Overcoming thermostability challenges in mRNA-lipid nanoparticle systems with piperidine-based ionizable lipids

mRNA delivery using lipid nanoparticles (LNPs) has shown great promise, particularly with advancements in ionizable lipids. These lipids, with their positively charged amino groups, are crucial for encapsulating negatively charged mRNA and facilitating endosomal escape. The successful development of mRNA-based vaccines against SARS-CoV-2 highlights the potential of this technology. However, a major hurdle remains: the long-term storage stability of mRNA/LNP formulations. mRNA is inherently unstable, susceptible to degradation through hydrolysis and oxidation, processes accelerated by thermal stress. Current practices involve storing mRNA/LNPs at -20°C or below, requiring extensive cryogenic infrastructure and logistics, increasing costs and limiting accessibility. Lyophilization offers an alternative, but this adds complexity and energy costs. The need for thermostable, liquid mRNA/LNP formulations is therefore substantial. A significant challenge arises from the unintended reaction of lipid impurities, particularly aldehydes generated from the oxidation and hydrolysis of tertiary amines in ionizable lipids, with mRNA nucleosides. These reactions compromise mRNA integrity and function. This study hypothesizes that by strategically designing the amine structure of ionizable lipids, the generation of these reactive aldehyde impurities can be controlled, leading to improved thermostability at higher temperatures. This represents, to the best of the authors' knowledge, a novel approach focusing on aldehyde production control based on lipid structural modification.

The literature extensively documents the challenges in mRNA stability and LNP formulation. Studies highlight the inherent instability of mRNA to hydrolysis and oxidation, with thermal stress exacerbating these processes. Existing approaches for enhancing stability include ultra-low-temperature storage and lyophilization, each presenting its own limitations in terms of cost, complexity, and potential for LNP damage during reconstitution. Recent work has pointed to the role of lipid impurities, specifically reactive aldehydes formed from the degradation of ionizable lipids, in mRNA inactivation. This degradation pathway involves N-oxidation of the ionizable lipid followed by hydrolysis which generates reactive aldehydes which react with mRNA nucleosides. These studies provided the impetus for this research to explore lipid engineering as a strategy to control aldehyde production and improve thermostability.

This research employed a multi-faceted approach involving lipid synthesis, LNP formulation, in vitro and in vivo efficacy testing, and detailed chemical analysis. A library of 23 piperidine-based ionizable lipids (CL15F) was synthesized, featuring branched structures to enhance mRNA delivery. These were compared to a previously developed library (CL4F). LNPs were formulated using a microfluidic device with a fixed molar ratio of ionizable lipid, cholesterol, DSPC, and DMG-PEG2000. In vitro efficacy was evaluated using FLuc mRNA transfection in HEK-293T cells, comparing CL15F LNPs with CL4F LNPs and a commercial transfection reagent (Lipofectamine MessengerMAX). In vivo efficacy, with a focus on the potential application of CL15F lipids for vaccination, was assessed using OVA mRNA immunization in mice, comparing the immune response elicited by CL15F LNPs to those generated by clinically successful lipids ALC-0315 and SM-102. The study further investigated the storage stability of mRNA/LNPs at different temperatures (-80°C and 4°C) over time, using a hEPO reporter system to assess in vivo efficacy. High-performance liquid chromatography (HPLC) with corona-charged aerosol detection (CAD) was used to assess lipid integrity during storage. A fluorescence-based microplate assay using NBD-H and LC-MS analysis with DNPH derivatization were used to quantify and identify aldehyde impurities. To investigate the mechanism of mRNA inactivation, the reactivity of aldehyde impurities with nucleosides and mRNA was assessed using HILIC and reversed-phase ion pair chromatography. Additional experiments explored the role of N-oxidation in aldehyde production using synthesized N-oxidized lipids. Finally, to assess the broader impact of cyclic amine structures on aldehyde generation and adduct formation, a series of additional lipids with varied cyclic amine moieties were synthesized and evaluated.

