Boosting LNP Performance: Higher Concentrations of Lipid Mixtures Improve In Vivo Gene Expression and Storage Stability

Abstract

Background: An efficient formulation of lipid nanoparticles (LNPs) is often considered crucial in the successful development of nucleic acid therapeutics. This study explores the impact of varying the lipid and payload concentrations as starting materials on key LNP properties.

Results: The outcomes of the study revealed that the desired particle properties could be retained even at a starting lipid mixture concentration of 70 mg/mL. Particle size remained largely unchanged despite changes in lipid mixture concentration, with polydispersity index values below 0.2. CryoTEM analysis revealed that LNPs prepared using higher lipid mixture concentrations were more uniform and more abundant in solid core morphologies. Buffer composition was shown to influence the LNP particle size, surface charge, and gene expression, as well as storage stability. In vivo studies in mice showed enhanced gene expression and biodistribution for LNPs formulated at higher lipid and RNA concentrations, with LNPs in Tris-sucrose eliciting superior gene expression compared to LNPs in PBS.

Conclusions: This study demonstrated that intensified mixing processes based on confined jet-impingement allow the use of elevated starting material concentrations in LNP formulations, resulting in improved biological performance and stability of mRNA-LNPs, as well as enhanced scalability and throughput

1. Introduction

The rapid advancement of novel messenger RNA (mRNA) vaccines for COVID-19 has drawn significant attention to lipid nanoparticles (LNPs) as a promising technology in drug formulation [1]. Market-approved LNP formulations have quickly emerged as the gold standard non-viral delivery system to encapsulate RNA material intended for applications in infectious diseases, as well as cell and gene therapies (CGTs) [2]. In contrast to other lipid-based nanoformulations—including liposomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) [3]—LNPs have assumed a more central role as delivery systems for biological molecules by offering adaptable and scalable manufacturing. Detailed comparisons of the structural features of LNPs versus other carriers, as well as the pharmacokinetics and the functional roles of individual lipid components, have been well described in the literature [3,4,5,6,7,8,9,10].

Besides the ability of LNPs to protect fragile biomolecules and ensure efficient cellular delivery, one of their most relevant advantages lies in enabling the rapid translation of therapies from lab to clinic, thus making them a key enabler in advancing novel therapies [11,12]. Consequently, current forecasts estimate that by 2035, 61% of the LNP market will be focused on oncological indications, largely due to their suitability for repeat dosing [13]. The recent success of the mRNA-based CRISPR therapy in treating hyperammonaemia in an infant further underlines the unique position of LNPs as effective delivery vehicles for nucleic acid-based therapies, highlighting their growing clinical potential [11]. However, despite the growing body of scientific research demonstrating the efficacy of novel therapeutics enabled by LNP technology [14], there is a clear consensus within the industry that the primary bottlenecks are no longer scientific [15]. Rather, challenges stem from unoptimized manufacturing processes, operational inefficiencies, and limited automation [16].

The principal technologies currently in use for mRNA-LNP manufacturing include microfluidic (MF) mixers, T-shaped mixers (often classified under confined impinging jet mixers (CIJM)), and CIJM systems with a dedicated mixing chamber [17]. MF mixers operate under laminar flow conditions, where mixing occurs predominantly through molecular diffusion. Some variations introduce secondary flow structures (e.g., herringbone grooves) to enhance mixing. MF mixers are ideal for low-volume formulations, but suffer from a high risk of clogging, poor throughput scalability, and limited adaptability to different formulation requirements [18,19]. T-mixers and traditional CIJM designs can support higher flow rates, yet their fixed geometries lead to shifts in mixing behavior when operating conditions change, resulting in inconsistent scale-up performance [19,20,21]. In contrast, the FR-JET® modular mixer provides a confined mixing environment with geometry-defined flow conditions that remain stable across scales (Figure 1).

Its modular architecture enables deliberate control of the mixing regime, reducing the variability typically encountered with conventional mixers during process transfer and scale-up. However, despite the variety of mixers and technologies available in the market today, challenges in LNP manufacturing persist, particularly in areas such as product throughput, scale-up, and automated workflows [22,23], which continue to demand further innovation and sustained investment to unlock the full clinical potential of LNP-based therapies. To address these bottlenecks, we explored a potential strategy for process intensification using the FR-JET® modular mixer with the aim of improving manufacturing efficiency.

In this study, we investigated process intensification as a strategy to improve product throughput and facilitate scale-up, specifically by examining the effects of increasing the initial lipid mixture and RNA concentrations during LNP formulation. Increasing the concentration of the starting materials during the formulation of LNPs has relevant implications in facilitating efficient and scalable manufacturing processes [24]. In the context of commercial-scale production, formulating LNPs using higher concentrations of starting materials may offer several key advantages in terms of cost, operational efficiency, and occupational safety. By increasing the concentrations of starting materials (i.e., lipids and RNA), the process requires less organic solvent for lipid dissolution and smaller volumes of both starting and final buffers, leading to a more efficient use of materials. Moreover, smaller buffer volumes simplify handling and reduce the logistical burden of managing large containers—an issue that becomes increasingly challenging at larger manufacturing scales. In addition, working with smaller volumes of organic solvents improves occupational safety by limiting operator exposure and reducing handling-related risks. Furthermore, the use of more concentrated starting materials results in a more concentrated LNP product after mixing, potentially shortening or eliminating the initial concentration step typically required during tangential flow filtration (TFF), thus allowing a direct transition to buffer exchange. The final LNP solution can subsequently be diluted to the desired target concentration. Therefore, process intensification achieved simply by formulating at higher starting material concentrations may offer a practical way to simplify and shorten the overall manufacturing process, enhancing both operational efficiency and scalability.

