Quantification of Intact MDa Poly(A)-Tailed and Non-Tailed mRNA Fractions with Charge Detection Mass Spectrometry

Abstract The clinical success of messenger ribonucleic acid (mRNA) therapeutics has driven the need for robust analytical techniques capable of characterizing mixtures of megadalton (MDa) constructs. A critical attribute of these therapeutics is the presence and length of the 3′ poly(A) tail, which governs mRNA stability and translational efficiency. Charge detection mass spectrometry (CDMS) was used to distinguish and quantify intact MDa mRNA variants differing only by the poly(A) content. Initial analysis under native-like conditions was limited by salt adduction, but a 75% methanol solution resulted in nearly a five-fold increase in charge (from ∼60e to ∼280e) and substantially less adduction. Measured masses of the intact mRNA were 1.054 MDa and 1.027 MDa for the tailed and non-tailed mRNA constructs, respectively, consistent with the theoretical 26.6 kDa mass difference. Mixtures of various compositions resulted in broad, unresolved peaks at intermediate mass owing to residual salt adduction. A constrained Gaussian deconvolution of these data enabled accurate quantification across seven different mole fractions (R2 = 0.978). Notably, an analysis of a subpopulation of the highest charge-state ions revealed separation into two distinct peaks, demonstrating that mass resolution for MDa mRNA analysis is limited by adduction rather than instrumental factors, motivating additional studies on methods to increase charging for more accurate quantification. These results establish CDMS as a powerful, top-down method for precise monitoring of mRNA heterogeneity and degradation in high molecular weight therapeutics.