Development of Biolayer Interferometry (BLI)-Based Double-Stranded RNA Detection Method with Application in...

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• In vitro-transcribed (IVT) mRNA is a key platform for therapeutics and vaccines.
• Double-stranded RNA (dsRNA) is a critical impurity in IVT mRNA, potentially causing adverse immune responses.
• Current dsRNA detection methods lack sensitivity, speed, or struggle with modified uridines.

Purpose of the Study:

• To investigate the reduced sensitivity of existing methods for modified dsRNA.
• To develop a rapid and sensitive assay for dsRNA detection in IVT mRNA.
• To enable real-time monitoring of dsRNA impurities during mRNA production.

Main Methods:

• Characterized binding affinities of anti-dsRNA antibodies to understand sensitivity limitations.
• Developed a biolayer interferometry (BLI) assay using Flock House Virus (FHV) B2 protein.
• Validated the BLI assay for various dsRNA types and modifications.

Main Results:

• The BLI assay detects dsRNA with uridine modifications (ψ, m1ψ, 5 moU) with high sensitivity.
• Quantified short, long, and hairpin-structured dsRNA impurities.
• Successfully monitored dsRNA removal during a purification process.

Conclusions:

• The BLI assay offers a sensitive and rapid method for dsRNA detection.
• This assay can ensure the quality and safety of IVT mRNA by monitoring impurities.
• Enables real-time impurity monitoring to prevent dsRNA-induced immunogenicity.