Molecular Therapy: Affinity Purification Combined with Vazyme ELISA Advances dsRNA Control in mRNA Manufacturing

Clark et al. (2025) demonstrated that optimized IVT and affinity purification reduced dsRNA to ~0.00007%. Enabled by Vazyme's ELISA, the study highlights a new benchmark for RNA purity and the importance of trace-level quantification in mRNA manufacturing.

In June2025, Clark et al. from Repligen [https://www.repligen.com/dsrna] and etherna published their findings in MolecularTherapy: Nucleic Acids [https://pubmed.ncbi.nlm.nih.gov/40487356/], revealing for the first time that a dsRNA specific affinity chromatography can reduce dsRNA byproducts by over 100fold, to as low as ~0.00007% w/w, while maintaining full mRNA integrity. This method tackles a critical manufacturing challenge for safer, more effective mRNA vaccines and therapeutics.

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Why dsRNA Matters

Double-stranded RNA is a major immunogenic impurity in IVT-derived mRNA and can trigger innate immune responses, complicating both efficacy and regulatory approval. Removing dsRNA is especially difficult because it shares size, sequence, and charge characteristics with single-stranded mRNA. Even residual levels as low as 0.1% can activate interferon pathways, underscoring the need for ultra-sensitive detection and efficient removal strategies.

Traditional Purification - Methods Fall Short

Standard dsRNA removal methods, e.g. cellulose-based chromatography or reverse-phase HPLC, only partially clear these impurities. In side-by-side tests, Clark et al. found that affinity purification removed dsRNA by ~270-fold, driving residual levels down to ~0.0003% of total RNA. In contrast, cellulose reduced dsRNA by only ~13-fold and RPHPLC by ~58-fold. This means even after HPLC or ethanol-cellulose purification, trace immunogenic dsRNAs can persist, posing manufacturing and regulatory risks.

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Figure 1G/H: dsRNA quantification by ELISA shows affinity resin reduces dsRNA to ~0.0003%, outperforming cellulose and RP-HPLC by 10-20 times . Powered by Vazyme's dsRNA kit.

Affinity Resin and Optimized IVT: New Insights

The new study highlights that specialized dsRNA binding resins and smarter IVT recipes can dramatically cut dsRNA. Clark et al. showed that a dsRNA-specific affinity resin (AVIPure) essentially eliminated dsRNA (to ~0.00007% of mRNA) without harming yield or integrity. Importantly, they also compared standard vs. optimized IVT protocols (WT T7 enzyme, but tweaked conditions). The optimized protocol yielded ~6-fold less dsRNA (0.016% vs. 0.09% w/w before resin). After affinity purification, both routes reached the 10-4% dsRNA range (standard IVT right 0.0006%, optimized right 0.00007%). In short, reducing dsRNA upstream (via IVT optimization) and downstream (via affinity chromatography) are both powerful. Notably, the Vazyme ELISA's LOD ~0.00007% w/w (50 pg/mL) allowed reliable detection and reproducible quantification of these trace levels. This underscores that quantitative dsRNA ELISA readouts can distinguish enzyme/protocol differences that dot blot might miss.

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Figure 3E/F: ELISA-based dsRNA quantification reveals large differences in dsRNA content between IVT approaches (standard vs. optimized). Red (optimized) and blue (standard) bars are percent dsRNA measured by a sensitive ELISA.

In A549-cell reporter assays, standard IVT mRNA triggered ~100fold luciferase induction, whereas affinitypurified mRNA reduced interferon signaling to just 30-50% at 24h and 48h. Remarkably, optimized IVT combined with affinity purification produced interferon levels indistinguishable from negative controls, fully eliminating immunogenicity. Concurrently, GFP expression peaked in samples with the lowest interferon signal, and only the optimized/purified RNA sustained enhanced GFP activity at 48h, correlating lower immunogenicity with increased efficacy and reduced toxicity.

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Figure 4 A/B/C/D: Transfection of standard and optimized RNA in A549 cells, with and without dsRNA affinity resin purification.

Vazyme's Two-Pronged Solution

Vazyme's mRNA quality control toolkit played a pivotal role in this study:

EasyAna dsRNA ELISA Kit (DD3509)

- LOD < 10pg/mL, LOQ almost equal to 47pg/mL, enabling precise quantification of trace dsRNA.

- M2/M5 antibody pair specifically recognizes dsRNA greater than or equal to 40bp, unaffected by mRNA modification or length.

- Readytouse kit completes the assay in 4hours.

- ICH Q2(R2) validated, with full validation dossier available to meet regulatory requirements.

T7 Turbo RNA Polymerase - GMP4120/T7 Low-dsRNA RNA Polymerase-DD4126(D13)

- Cuts dsRNA ~100-fold versus wild-type T7, leaving residual dsRNA at only a few ppm

- Maintains high mRNA yield and integrity. In other words, yo- "fix the tap" without sacrificing product.

- GMPgrade quality, supporting clinical pipelines including FDA submissions.

Together, these tools form a complete solution: upstream enzyme engineering to suppress dsRNA formation, and downstream precision detection to monitor and validate its removal.

Vazyme is committed to empowering mRNA developers with robust, regulatory-ready tools for cleaner, safer, and more effective RNA therapeutics.

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