Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics

Abstract

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic—DOTAP and DDAB; ionisable—MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics.

Continuous Microfluidic Production of Citrem-Phosphatidylcholine Nano-Self-Assemblies for Thymoquinone Delivery

“…However, we observed a slight decrease in the mean (±SD) nanoparticle sizes of TQ-free and TQ-loaded nano-self-assemblies on increasing TFR from 50 to 100 µL/min (Table 1). This is consistent with previous findings [40,45,[59][60][61], and most likely attributed to presence of a relatively higher shear stress at TFR of 100 µL/min, leading to self-assembly of both lipids in the presence of relatively higher concentration of ethanol at the intersection of the center and side microfluidic channels. This is associated with a provision of relatively shorter time for ethanol to diffuse out and mix with excess buffer on increasing TFR from 50 to 100 µL/min [40,45,59].…”

Resources, Production Scales and Time Required for Producing RNA Vaccines for the Global Pandemic Demand

“…The bottleneck for drug substance production is the formulation step, whereby the RNA is encapsulated into lipid nanoparticles (LNPs) using a controlled mixing process [ 8 , 35 ]. For this purpose, microfluidic mixing devices are commonly used to mix an aqueous stream containing the RNA with an ethanol stream containing the following four lipid components: phospholipids, polyethylene glycol (PEG) lipids, cholesterol and a proprietary ionisable lipid [ 35 , 36 , 37 , 38 , 39 , 40 ]. This bottleneck can be addressed by operating multiple microfluidic mixing devices in parallel or by using larger devices.…”

Self-assembled mRNA vaccines

“…This observation prompted extensive research about the structure-activity relationship (SAR) of novel lipid materials tailored for efficient mRNA delivery [ 4 , 139 , [148] , [149] , [150] ]. While the question remains just what parameters must be customized precisely to maximize the delivery of a particular nucleic acid payload, it is clear that the structure of LNPs is different depending on the nucleic acid payload [ 147 , 151 , 152 ] and is determined by the initial stages of self-assembly [ 153 , 154 ].…”