mRNA Purification Using Anion Exchange Chromatography At Ambient Temperature

Efficient mRNA purification is critical for manufacturing mRNA-based therapeutics and vaccines. Anion exchange chromatography (AEX) in bind/elute mode is effective for separating negatively charged nucleotide products like mRNA. However, the purification of larger mRNA molecules (>500 nucleotides) poses challenges due to strong multi-point binding and complex secondary structures. Traditional methods often require elevated temperatures or chaotropic reagents, which can be difficult to implement in GMP environments.

This study demonstrates a scalable, ambient-temperature purification method using strong anion exchange membranes and resins with quaternary amines. Dual gradient elution, which simultaneously increases pH and salt concentration in the presence of chaotropic salts, proved highly effective for mRNA elution. Key findings include high yield and purity for both 858-nt and 1,929-nt mRNA molecules. The approach also efficiently removes impurities such as dsRNA, aggregates, and DNA/RNA hybrids, which are critical for meeting quality specifications.

Compared to traditional salt gradient methods, the dual gradient technique achieved superior elution recovery and selectivity without compromising product integrity. Step elution experiments further validated the method’s scalability for pilot and commercial manufacturing, demonstrating yields of over 90% for shorter mRNA and approximately 75% for longer mRNA.

This optimized process offers a practical, GMP-compliant solution for large-scale mRNA purification, supporting the growing demand for mRNA therapeutics. By eliminating the need for elevated temperatures, this innovative method enhances efficiency and maintains product stability, ensuring reliable manufacturing outcomes.