Single-use bags as a viable solution for long-term stability of lipid nanoparticles

In recent years, therapeutics based on ribonucleic acid (RNA) have gained significant attention.^1–3 Lipid-based formulations, such as liposomes and nanoparticles, have been extensively researched over the decades.⁴ A notable milestone was achieved in 2018, when the FDA approved Onpattro, the first small interfering RNA (siRNA) drug utilizing lipid nanoparticles (LNPs). The development of ionizable lipids has significantly enhanced the intracellular delivery of RNA into the cytoplasm.⁵ The successful advancement of messenger RNA (mRNA) vaccines using LNPs against COVID-19 has marked the beginning of a new era in genetic medicines. This advancement has enabled targeted delivery to diverse tissues, such as the lungs, immune system, and brain, and has spurred progress in cancer vaccines and gene editing. These therapeutic applications of LNPs and liposomes are currently under development and are expected to undergo clinical testing in the coming years.^2,3,6–10

Ensuring the stability and efficacy of LNPs after long-term storage is crucial to prevent degradation that could impair the immune response.¹¹ However, achieving long-term storage of LNPs and mRNA remains a challenge for their widespread application. Hermosilla et al. reported aggregation or degradation of Comirnaty, the mRNA-LNP vaccine against COVID-19 developed by Pfizer and BioNTech, after its expiration.¹¹ Buffers and cryoprotectants are known to be critical factors for the cryopreservation of nanoparticles.^12,13 Currently, the mRNA and LNPs used in COVID-19 vaccines require storage in ultra-low temperature freezers, although recent research suggests alternative storage methods such as lyophilization and spray-drying.^12,13

Single-use (SU) technology has been implemented in the manufacture of mRNA and LNPs for COVID-19 vaccines.¹⁴ SU bags are widely utilized for the storage, sampling, and transfer of mRNA and LNPs, as well as other biopharmaceutical fluids like cell culture media and buffers, across various production steps. These SU bags are essential for ensuring efficiency, sterility, and flexibility, as they eliminate the need for cleaning and validation processes associated with traditional stainless steel systems [Fig. 1(A)].^14,15 The adoption of SU setups significantly reduces capital expenditure compared to traditional stainless steel-based setups. They offer the necessary flexibility to scale production from small-scale laboratory settings to large-scale manufacturing. For instance, Sartorius provides SU bags made of plastics such as polyethylene (PE) and ethylene-vinyl acetate (EVA), available in sizes ranging from 5 ml to more than 50 L [Figs. 1(B) and 1(C)]. This scalability is particularly crucial for RNA vaccines, where rapid scale-up is often needed to respond to pandemics. Pre-sterilized SU bags minimize the risk of contamination and do not require the development and validation of cleaning methods. Consequently, SU technology has been shown to reduce the environmental impact, compared with reusable equipment, as it decreases water and energy consumption associated with cleaning processes.¹⁴ Additionally, their smaller cross-sectional area compared to bottles or vials facilitates a faster freeze–thaw workflow,¹⁶ which is crucial as slower thawing can lead to antibody aggregation and reduced cell viability.¹⁷ 

While SU bags have been utilized as a storage solution for mRNA and LNPs, there are currently no reports available on the long-term stability of LNPs in SU bags. In this study, we assessed the long-term stability of LNPs and mRNA in SU storage bags. To the best of our knowledge, this is the first report demonstrating that LNPs remain stable in SU bags for six months at −80 °C. Our findings indicate that while particle size and RNA concentration after storage were stable, the particle concentration, as measured by dynamic light scattering (DLS), decreased within an hour, suggesting potential particle adsorption to the storage containers. We further investigated the factors contributing to this adsorption.

Lipid nanoparticles (LNPs) were formulated using ALC-0315 (Avanti Polar Lipids), cholesterol (Sigma), DSPC (Lipoid), and ALC-0159 (Avanti Polar Lipids) lipids, with molar ratios of ALC-0315:DSPC:Cholesterol:ALC-0159 at 46.3:9.4:42.7:1.6, using a microfluidic mixer chip (PreciGenome) with a flow rate ratio of lipid to aqueous at 1:3. EGFP mRNA (TriLink BioTechnologies) was encapsulated within the LNPs. For the long-term stability study, samples underwent dialysis against Tris-buffered saline (Quality Biological) containing 10% sucrose (VWR) and were subsequently sterile-filtered using a syringe filter (VWR). For the short-term stability study using Flexsafe bags, LNPs were dialyzed against PBS (Boston Bioproducts). Particle size and derived mean count rate were measured using a Zetasizer Pro (Malvern Panalytical), and mRNA loading was quantified using RiboGreen assay (Thermo Fisher). Encapsulation efficiency (EE) was calculated using the formula $EE=(1−CfCt)×100$ (%), where C_f represents the free mRNA concentration and C_t the total mRNA concentration. The total mRNA concentration was measured by disrupting the lipid membrane with Triton X-100 (Sigma), while the free mRNA concentration was measured without addition of Triton X-100.

