent over one year, although the other assays, such as cryo-TEM, mRNA purity, and transfection efficiency, clearly indicated the stability failures in some of the formulations (Figure 6). As expected, at long-term storage and across all formulations, sub-zero temperature was essential to preserve the LNP drug products’ quality and function. Particle counts, lipid composition, and mRNA integrity remained essentially unchanged at −80 °C, and only modest losses (<10%) were detected at −20 °C for nanoparticle counts.

In contrast, refrigeration (5 °C) triggered up to 70% nanoparticle count loss (Figure S9) and a parallel rise in subvisible aggregates (Figure 4), while ambient temperature (25 °C) accelerated disassembly by ~90% and markedly reduced in vitro and in vivo potency by six months. Notably, for subvisible particle counts, 20 mM Tris worked slightly better in reducing the aggregations compared to 30 mM for most of the formulations.

Based on our data, the observed physical characterizations did not fully confirm the chemical stability readouts. For instance, DSPC’s hydrolyzed species (DSPC-C_18_H_34_O) remained unchanged at −80 °C and −20 °C over one year but surged ≥4-fold at 5 °C and ≥10-fold at 25 °C. Oxidized MC3 in MC3-containing LNPs was not affected by storage at frozen conditions, N-oxidized MC3 accumulated by ~6-fold at 25 °C over one-year of storage. The oxidized ALC-0315 remained largely unchanged during one-year storage except at 25 °C with up to 40% increase. This finding, in line with other previous studies, may suggest the superior oxidative robustness of the ALC-0315 structure compared to MC3 formulations.

The oxidized lipids and, most importantly, ionizable lipid N-oxides are the major sources of mRNA instability [8]. Ionizable lipids’ N-oxide species can specifically create a reactive aldehyde, which can have an electrophilic attack on the mRNA backbone, resulting in an mRNA-lipid adduct formation for the non-frozen formulations [8]. Although our findings (Figure 5) indicated that over one year of storage at frozen state, MC3 and ALC-0315 have undetectable levels of N-oxide, further analysis would be useful to quantify any generated mRNA-lipid adduct level to confirm the adduct-free mRNA–LNP product quality at frozen conditions, as well.

As mentioned, the buffer’s ionic strength may impact the LNP stability. Reducing Tris from 30 mM to 20 mM consistently generated fewer ≥10 µm and ≥25 µm particles after 12 months at the majority of storage temperatures, suggesting that lower ionic strength may mitigate ice-crystal-induced fusion during freezing [18] and thus reduce the stability risk associated with the long-term storage. Interestingly, the same buffer ionic strength trend extended to mRNA structural stability. For example, 5′-cap retention exceeded 95% for all formulations stored in 20 mM Tris at −80 °C and −20 °C, but decreased by 5–15% in 30 mM Tris, indicating a mild 5′-cap hydrolysis at higher ionic strength even at the frozen state (Table 3). The 5′-cap integrity is essential for efficient translation and influences intracellular mRNA turnover [19]. In cells, mRNA is typically cleared through regulated pathways that include deadenylation followed by decapping and exonucleolytic degradation (e.g., Xrn1-mediated 5′ to 3′ decay, [20]).

Accordingly, the 5′-cap hydrolysis we monitor here is treated as a drug product degradation liability because it can reduce translational competence and increase susceptibility to intracellular exonucleases after dosing. Importantly, our formulation and storage optimization is intended to maintain product quality during shelf-life prior to administration and it does not aim to prolong uncontrolled persistence in vivo, where endogenous RNA surveillance and decay mechanisms remain active. Recent literature has also explored broader biological roles of RNA processing/turnover in specialized contexts (e.g., RNA-templated DNA repair), underscoring the importance of preserving intended mRNA quality attributes and continuing to evaluate safety in the appropriate translational setting [21].

Nonetheless, these 5′-cap losses and modest differences in particle concentration did not translate into measurable divergences in the in vitro gene expression and stayed within 0.2 log unit variations for 20 mM versus 30 mM Tris under frozen conditions. This highlights that a sizeable transfection efficiency reserve may mask mild chemical changes during the shelf-life storage.

