Nanotechnology-assisted RNA delivery has gotten a tremendous boost over the last decade and made a significant impact in the development of life-changing vaccines and therapeutics. With increasing numbers of emerging lipid- and polymer-based RNA nanoparticles progressing towards the clinic, it has become apparent that the safety and efficacy of these medications depend on the comprehensive understanding of their critical quality attributes (CQAs). However, despite the rapid advancements in the field, the identification and reliable quantification of CQAs remain a significant challenge. To support these efforts, this review aims to summarize the present knowledge on CQAs based on the regulatory guidelines and to provide insights into the available analytical characterization techniques for RNA-loaded nanoparticles. In this context, routine and emerging analytical techniques are categorized and discussed, focusing on the operation principle, strengths, and potential limitations. Furthermore, the importance of complementary and orthogonal techniques for the measurement of CQAs is discussed in order to ensure the quality and consistency of analytical methods used, and address potential technique-based differences.

Lipid nanoparticles (LNPs) have emerged as a groundbreaking delivery platform, revolutionizing the development of nucleic acid-based medicines for gene delivery and gene therapy. This review provides an insightful industrial perspective on the production process of LNPs, focusing on cutting-edge manufacturing equipment, downstream processing and the crucial transition from laboratory to large scale. While LNP production in the discovery phase relies on a small scale (µL to mL) for screening various LNP formulation candidates, transferring to preclinical (up to hundreds of mL) and clinical/commercial scales (up to liters) requires a robust and reproducible manufacturing process. Thus, mixing technologies throughout these scales must be carefully selected and require precision, scalability and high reproducibility to meet the target quality of the LNP drug product. Key mixing technologies in mRNA-LNP production primarily include microfluidic systems and impinging jet mixers (IJMs). In this review, we discuss key critical process parameters (CPPs) in LNP preparation, including flow rate ratio (FRR) or total flow rate (TFR), in relation to associated critical quality attributes (CQAs) across multiple manufacturing scales. We further assess the impact of downstream processing, specifically tangential flow filtration (TFF), on the formulation’s CQAs. In particular, the review highlights the importance of maintaining CQAs along each step of the process and emphasizes the role of robust analytical methods in ensuring product quality and safety. Additionally, we touch on current challenges associated with these advanced delivery vehicles, such as their long-term stability, and introduce the readership to innovative stabilization strategies aimed to extent LNP shelf-life.

Safe and functional delivery of delicate mRNA molecules to target tissues is a crucial step in the development of effective vaccines and therapeutic interventions. Lipid nanoparticles (LNP) are the most clinically advanced delivery vehicles for mRNA drugs and crucially rely on ionizable cationic lipids. However, the structure‐function relationships between ionizable lipids and efficient in‐vivo mRNA delivery remain poorly understood. Here, we focus on the rational design and sequential structural optimization of our ionizable lipids that performed well in vitro, but not in vivo. Through two distinct iterative optimization cycles—targeting the lipid tail and the headgroup—we studied how fusogenicity and pKa of ionizable lipids contribute to LNP delivery performance, in vivo. By engineering lipids with both unsaturated tails and more hydrophobic amino headgroups, we achieved both significant improvement of protein expression in vitro, reduced hemolysis risk, and more than 200‐fold improvement of in vivo mRNA delivery. When compared head‐to‐head to a market‐approved LNP benchmark, the newly developed ionizable lipids/LNP resulted in equally highly efficient in vivo mRNA delivery, with strong liver and spleen tropism upon intravenous injection, while matching the safety of the approved platform. Our findings are pivotal for the development of next‐generation mRNA‐LNP therapies and vaccines.

Background/Objectives: This study investigated the effect of storage conditions on mRNA-LNPs in situ via identification of the formulation traits necessary for improving storage stability. Methods: We synthesized an ionizable lipid, namely TOT-28, which has a hydrolysis-susceptible ester bond in its hydrophilic head group that allows it to act as an indicator of the hydrophilic environment within the mRNA-LNPs. LNPs were stored either at 4 or 25 °C for up to 8 weeks to investigate the effect of pH and temperature on ester hydrolysis, internal mRNA integrity, physicochemical properties of the LNPs, and mRNA gene expression. Results: The results indicate that, at 25 °C, a lower buffer pH increases ester hydrolysis, whereas an opposite trend slightly occurs in ester hydrolysis with storage at 4 °C. We also found that TOT-28-based LNPs were less hydrated and microviscosity was higher at 4 °C compared with storage temperature at 25 °C. Therefore, TOT-28-based LNPs seem less sensitive to external buffer solutions because of a higher-order structure when stored at lower temperatures. In addition, we found that LNPs with different ionizable lipid structures exhibit distinct responses to pH changes at specific storage temperatures. Conclusions: Our findings provide novel insights into the appropriate conditions for long-term storage of the mRNA-LNPs as a liquid formulation.