Technical Review: Single-use bags as a viable solution for long-term stability of lipid nanoparticles
- aprilt97
- Mar 16
- 4 min read
Updated: Apr 10
Title: Single-use bags as a viable solution for long-term stability of lipid nanoparticles
Authors: Takeda, Y., Wang, F., Perier, N., Gu, X., Delaunay, L., Akbari, S.
Affiliations: Sartorius Stedim North America Inc. and Sartorius Stedim FMT S.A.S.
Background Introduction
This paper sits at the intersection of pharmaceutical technology, nanomedicine, and RNA therapeutics – fields that have gained tremendous attention following the success of mRNA-based COVID-19 vaccines. Lipid nanoparticles (LNPs) have emerged as critical delivery vehicles for mRNA therapeutics, offering protection from degradation and facilitating cellular uptake. A significant challenge in the widespread adoption of these therapies involves ensuring the long-term stability of both mRNA and LNPs during storage and transportation.
Traditional approaches to LNP storage typically involve specialized containers or glass vials, which present challenges in terms of cost, scalability, and handling. These limitations become particularly pronounced at commercial manufacturing scales. The paper under review explores single-use (SU) bags as an alternative storage solution, addressing a critical need in the biopharmaceutical industry for effective, scalable storage methodologies for genetic medicines.
Fig 2. (A) Advantages of single-use bags: Highlighting benefits such as efficient storage, sterility, scalability, and reduced capital expenditure. (B, C) Examples of single-use bags: (B) Flexsafe Bag: Composed of a polyethylene (PE)-based film structure, suitable for intermediate processes above 0°C, including storage and mixing. (C) Flexboy Bag: 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.
Materials and Methodology
The researchers conducted a comprehensive investigation into the stability of mRNA and mRNA-loaded LNPs stored in single-use bags, specifically Flexboy® bags manufactured by Sartorius. For the preparation of the lipid nanoparticles, the study utilized PreciGenome mixer chips, which employ microfluidic technology to achieve controlled mixing of lipid and aqueous phases, resulting in uniform nanoparticle formation. This preparation method ensured consistent particle size distribution and encapsulation efficiency prior to storage testing.
The single-use bags featured a multilayer film construction with inner and outer layers of ethylene-vinyl acetate (EVA) and a structural core layer. The researchers stored mRNA-LNP formulations under two main conditions: long-term at -80°C for up to six months, and short-term at 4°C for up to two weeks.
Throughout the storage period, the team monitored several critical parameters including particle size and polydispersity index using dynamic light scattering (DLS), mRNA concentration, in vitro transfection efficiency, and evidence of particle adsorption to container surfaces. Comparative analyses between samples stored in SU bags versus conventional glass vials provided valuable baseline data for interpreting the results.
Fig 3. PreciGenome MIX-4 chip
Results
The study yielded several significant findings regarding LNP stability in single-use bags:
First, mRNA-loaded LNPs maintained remarkable stability for at least six months when stored at -80°C in SU bags. Throughout this extended period, no significant changes were observed in particle size or RNA concentration, indicating preservation of structural integrity. This finding is particularly noteworthy as long-term stability at ultra-low temperatures represents a critical requirement for mRNA therapeutic products.
Fig 4. Long-term stability of LNPs in SU bags. (A) Particle size remained stable for 6 months at -80°C. (B) mRNA concentration was stable, and mRNA encapsulation was maintained during storage. (C, D) Transfection assay for HEK293T cells after 3-month storage of LNP at -80°C. The percentage of EGFP-positive cell was assessed using IncuCyte. mRNA-loaded LNPs effectively transfected cells after cryostorage in both SU bags and glass vials. Scale bar, 400 lm.
Second, the researchers confirmed functional stability through in vitro transfection tests. The LNPs maintained consistent transfection efficiency after the six-month storage period, demonstrating that not only physical but also biological properties were preserved. This functional validation is crucial, as physical stability alone does not guarantee therapeutic efficacy.
Third, for short-term applications, mRNA-encapsulated LNPs showed acceptable stability when stored at 4°C for up to two weeks in SU bags, offering flexibility for temporary storage during manufacturing or distribution processes.
Fourth, the researchers observed a notable decrease in particle concentration within the first hour of storage, suggesting particle adsorption to the storage container walls. Further investigation revealed that both the LNP production procedure and the integrity of the LNP structure significantly impacted the degree of adsorption. Specifically, a smooth production process and robust LNP architecture minimized adsorption issues.
Conclusion
The study demonstrates the suitability of SU bags as an alternative to traditional methods for long term storage of mRNA LNPs. Comparative analyses between SU bags and glass vials provide a valuable reference point, and the extended storage period of six months showed promising results with realistic insights into potential commercial applications.
A particular strength lies in the investigation of particle adsorption, a phenomenon that can significantly impact product potency and dosing accuracy. By identifying factors that influence adsorption, the study provides practical guidance for manufacturers seeking to optimize their LNP storage processes. Further investigation will be vital for preventing adsorption during initial storage conditions.
A smooth production process was identified as a key factor in preventing both adsorption and aggregation, and microfluidic mixing has the strong ability to offer this. The PreciGenome Mix-4 Chip which was used in this experiment has the high mixing efficiency required to address this requirement.
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Interesting research.