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

Christine Vo¹, Raymond Lieu¹, Yuchen Fan¹

¹Synthetic Molecule Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Journal of Chromatography. A

|March 31, 2026

Summary

Researchers discovered rapid separation of mRNA and oligonucleotides using ion-exchange chromatography (IEX). This breakthrough enables high-throughput analysis of mRNA therapeutics, improving quality control and process development.

Keywords:

High-throughput analysis Ion-exchange chromatography Linear ionic strength model On-off retention Poly(A) tail mRNA therapeutics

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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.