Storage bags, Flexboy and Flexsafe, were gamma-irradiated at 50 kGy and were supplied by Sartorius Stedim FMT.

LNPs were stored in Flexboy and Flexsafe storage bags (3 ml each) and sterile glass vials (ALK Life Science Solutions) (1 ml each). Samples were initially stored at 4 °C for 1 week, followed by transfer to a freezer at −80 °C for 6 months.

mRNA-loaded liposomes were prepared using in vivo-jetRNA+ (Polyplus) according to the manufacturer's instructions. EGFP mRNA was encapsulated in the liposomes, and the mRNA concentration was 4 μg/mL, with a lipid/mRNA ratio of 2 μL/1 μg. Samples were stored at 4 °C for 2 weeks.

mRNA was stored in Flexboy bags and cryovials at a concentration of 100 μg/mL in 1 mM sodium citrate (pH 6.5, Fisher) at −80 °C for 6 months. mRNA concentration was measured using Nanodrop One (Thermo Fisher), and RNA integrity was evaluated through agarose gel electrophoresis. For the transfection study to confirm mRNA functionality, the stored mRNA was encapsulated in in vivo-jetRNA+ prior to dosing.

Cells were sourced from the American Type Culture Collection (ATCC). HeLa cells were cultured in EMEM (Quality Biological) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMax (Gibco), and 1% PenStrep (Gibco). HEK293T cells were cultured in DMEM (Cytiva) with the same supplements. Cells were seeded in a 96-well plate one day prior to dosing. Fluorescent images were captured hourly for 3 days post-dosing using IncuCyte S3 (Sartorius). Cell viability, live cell count, and the percentage of GFP-positive cells were analyzed using IncuCyte AI Cell Health Analysis Software Module.

The aim of this study was to evaluate the long-term stability of lipid nanoparticles (LNPs) in single-use (SU) storage bags. We prepared an LNP formulation similar to Comirnaty by following the ingredient information provided on its product label. Flexboy bags, constructed with a multilayer film featuring inner and outer layers of ethylene-vinyl acetate (EVA) and a core layer of ethylene-vinyl alcohol (EVOH) for enhanced gas barrier properties, were selected for the long-term storage study [Fig. 1(C)]. Sartorius offers a freeze and thaw (F&T) product line called Celsius, which utilizes the same EVA and EVOH films. The choice of Flexboy bags was influenced by their construction, which is similar to the Celsius products. No particulate contamination was detected in the SU bags used for this study, as assessed by dynamic light scattering (DLS) (data not shown). As shown in Figs. 2(A) and 2(B), we confirmed the stability of LNPs in Flexboy bags at −80 °C for 6 months, with particle size and mRNA concentration remaining stable over time. Additionally, we assessed the in vitro transfection activity of LNPs stored in Flexboy bags for 3 months. Figures 2(C), 2(D), 3, and 4 demonstrate that HEK293T cells treated with cryopreserved LNPs began expressing enhanced green fluorescent protein (EGFP) within 3 h, with over 80% of the cells expressing EGFP after 8 h (Multimedia view). No significant difference in the number of live cells was observed between untreated cells and LNP-treated cells, as counted by IncuCyte. We also verified the short-term stability of particle size and encapsulated mRNA concentration in LNPs at 4 °C in Flexsafe (PE) bags for 1 week. Overall, there was no significant difference in particle size, mRNA concentration, and in vitro activity. Although in vivo activity and biodistribution studies after storage were beyond the scope of this research, such studies could further confirm the stability of LNPs.

