Reverse-phase chromatography removes double-stranded RNA, fragments, and residual template to decrease immunogenicity...

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.