pSIM: A next generation in vitro mRNA potency assay.

Potency testing for mRNA-lipid nanoparticle (LNP) vaccines remains challenging due to complex cell-based mechanisms and dependence on strain-specific antibodies. Here, we introduce a next-generation in vitro mRNA potency assay named as pSIM, pulsed stable isotope labeling by amino acids in cell culture (SILAC) internal dosing control (IDC) mass spectrometry (MS). The pSIM assay co-processes test and reference LNPs enabling effective concentration (EC50)-based or dosing-control-normalized linear slope readouts. We demonstrate its applicability across monocistronic and multivalent self-amplifying mRNA constructs for influenza and SARS-CoV-2, show an exploratory correlation between in vitro potency and in vivo neutralization in mice, and establish stability-indicating performance under heat stress, real-time storage, freeze-thaw, and RNA-lipid adduct formation. The assay is co-qualified for early phase quality control implementation to support phase-appropriate release and stability testing.