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.

Background/Objectives: Lipid nanoparticles (LNPs) are a prominent example of delivery systems that are used to prevent the degradation of messenger ribonucleic acid (mRNA) and facilitate cell uptake. Improving LNP transfection efficiency requires careful selection of key formulation components, including the ionizable lipid and the coding sequence of the nucleic acid. Therefore, it is crucial to assess various options for the target cells, as results can differ significantly between cell types. Building on previous work investigating the effect of apolipoprotein E4 on LNP transfection of human monocyte-derived dendritic cells, we assess the impact of different ionizable lipids and compare modifications in the mRNA uridine to further optimize the delivery to these cells. Methods: LNPs containing eGFP mRNA with different uridine modifications were produced via microfluidic mixing and investigated for their in vitro transfection efficiency of human monocyte-derived dendritic cells. Transfection occurred in the presence of apolipoprotein E4 for different encapsulated mRNA concentrations. Delta mean fluorescence intensity and eGFP positive cells were measured by flow cytometry 48 h after transfection. Cell viability was assessed via AnnexinV/7-AAD staining, after comparing this method to LIVE/DEAD^TM Fixable Near-IR staining. Results: This study shows that a combination of SM-102 as the ionizable lipid with eGFP mRNA containing N1-methylpseudouridine enabled the transfection of human monocyte-derived dendritic cells with very high efficiency at low concentrations, allowed for dose sparing, and even led to the LNPs outperforming a specifically tailored electroporation protocol. Conclusions: Improvement of nucleic acid delivery to human monocyte-derived dendritic cells, known for their difficulty to be transfected, was achieved by LNP formulation tuning.