O'Shea, A. R. | Search bioRxiv

bioRxiv2026

Double-Stranded RNA Profiling with Mass Photometry

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.

biophysics↗

bioRxiv2025

Mass photometry reveals stoichiometry and binding dynamics of bispecific tetravalent anti-VEGF-PD-1 antibody ivonescimab

BackgroundThe bispecific antibody ivonescimab targets programmed cell death protein 1 (PD-1) and vascular endothelial growth factor (VEGF). Recent clinical trials have shown it has greater efficacy against PD-L1 positive non-small cell lung cancer than pembrolizumab (Keytruda), a frequently prescribed anti-PD-1 monoclonal antibody. Ivonescimab binds to two VEGF and two PD-1 molecules, with complex formation through higher-order structure formation (or daisy chain binding). However, the binding stoichiometries and interaction dynamics of ivonescimab with VEGF and PD-1 have not been characterized in depth. MethodsWe used mass photometry (MP) and kinetic modelling to analyze these interactions, quantifying the complexes formed and their affinities. Dissociation constants (KD) for ivonescimabs binding to VEGF and PD-1 were calculated from equilibrium counts and real-time measurements, respectively. ResultsVEGF drove oligomerization of ivonescimab, which bound VEGF predominantly in a 2:2 stoichiometry, with KD=0.08 nM. Higher-order oligomeric complexes, present only at low abundance, displayed markedly weaker affinities (3.17 nM; 1.29 nM). Ternary complexes of ivonescimab with its two targets consistently presented two PD-1 antigens for each ivonescimab molecule, with a 1.66 nM KD for the binding of the first PD-1 and the slightly stronger 0.89 nM for the second PD-1 molecule. ConclusionsMP confirmed VEGF-induced ivonescimab oligomerization and revealed that dimers, not higher-order structures, were the most stable stoichiometry. MP enables detailed analysis of antibody-antigen interactions, even for bispecific antibodies that interact with antigens with complex stoichiometries. Statement of significanceWe report the first application of mass photometry to characterize the binding of the clinically promising bispecific antibody ivonescimab to its targets, VEGF and PD-1. The most stable assembly is a dimeric complex, not higher-order, as expected - advancing understanding of ivonescimab and demonstrating mass photometrys value for complex biologics analysis.

biophysics↗