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Updated: Jan 27, 2026

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
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Published on: November 9, 2019

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A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA

Markus Baiersdörfer1, Gábor Boros1, Hiromi Muramatsu1

  • 1BioNTech RNA Pharmaceuticals, 55131 Mainz, Germany.

Molecular Therapy. Nucleic Acids
|April 2, 2019
PubMed

Insights

Manufacturing pure in vitro-transcribed (IVT) mRNA requires removing double-stranded RNA (dsRNA) contaminants. A new cellulose-based method efficiently purifies IVT mRNA, enhancing its suitability for therapeutic applications in vivo.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • RNA Therapeutics

Background:

  • In vitro-transcribed (IVT) mRNA is crucial for in vivo protein synthesis.
  • Double-stranded RNA (dsRNA) is a common contaminant in IVT mRNA production.
  • Existing dsRNA removal methods are complex, costly, and generate hazardous waste.

Purpose of the Study:

  • To develop a simple, fast, and cost-effective method for purifying IVT mRNA.
  • To eliminate dsRNA contaminants from IVT mRNA preparations.
  • To assess the efficacy of purified IVT mRNA for in vivo applications.

Main Methods:

  • A novel purification strategy utilizing the selective binding of dsRNA to cellulose in an ethanol-based buffer.
  • Standard laboratory techniques adaptable for scalable purification.
  • In vivo assessment of purified mRNA's translational efficiency and immunogenicity in mice.

Main Results:

  • The cellulose-based method effectively removes at least 90% of dsRNA contaminants.
  • High recovery rates (>65%) of purified IVT mRNA were achieved.
  • Purified IVT mRNA, including modified variants, showed enhanced in vivo translation and reduced interferon-alpha induction.

Conclusions:

  • Cellulose-based purification offers an efficient, reliable, and safe alternative for removing dsRNA from IVT mRNA.
  • This method yields highly pure IVT mRNA suitable for therapeutic applications.
  • The purification process is scalable for both microgram and milligram quantities.
Keywords:
RNA immunogenicityRNA purificationcellulose-based purificationdouble-stranded RNAin vitro transcriptionmessenger RNAnucleoside-modified RNA

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