A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA
Open accessAbstract
The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications.
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dsRNA removal through HPLC or cellulose methods drastically enhances the translational efficiency of IVT mRNAs.
“It has been shown that dsRNA removal through HPLC or cellulose methods drastically enhances the translational efficiency of IVT mRNAs.13,14”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [14]
The Results section (“Cellulose-Purified IVT mRNA Translates Better In Vivo”; page unavailable) reports substantially higher protein levels after purification of U-containing mRNA and says cellulose and HPLC similarly increase its translational capacity. But for m1Ψ-containing mRNA, the increase after either method was minor and not statistically significant. The unqualified claim that purification drastically enhances translation of IVT mRNAs overstates the finding. (LLM, conf=0.88)
Experimental — accuracy not yet measureddsRNA depletion leads to a decrease of type I IFN and a translational enhancement of the mRNA into transfected DC cells.
“This dsRNA depletion leads to a decrease of type I IFN and a translational enhancement of the mRNA into transfected DC cells.”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [14]
The Results section reports that removing dsRNA by HPLC had been shown to eliminate IFN-α secretion by transfected dendritic cells. It also reports enhanced translation after purification, but that experiment measured protein production in mice, not in transfected dendritic cells. The paper further limits the IFN finding: cellulose-purified m1Ψ-containing mRNA no longer induced IFN-α, whereas U-containing mRNA still did. Page numbers are not provided in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measureddsRNA contaminants can be purified by high-performance liquid chromatography or cellulose chromatography.
“dsRNA contaminants can be purified by high-performance liquid chromatography (HPLC) or cellulose chromatography13,14”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [14]
Purification of IVT mRNA using cellulose, HPLC, FPLC, oligo(dT) purification, or TFF can further improve mRNA quality.
“Further improvement of mRNA quality can be made with purification of IVT mRNA using cellulose,39 high-pressure liquid chromatography (HPLC),40 fast protein liquid chromatography (FPLC),41 oligo(dT) purification,3 or tangential flow filtration (TFF).42,43”
From COVID-19 mRNA vaccines: Platforms and current developments (2022) · cites this paper as [39]
Removing double-stranded RNA contaminants further ablates in vitro transcribed RNA's immunostimulatory activity.
“Additional efforts to remove double-stranded (ds)RNA contaminants even further ablated IVT RNA's immunostimulatory activity [8,9]”
From The dawn of mRNA vaccines: the COVID-19 case (2021) · cites this paper as [8]
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“Plasmids were linearized with restriction enzymes, mRNA was produced using the MEGAscript T7 Transcription Kit (Ambion, USA), and purified using cellulose-loaded column (28).”
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- Authors
- Baiersdörfer, M., Boros, G., Muramatsu, H., Mahiny, A., Vlatkovic, I., Sahin, U., & Karikó, K.
- Affiliations
- BioNTech (Germany) (DE)
- Corresponding author
- Baiersdörfer, Markus
- Venue
- Molecular Therapy — Nucleic Acids
- Publisher
- Elsevier BV
- Published
- 2019-04
- Volume
- 15
- Pages
- 26-35
- ISSN
- 2162-2531 (ISSN-L)
- PMC
- PMC6444222
- OpenAlex
- W2915388137
- Cited by
- 569
- References
- 36
- Citations by year
- 2026: 106 · 2025: 112 · 2024: 107 · 2023: 83 · 2022: 81 · 2021: 55 · 2020: 21 · 2019: 3
- FWCI
- 13.24
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Nucleic Acids (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- BioNTech RNA Pharmaceuticals
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- in vitro transcribed mRNA, mRNA purification, interferon-alpha induction, in vivo gene delivery, mRNA translation
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