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Updated: May 16, 2025
Isolation of Small Noncoding RNAs from Human Serum
Published on: June 19, 2014
Reverse-phase chromatography removes double-stranded RNA, fragments, and residual template to decrease immunogenicity
Andreja Krušič1, Nina Mencin1, Marta Leban1
1Sartorius BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
Abstract:
mRNA is produced by in vitro transcription reaction, which also leads to formation of immuno-stimulatory impurities, such as double-stranded RNA (dsRNA). dsRNA leads to activation of innate immune response linked to inhibition of protein synthesis. Its removal from mRNA preparations increases efficiency of protein translation. Previous studies identified ion-pair reverse-phase high-performance liquid chromatography as a highly efficient approach for dsRNA removal. Here, we present a comprehensive study of IP-RP LC purification on monolith chromatographic supports for mRNA polishing, demonstrating its ability to remove dsRNA, as well as hybridized RNA fragments and residual DNA template, which are not fully removed by mRNA capture methods. We develop step elution methodology, including at microgram scale with novel spin columns operated by centrifugation. We demonstrate SDVB efficiency across a range of molecular sizes and explore the necessity for temperature control for effective dsRNA removal from self-amplifying RNA. SDVB-purified mRNA and saRNA showed significantly increased transgene expression in cell-based assays and reduced the activation of cell autonomous innate immunity in A549 at early time points. Our findings highlight the importance of IP-RP purification for high-quality mRNA production, while simplifying the technological requirements for its adoption in clinical mRNA and saRNA manufacturing processes.
Insights
Purifying messenger RNA (mRNA) with ion-pair reverse-phase (IP-RP) chromatography effectively removes impurities like double-stranded RNA (dsRNA). This enhances protein translation and reduces innate immune responses for improved mRNA therapeutics.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Messenger RNA (mRNA) production via in vitro transcription generates impurities, notably double-stranded RNA (dsRNA).
- dsRNA activates innate immune responses, inhibiting protein synthesis and reducing mRNA therapeutic efficacy.
- Current mRNA purification methods may not fully remove all critical impurities like dsRNA, hybridized fragments, and DNA templates.
Purpose of the Study:
- To comprehensively study ion-pair reverse-phase (IP-RP) liquid chromatography (LC) purification on monolith supports for mRNA polishing.
- To demonstrate the removal of dsRNA, hybridized RNA fragments, and residual DNA template using IP-RP LC.
- To evaluate the impact of this purification method on mRNA and self-amplifying RNA (saRNA) quality and function.
Main Methods:
- Development of a step elution methodology for IP-RP LC purification on monolith supports.
- Application of the method at microgram scale using novel spin columns operated by centrifugation.
- Assessment of SDVB (a type of monolith support) efficiency across various molecular sizes and investigation of temperature control for dsRNA removal from saRNA.
Main Results:
- IP-RP LC effectively removed dsRNA, hybridized RNA fragments, and residual DNA template, surpassing standard mRNA capture methods.
- SDVB-purified mRNA and saRNA demonstrated significantly increased transgene expression in cell-based assays.
- Purified mRNA and saRNA showed reduced activation of innate immunity in A549 cells at early time points.
Conclusions:
- IP-RP purification on monolith supports is crucial for producing high-quality mRNA and saRNA.
- This purification strategy simplifies technological requirements for clinical mRNA and saRNA manufacturing.
- Effective impurity removal via IP-RP LC enhances mRNA translation efficiency and reduces immunogenicity.
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