LNPs are currently prominent in mRNA vaccine manufacturing. However, liposomes also present significant potential in mRNA therapy. Traditionally, liposomes have been used to deliver small molecules and proteins, such as in the cancer therapy drug Doxil and the hepatitis vaccines Epaxal.³ Recent advancements, like the lipoplex approach, have facilitated the delivery of mRNA using liposomes. Several research groups have recently reported the assembly of lipoplexes at the point of use, where mRNA is diluted and mixed with cationic liposomes just before administration.¹³ This approach eliminates the need for long-term stability studies, as mRNA and empty liposomes can be stored separately. A notable example is BioNTech's Lipo-MERIT (BNT111), a lipoplex for cancer vaccines, which has shown positive results in Phase 1 and 2 clinical trials (NCT02410733 and NCT04526899).^18,19 Given the potential for transfer and short-term storage of lipoplexes after mRNA loading, we evaluated the short-term stability of mRNA-loaded liposomes in SU bags. We loaded mRNA into liposomes (in vivo-jetRNA+) just before testing. The particle size and mRNA concentration of liposomes stored in both Flexsafe and Flexboy bags were confirmed to be stable at 4 °C for at least 2 weeks (Fig. 5). The RiboGreen assay confirmed that encapsulation efficiency was higher than 95%.

Storing and transferring larger volumes of mRNA in appropriate containers is crucial for the distribution of mRNA drug products. We assessed the long-term stability of mRNA in Flexboy bags (Fig. 6). Throughout the storage period, the mRNA concentration remained stable, and agarose gel electrophoresis confirmed the integrity of the mRNA. We evaluated the functionality of the stored mRNA in vitro by transfecting HeLa cells using liposomes [Figs. 6(C) and 6(D)]. Over 80% of the cells treated with mRNA stored for 4 months in both Flexboy bags and cryovials exhibited EGFP expression within 4 h, and the EGFP signal was maintained for 72 h. However, mRNA samples stored for six months both in Flexboy bags and cryovials demonstrated reduced potency; the percentage of EGFP-positive cells was lower and decreased over time. Although we did not detect any major degradations in agarose gel electrophoresis, there may be minor changes in the mRNA after long-term storage. Analyses with higher resolution could help in understanding the changes in mRNA over time.

No significant differences were observed between SU bags and vials in all tests. In this study, we stored small sample volumes (3 ml for bags, 1 ml for vials) in storage containers. However, in actual manufacturing situations, more than 3 ml of materials are typically stored in SU bags. Although we did not observe any difference between storage containers, the faster freeze–thaw workflow, attributed to the smaller cross-sectional area, could enhance product quality after storage and improve the overall manufacturing operation, particularly when larger volumes of materials are stored.

Nanoparticles are susceptible to adsorption due to their high surface area-to-volume ratio.²⁰ The adsorption of proteins and culture medium components to SU bags has been previously studied and shown to affect their potency during storage.²¹ For example, lipid adsorption from the culture medium onto certain films has been found to impact cellular growth.²² In dynamic light scattering (DLS), the mean count rate refers to the average number of photons detected per second by the photodetector.²³ This rate measures the intensity of scattered light from the particles in the sample. Although the mean count rate is influenced by several factors, it can be used for particle concentration assessment as long as particle size and the refractive indices of the solvent and lipids remain unchanged.²⁴ We observed a 15% reduction in particle concentrations for lipid nanoparticles (LNPs) and liposomes stored in both Flexboy bags and glass vials within the first hour [Figs. 7(A) and 7(B)]. A similar trend was observed with blank LNPs (Fig. S1). To determine if the decrease in particle concentration was due to particle adsorption to the storage containers, we conducted an additional experiment. After completing the storage study, we transferred the collected LNPs into both new and original Flexboy bags. One hour later, we retrieved the LNPs from the bags. The particle concentration for the samples in the original bag remained unchanged, while the particle concentration for the samples in the new bag decreased by an additional 7% [Fig. 7(C)]. This result suggests that the decrease is likely due to adsorption rather than material loss from transferring in and out of the bags.

