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Purification of Double-Stranded RNA Impurities From In Vitro-Transcribed mRNA Using Size-Exclusion Chromatography
Kyle J Tynan1,2, Trina Mouchahoir1,2, Mark S Lowenthal1
1National Institute of Standards and Technology (NIST), Gaithersburg, Maryland, USA.
Abstract:
dsRNA is a highly immunogenic byproduct generated during in vitro transcription of mRNA, leading to unwanted immunostimulatory responses and reduced vaccine efficacy. Removal of dsRNA impurities from mRNA is challenging, as they share similar physicochemical properties. Here, we present a lab-scale size-exclusion chromatography method that effectively separates dsRNA impurities from mRNA. This method achieves baseline resolution between dsRNA impurities and mRNA (resolution factor of 1.6) while maintaining high recovery of the target mRNA (81.8%). This work introduces a novel chromatographic strategy for the removal of dsRNA from mRNA therapeutics.
Insights
Double-stranded RNA (dsRNA) impurities are removed from messenger RNA (mRNA) using a novel size-exclusion chromatography method. This technique enhances vaccine efficacy by purifying mRNA therapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Double-stranded RNA (dsRNA) is a byproduct of in vitro mRNA transcription.
- dsRNA triggers immunogenic responses, reducing mRNA vaccine efficacy.
- Separating dsRNA from mRNA is difficult due to similar physicochemical properties.
Purpose of the Study:
- To develop an effective method for removing dsRNA impurities from mRNA.
- To improve the purity and efficacy of mRNA therapeutics.
Main Methods:
- A lab-scale size-exclusion chromatography (SEC) method was developed.
- The SEC method was optimized for separating dsRNA from mRNA.
Main Results:
- Baseline resolution between dsRNA and mRNA was achieved (resolution factor = 1.6).
- High recovery of mRNA was maintained (81.8%).
- The method effectively removes immunogenic dsRNA impurities.
Conclusions:
- The developed SEC method provides a novel chromatographic strategy for purifying mRNA.
- This purification strategy is crucial for enhancing the safety and efficacy of mRNA therapeutics.
- This technique addresses a key challenge in the manufacturing of mRNA-based medicines.
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