High-throughput mRNA poly(A) tail characterization through on-off retention ion-exchange chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• High-throughput analysis of large nucleic acids like mRNA is crucial for therapeutic development.
• Current chromatographic methods face limitations in speed and resolution for mRNA characterization.
• Understanding the chromatographic behavior of mRNA under ion-exchange (IEX) conditions is essential.

Purpose of the Study:

• To investigate the salt-displacement sensitivity of oligonucleotides and mRNA under strong IEX conditions.
• To develop rapid, high-throughput IEX methods for characterizing mRNA poly(A) content and tail length.
• To establish a unified platform for efficient mRNA analysis supporting therapeutic development.

Main Methods:

• Utilized a normalized linear ionic strength model to redefine solvent-strength scaling in IEX.
• Systematically investigated the impact of mobile phase pH and column temperature on separation.
• Developed two complementary high-throughput IEX methods using ultra-short (5 cm) columns.

Main Results:

• Demonstrated pronounced salt-displacement sensitivity and 'on-off' elution behavior for oligonucleotides and mRNA.
• Achieved baseline separation and quantification of non-poly(A)- and poly(A)-mRNA species in 6.5 minutes.
• Separated poly(A) ladders (20-120 nt) in 4 minutes, enabling high-throughput poly(A) tail length determination.

Conclusions:

• Established 'on-off' elution behavior as a defining retention regime in IEX for nucleic acids.
• Developed a powerful strategy for high-throughput characterization of mRNA poly(A) content and tail length.
• The method shows strong potential for supporting process development and quality control of mRNA therapeutics.