The study identified piperidine-based lipids (CL15F) that significantly improved the long-term storage stability of mRNA/LNPs at 4°C as liquid formulations. In vitro transfection studies demonstrated that most CL15F LNPs displayed superior or comparable efficacy to CL4F LNPs and Lipofectamine MessengerMAX. In vivo immunization studies revealed comparable anti-OVA IgG titers between CL15F, SM-102, and ALC-0315 LNPs. Importantly, CL15F LNPs, particularly those with shorter tails, exhibited significantly stronger antigen-specific cellular responses (IFN-γ secretion) compared to SM-102 and ALC-0315 LNPs. The in vivo efficacy of hEPO mRNA-loaded CL15F LNPs persisted even after five months of refrigerated storage, unlike CL4F LNPs and other control LNPs which showed substantial loss of efficacy. HPLC analysis confirmed that lipid components remained largely intact during long-term storage with CL15F LNPs, even at 4°C. Crucially, NBD-H assays revealed a significant reduction in aldehyde impurities in CL15F lipids compared to CL4F lipids. DNPH derivatization and LC-MS identified the aldehyde impurities as fatty aldehydes derived from the oxidation and hydrolysis of ionizable lipids. Further investigations demonstrated that N-oxidation of CL4F lipids was the primary driver of aldehyde production, while CL15F lipids, despite also undergoing N-oxidation, produced considerably less aldehyde impurities. The study found that these aldehyde impurities reacted with nucleosides and mRNA, forming adducts which greatly contributed to the decreased efficacy of mRNA. Furthermore, removal of aldehyde impurities from CL4F lipids using a scavenger resin significantly reduced adduct formation with mRNA. The study also investigated other cyclic amines. CL17F lipids, similar to CL15F, showed improved stability while CL16F showed reduced aldehyde impurities but did not improve storage stability as much, highlighting the importance of both minimizing aldehydes and the overall lipid structure in mRNA stability. Finally, a correlation was observed between the reduction in mRNA activity and the amount of aldehyde impurities present in the lipids.

The findings demonstrate that the chemical structure of the ionizable lipid is crucial for controlling the generation of reactive aldehyde impurities and, consequently, the long-term stability of mRNA/LNP formulations. The superior performance of piperidine-based lipids, particularly CL15F and CL17F, is attributed to the cyclic amine structure which limits the production of reactive aldehydes even in the presence of N-oxides. The formation of cyclic imines via intramolecular reactions prevents the release of free, reactive fatty aldehydes, preserving mRNA integrity and function. These results offer a new strategy to overcome the thermostability challenges of mRNA/LNP systems, potentially simplifying storage and distribution by reducing the dependence on cryogenic temperatures. The improved immunogenicity observed with CL15F lipids further suggests their potential in vaccine development, especially for applications requiring strong cellular immunity. This approach offers a significant advantage by addressing a fundamental issue in mRNA delivery – the degradation of lipids which results in the formation of reactive aldehyde impurities that inactivate the mRNA.

This study successfully demonstrated that incorporating piperidine-based ionizable lipids, particularly CL15F and CL17F, significantly enhances the thermostability of mRNA/LNP systems, allowing for long-term storage at 4°C. The mechanism involves controlling aldehyde impurity generation through the unique properties of the cyclic amine structure. This approach offers substantial implications for cost-effective and widespread distribution of mRNA therapeutics. Future research should focus on further optimizing lipid structure and composition to achieve even greater stability and exploring the application of these lipids in various therapeutic settings. Further investigation on the role of mRNA structure and sequence on adduct formation is warranted.

The study acknowledges several limitations. First, precisely quantifying the impact of small aldehyde impurities on mRNA was challenging due to the difficulty in detecting small aldehyde-mRNA adducts. Second, the study investigated a limited number of lipids. Third, mRNA sequence and modification patterns and their impact on adduct formation efficiency were not comprehensively studied. Fourth, there are other mechanisms that result in the loss of mRNA stability beyond aldehyde adduct formation. The observed reduction in efficacy cannot be solely attributed to aldehyde impurities.