Double-Stranded RNA Profiling with Mass Photometry
Year: 2026
Type: preprint
Abstract: ABSTRACT Double-stranded RNA (dsRNA) is a potent immunogenic impurity and its detection is a critical quality attribute in characterizing mRNA therapeutics. Standard analytical methods are only able to resolve the bulk presence of dsRNA and cannot characterize the different sub-species that may be present within a mRNA sample. In this study, we use mass photometry (MP) as a single-molecule analytical platform for the simultaneous detection and characterization of dsRNA impurities in mRNA samples. We demonstrate how ionic strength can interfere with the stability of the mAb/dsRNA complex and measure the binding affinity (1 nM) under a set of parameters for reproducible characterization of the complex. We leverage the J2 antibody to identify antibody/dsRNA complexes that resolve dsRNA-positive species within an mRNA sample based on discrete molecular weights. Furthermore, we introduce a novel MP assay that harnesses the repulsive surface chemistry of uncoated glass to exclude the bulk mRNA analyte for higher loading concentrations to sensitively profile trace dsRNA impurities as antibody-bound species. The semi-quantitative MP aligns with the bulk values determined for ELISA and dot blot detection of dsRNA. This work establishes MP as a valuable next generation mRNA analytical tool for analyzing dsRNA byproducts within mRNA samples. GRAPHICAL ABSTRACT
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7161282429