Additionally, our data indicates that the lipid composition can determine the main pathways responsible for LNP instability. For instance, the MC3/ALC-0159 blend (F3) was most aggregation-prone, generating the highest subvisible particle counts at −20 °C, whereas the ALC-0315/ALC-0159 system (F4) preserved morphology even at −20 °C (Figure 2) and showed the lowest N-oxidant burden (Figure 5). Moreover, cryo-TEM reaffirmed the morphology preservation of F4 formulation at frozen state (both −80 °C and −20 °C). Conversely, MC3-based LNPs retained encapsulation efficiency better than ALC formulations, implying a possibly weaker electrostatic interaction between mRNA and cationic head group of ionizable lipid in ALC-0315 systems due to the more branched structure of the lipid, thus facilitating gradual mRNA leakage despite intact particles. These findings echo recent reports that head group ionization and carbon tail structure (length and saturation) of ionizable lipid can significantly modulate both physical and functional stability of LNPs [14].

Integrating all critical quality attributes, two mechanistic theories may emerge for the tested LNPs’ compositions: (i) Above the glass transition temperature (Tg’) of LNPs (in the presence of 10% sucrose, Tg’ reaching around <−30 °C) at the liquid state storage (5–25 °C), oxidative lipid chemistry and potential mRNA degradation may dominate the other instability pathways. Therefore, constituting total parent lipid loss (Figure S11) and reactive DSPC degradant in addition to N-oxide/aldehyde formations (Figure 5) correlate with mRNA degradation (Figure 6) and significant 5′-capping loss (Table 3). These events could eventually result in in vitro and in vivo potency collapses (Figure 1 and Figure 7). (ii) At frozen conditions, but still above the glass transition temperature of LNPs, for example, at −20 °C storage temperature [22], physical attrition and particle fusion or lipid disassembly become the principal failure mode, most observable in subvisible particle count and cryo-TEM assays (Figure 2 and Figure 4), yet largely silent in chemical degradation metrics (such as oxidized or hydrolyzed lipids) and transfection efficiency until advanced stages at longer storage times, for instance, at over 12 months.

Based on the observations, LNP should be stored at frozen conditions below their glass transition temperature (e.g., −80 °C or −60 °C) to maintain long-term stability. If maintaining stable deep-cold storage is not feasible (e.g., storage at −20 °C is inevitable), a low-ionic-strength buffer such as 20 mM Tris with proper lipid compositions can be used for improved stability. Also, maintaining ≥ 10% sucrose is necessary to mitigate freeze–thaw excursions throughout the cold chain storage, handling, and transport. Lastly, while the luciferase mRNA used in this study is a tractable model, formulation screening efforts must confirm that similar excipients and lipid types do apply to longer mRNA sequences, chemically modified mRNA, and/or to co-delivery products such as guide RNA/mRNAs [23].

5. Conclusions

Overall, our year-long, multivariate study showed that the tested clinically relevant mRNA–LNP drug products can retain complete physicochemical and functional integrity when formulated with ≥10% sucrose in low-ionic-strength (20 mM) Tris and stored at or below –20 °C. Across four clinically relevant lipid compositions, frozen storage suppressed DSPC hydrolyzation and ionizable lipid oxidation, preserved ≥95% mRNA 5′-cap integrity, maintained nanoparticle and subvisible particle counts, and sustained in vitro and in vivo transfection. In contrast, 5 °C provoked aggregation, mRNA degradation, and a >10-fold potency loss by month 12, while 25 °C showed a complete loss of expression within six months. Notably, raising Tris to 30 mM accelerated 5′-cap hydrolysis. In summary, these findings re-emphasize the requirement for a comprehensive testing of the LNP drug product quality attributes as an indispensable and invaluable tool for the manufacturing and stability evaluations. Moreover, our results underscored sub-zero temperature with low-ionic-strength storage buffer, tailored to the distinct chemistries of ionizable and PEG-lipids, as the essential design principle for next-generation and long-shelf-life mRNA–LNP therapeutics.