Double-Stranded RNA Profiling with Mass Photometry
- 1Refeyn
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 (e.g., sandwich ELISA) 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 then leverage the J2 antibody to identify antibody/dsRNA complexes that then resolve dsRNA-positive species within an mRNA sample based on discrete molecular weight profiles. Furthermore, we introduce a novel MP assay that harnesses the repulsive surface chemistry of uncoated glass to exclude the bulk mRNA analyte to enable the use of higher loading concentrations to sensitively profile trace dsRNA impurities as antibody-bound species. This work establishes MP as a valuable next generation mRNA analytical tool for analyzing dsRNA byproducts within mRNA samples.
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BibTeX
@misc{ranaghan2026doublestranded,
title = {{Double-Stranded RNA Profiling with Mass Photometry}},
author = {M. J. Ranaghan and N. E. Clark and K. Fay and A. R. O'Shea and N. Markova and S. Cheeseman and G. Kiss},
howpublished = {bioRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.15.725554},
url = {https://www.biorxiv.org/content/10.64898/2026.05.15.725554v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Double-Stranded RNA Profiling with Mass Photometry
AU - M. J. Ranaghan
AU - N. E. Clark
AU - K. Fay
AU - A. R. O'Shea
AU - N. Markova
AU - S. Cheeseman
AU - G. Kiss
PY - 2026
DA - 2026/05/15
PB - bioRxiv
DO - 10.64898/2026.05.15.725554
UR - https://www.biorxiv.org/content/10.64898/2026.05.15.725554v2
AB - Double-stranded RNA (dsRNA) is a potent immunogenic impurity and its detection is a critical quality attribute in characterizing mRNA therapeutics. Standard analytical methods (e.g., sandwich ELISA) 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 then leverage the J2 antibody to identify antibody/dsRNA complexes that then resolve dsRNA-positive species within an mRNA sample based on discrete molecular weight profiles. Furthermore, we introduce a novel MP assay that harnesses the repulsive surface chemistry of uncoated glass to exclude the bulk mRNA analyte to enable the use of higher loading concentrations to sensitively profile trace dsRNA impurities as antibody-bound species. This work establishes MP as a valuable next generation mRNA analytical tool for analyzing dsRNA byproducts within mRNA samples.
ER -