mRNA-Lipid Nanoparticles: Quality Control Releasing Testing And Characterization

The successful introduction of Pfizer's Comirnaty® and Moderna's Spikevax® COVID-19 vaccines pioneered mRNA-LNP technology, accelerating the path to approval for other mRNA therapeutics. This achievement, though rapidly authorized, was the culmination of about three decades of development focused on: (1) synthetic mRNA production and (2) LNP delivery systems protecting mRNA and enabling cell entry.

mRNA-LNPs contain mRNA with the gene of interest, flanked by UTRs, a 5' cap, and a polyA tail, all influencing stability and translation. Synthetic mRNA is produced via in vitro transcription from a DNA template, often incorporating modified nucleotides for stability. Capping at the 5' end and polyadenylation at the 3' end, crucial for translation and stability, can occur during or after transcription. The resulting mRNA undergoes purification and buffer exchange.

LNPs are formed using an ionizable lipid, cholesterol, a helper phospholipid, and PEG, typically through microfluidic mixing of mRNA and lipids. This process leads to electrostatic interactions and self-assembly into nanoparticles encapsulating the mRNA. Particle size and encapsulation efficiency are controlled by process parameters. Ethanol is subsequently removed via buffer exchange.

As a novel modality, mRNA-LNP quality control and characterization are evolving. While existing analytical methods address current regulatory needs, new characterization techniques are continuously being developed. Comprehensive characterization is vital for demonstrating product comparability throughout development, ensuring no significant changes in safety or efficacy occur from early stages to commercialization, as even minor structural changes can have significant impacts.

In this paper, we outline the regulatory expectations for pharmaceutical QC release testing and discuss which additional characterization analytics that we anticipate will be required to comprehensively characterize the mRNA-LNP product.