Although introducing higher starting material concentrations in the manufacturing process can support process intensification, this approach is seldom reported in the literature. A likely limiting factor is the lack of efficient mixing technologies that can preserve product quality at elevated concentrations and prevent material loss. For example, the preparation of LNPs at higher lipid mixture concentrations using a microfluidics-based mixing technology has been shown to result in increased particle size and reduced encapsulation efficiency (EE) [25]. Moreover, based on published patents and patent applications describing the use of conventional T-mixing in the commercial manufacturing of LNP-based drug products—specifically Comirnaty®, Onpattro®, and Spikevax®—the lipid mixture concentrations used in these processes were 49.5 mg/mL, 30 mg/mL, and below 25 mg/mL, respectively [26,27,28]. Meanwhile, most peer-reviewed studies from academic research report the preparation of LNPs using even lower initial lipid mixture concentrations, typically below 15 mg/mL [29,30,31,32,33,34].

Therefore, the aim of this study was to explore the feasibility of formulating LNPs with a lipid mixture concentration of above 50 mg/mL and evaluate the effect of this process parameter on the physico-chemical properties of the resulting particles as well as their biological performance. Moderna’s Spikevax® formulation was selected as the LNP model, as previous studies have demonstrated that SM-102-containing LNPs exhibited higher gene expression and better long-term stability compared to LNPs containing other ionizable lipids (e.g., ALC-0315, CKK-E12) [35,36]. The study was conducted in two parts. In the first part, feasibility experiments were performed to evaluate the formulation of LNPs across a range of lipid mixture concentrations (from 5 to 70 mg/mL), using polyadenosine (poly(A)) as a surrogate payload. In the second part, LNPs encapsulating Firefly luciferase mRNA (Fluc-mRNA) were prepared at lipid mixture concentrations of 15, 45, and 70 mg/mL, corresponding to 0.2, 0.7, and 1.0 mg/mL mRNA, respectively. LNPs were formulated at a total flow rate (TFR) of 30, 60, and 80 mL/min and dialyzed in two different buffers—phosphate-buffered saline (PBS) and Tris-sucrose. The resulting formulations were investigated for particle size distribution, EE, and particle morphology, as well as in vitro and in vivo gene expression. In addition, the effect of lipid mixture concentration and buffer composition on storage stability at 2–8 °C and 20 °C was assessed by monitoring changes in particle size and polydispersity index (PDI) over a period of six months. Our findings indicate that enabling LNP formulation at higher lipid mixture concentrations not only supports process intensification but also improves particle properties and their biological performance in vivo.

Materials

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Lipoid (Ludwigshafen, Germany). 1,2 dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) was obtained from Avanti polar lipids (Alabaster, AL, USA). Cholesterol, Poly(A), and sodium citrate tribasic dihydrate were obtained from Sigma-Aldrich (Gillingham, UK). The ionizable lipid heptadecan-9-yl 8-((2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino) octanoate (SM-102) was purchased from Broadpharm (San Diego, CA, USA). EZ Cap firefly luciferase mRNA (5-moUTP) was obtained from Apexbio (Houston, TX, USA). Ethanol and sucrose were obtained from VWR chemicals. Tris-HCl buffer (1 M), phosphate-buffered saline (100 mM; pH 7.4), MOPS buffer, Alamar blue cell viability reagent, SYBR green RNA gel stain, and Ultrapure Agarose were procured from Fisher scientific (UK). VivoGlo Luciferin in vivo grade and the One-glo luciferase assay system were purchased from Promega (Southampton, UK). The HEK 293 cell line was obtained from ATCC (Manassas, VA, USA). Lipofectamine 3000, 1,1′-dicotadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodide (DiR), and trypsin EDTA (0.25%) were purchased from Thermo Fisher (Waltham, MA, USA). Amicon® Ultra-15 Centrifugal filter units (100 K) and dialysis tubing cellulose membrane (14,000 K) were purchased from Merck, KGaA (Darmstadt, Germany). The other solvents and chemicals utilized were of analytical grade, and deionized water was provided by an in-house system.

Shkodra, B.; Muglikar, A.; Thangapandian, J.; Schumacher, M.; Binici, B.; Perrie, Y. Boosting LNP Performance: Higher Concentrations of Lipid Mixtures Improve In Vivo Gene Expression and Storage Stability. Pharmaceutics 2026, 18, 50. https://doi.org/10.3390/pharmaceutics18010050

Read the full article here.

Download the PDF Document here.