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Purifying circular RNA by ultrafiltration
Biorxiv : the Preprint Server for Biology
|December 16, 2024
Summary
Circular RNA (circRNA) offers enhanced stability for therapeutics. This study demonstrates ultrafiltration effectively purifies circRNA, achieving 86% purity, surpassing traditional methods for scalable manufacturing.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Messenger RNA (mRNA) vaccines have spurred interest in mRNA therapeutics.
- Linear mRNA's short lifespan due to degradation limits its therapeutic potential.
- Circular RNA (circRNA) offers superior stability but requires high purity, which is challenging to achieve.
Purpose of the Study:
- To explore ultrafiltration for purifying protein-encoding circRNA from in vitro transcription (IVT) mixtures.
- To assess the efficacy of ultrafiltration compared to size-exclusion high-performance liquid chromatography (SE-HPLC).
Main Methods:
- Investigated ultrafiltration using polyethersulfone membranes (30–300 kDa MWCO).
- Measured sieving coefficients for circRNA, linear precursor RNA, and nicked RNA conformers.
- Determined critical fluxes and optimal operating conditions for RNA purification.
Main Results:
- Achieved 86% purity and >50% yield using ultrafiltration.
- Size-exclusion high-performance liquid chromatography (SE-HPLC) yielded 41% purity and 45% yield.
- Ultrafiltration demonstrated superior performance in circRNA purification.
Conclusions:
- Ultrafiltration is a highly effective method for purifying circRNA at the research scale.
- The scalability of ultrafiltration suggests its potential for large-scale circRNA therapeutic manufacturing.
- This purification method addresses a key bottleneck in developing stable circRNA-based therapies.
