Encapsulation of mRNA in lipid nanoparticles by membrane micromixing

Abstract

Messenger RNA encapsulated in lipid nanoparticles (mRNA-LNPs) are successfully used worldwide for vaccination against COVID-19. mRNA-LNPs are manufactured by mixing an ethanolic phase (containing an ionizable lipid, a phospholipid, a PEGylated lipid and cholesterol) and an aqueous phase (citrate buffer at pH 4.5 containing mRNA). The characteristics of the mRNA-LNPs obtained (size, internal structure, mRNA functional delivery in vivo, etc) depend on several parameters and among them are the mixing conditions of the ethanolic and aqueous phases. While the impact of these parameters is well-recognized for small scale preparations (some microliters), their effect at a larger scale is less well-known. The purpose of this study is to investigate the production of some hundreds of millilitres of mRNA-LNP suspension using a ceramic membrane (SPG Technology) as a new mixing device. Summarily, in this technique, the ethanolic phase permeates through the membrane pores and mixes with the aqueous phase flowing in a small annular pipe between the tubular membrane and an inside rod. First, empty LNPs were prepared to determine the effect of membrane pore size, flow rate, and the ratio between the ethanolic and aqueous phase volumes. The experimental conditions tested did not change the characteristics of the LNPs obtained. Second, mRNA-LNPs were prepared and the suspensions obtained were then submitted to buffer exchange/concentration/ sterile filtration dialysis. The resulting mRNA-LNPs had a particle size of 103 ± 5 nm, a polydispersity index of 0.15 ± 0.005, and encapsulation efficiency of 96 ± 0.0 %. mRNA-LNPs with similar characteristics were obtained using ethanol ratios of 1/4 (20 % ethanol in the final preparation) and 1/6 (14 % ethanol). Overall, membrane micromixing using a ceramic membrane was shown to be a suitable technology for the preparation of mRNA-LNP suspensions.