The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case

Abstract

The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles.

Keywords: Degradation mechanisms; Formulation; Impurities; Ionizable lipid; LNP; Lipid nanoparticles; Physical and chemical analysis; Shelf life; Stability; Vaccine; mRNA structure.

Fig. 1

Overview of the nucleotides found in natural and in vitro transcribed (IVT) mRNA molecules. While natural mRNA contains uridine, IVT mRNA instead may contain modified pseudouridine to increase the safety and efficacy of mRNA vaccines.

Fig. 2

Schematic diagram of IVT mRNA primary structure (5’ terminal cap, untranslated regions, antigen coding region, 3’ poly-A tail) including chemical structure of the 5’-cap dinucleotide caps. (UTR, untranslated regions)

Fig. 3

Mechanism of the degradative transesterification reaction in mRNA strands leading to strand cleavage. B denotes a Brönsted base and BH⁺ is the corresponding conjugate acid.

Fig. 4

Schematic of theoretical mRNA transesterification mechanisms and their sites on the mRNA molecule. [O]: oxidant. The R (lipid)-aldehyde forming the lipid-mRNA adduct is the result of lipid oxidation reactions of the ionizable lipid.

Fig 5

Tris buffer acting as an “aldehyde sink” by formation of an imine adduct (adapted from Moderna Science and Technology day, May 2022, slide 76)

Fig 6

Schematic of ‘ready-to-use’ mRNA drug product formulations currently marketed versus an approach using ‘at point of use’ mRNA drug product formulation by employing a ‘kit-based approach’.