In a separate experiment, we did not observe a decrease in the mean count rate for the LNPs stored in Flexsafe bags and glass vials [Fig. 7(D)]. Since there were no changes in particle concentration even when stored in glass vials, we hypothesized that the observed differences in mean count rate were due to variations in the LNP preparation procedure rather than the storage bag material. For the LNP used in the stability study with Flexboy bags, we utilized a lab-made LNP production setup that produced 1 ml of LNP at a time, requiring 2 h to prepare LNP samples due to equipment limitations. In contrast, for the LNP used in the stability study with Flexsafe bags, we utilized a dual-channel syringe pump (Chemyx), which allowed for the preparation of LNPs in just a few minutes. Since LNPs are unstable in a buffer solution containing 25% ethanol,²⁵ leaving the LNP samples at room temperature for an extended period before dialysis could have induced changes, such as compromising the integrity of the LNP structure and increasing particle size, potentially leading to particle adsorption to the storage bags. To test this hypothesis, we conducted an additional experiment by filling SU bags with blank LNPs under two conditions: immediate dialysis and delayed dialysis, where the samples were left at room temperature for 2 h without dilution in the dialysis buffer. We observed that the particle size of the samples after delayed dialysis increased by 8 nm (from 112 to 120 nm), suggesting potential particle fusion or aggregation. Additionally, these LNPs exhibited a 9% decrease in the mean count rate within one hour in both Flexboy and Flexsafe bags [Fig. 7(E)]. In another experiment, we observed that delayed dialysis of RNA-loaded LNPs caused aggregation after dialysis, increasing the particle size by 19 nm (from 107 to 126 nm), while these aggregated LNPs did not adsorb to glass vials. This suggests a competition between particle–surface and particle–particle interactions, leading to either adsorption or aggregation.^26,27 Although aggregation and adsorption are often associated and are complex phenomena,²⁷ they are related to particle formation and stabilization issues rather than the use of storage bags. This finding highlights the importance of a smooth LNP production procedure and maintaining the integrity of the LNP structure to minimize particle adsorption or aggregation. Diluting the produced LNPs immediately in the downstream buffer could help stabilize the LNPs and reduce particle aggregation and adsorption to the storage containers.²⁸ Given the stable mRNA concentration encapsulated in LNPs during the storage period [Fig. 2(B)], it is possible that instead of the entire particle being adsorbed, a component of the LNPs may have adhered to the storage containers. The adsorption of LNP components could impact the effectiveness of dosing. For example, PEG shedding from the LNP is known to affect the fate of LNPs, including cellular uptake and clearance by the immune system.²⁹ The effects of particle adsorption, possibly caused by small changes in LNPs, such as increases in particle size and changes in structural integrity, on the efficacy and fate of LNPs should be studied in the future.

This study demonstrated that SU bags are suitable for the long-term storage and transfer of LNPs and mRNA, providing valuable insights for the bioprocessing and manufacturing of genetic medicines. We identified potential lipid adsorption to storage containers and suggested that particle adsorption is influenced by the integrity of the LNP structure. Variations caused by particle adsorption could lead to inconsistencies in product quality and efficacy. Understanding the effect of adsorption on the fate of the LNPs and developing strategies to prevent it remain crucial areas for future research. Exploring surface modification of SU bags and alternative storage materials compatible with cryopreservation could help minimize particle adsorption. Additionally, reducing particle adsorption to storage containers, packaging, and syringes could further enhance the efficacy and safety profile of LNPs.

See the supplementary material for details on particle concentration changes of blank LNPs stored in Flexboy bags.

The authors appreciate the Sartorius Corporate Research Group and the Sartorius Stedim FMT team for their insightful discussions. We also thank the Polyplus-transfection team for generously providing materials to us. The Highlight Image was created with https://BioRender.com/h09g908.

Y.S.T., F.W., X.G., and S.A. are employees of Sartorius Stedim North America (SSNA) and receive their salaries from SSNA. L.D. is an employee of Sartorius Stedim FMT and receives a salary from the company. Y.S.T., F.W., and S.A. have a patent pending (WO2024013149A1).

Yuji S. Takeda: Investigation (lead); Methodology (lead); Visualization (lead); Writing – original draft (lead); Writing – review & editing (lead). Fujun Wang: Investigation (supporting); Methodology (supporting); Resources (supporting); Writing – review & editing (supporting). Xin Gu: Investigation (supporting); Writing – review & editing (supporting). Lucie Delaunay: Conceptualization (equal); Funding acquisition (equal); Project administration (equal); Resources (lead); Supervision (equal); Writing – review & editing (supporting). Samin Akbari: Conceptualization (equal); Funding acquisition (equal); Methodology (supporting); Supervision (equal); Writing – review & editing (supporting).

The data that support the findings of this study are available within the article and from the corresponding author upon reasonable request.