Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns

Area of Science:

• Biopharmaceutical analysis
• Chromatographic techniques
• Nucleic acid characterization

Background:

• Messenger ribonucleic acid (mRNA) therapies offer significant potential for treating various diseases.
• Product quality and safety are paramount for mRNA therapeutics, with impurities like fragments and aggregates impacting efficacy.
• Robust analytical methods are essential for characterizing mRNA products and ensuring their quality.

Purpose of the Study:

• To explore the utility of size exclusion chromatography (SEC) for characterizing messenger ribonucleic acid (mRNA) products.
• To identify optimal chromatographic conditions for the analysis of mRNA size variants and impurities.
• To establish a reliable SEC method for quality control of diverse mRNA samples.

Main Methods:

• Utilized state-of-the-art ultra-wide pore size exclusion chromatography (SEC) columns (1000 Å and 2500 Å).
• Investigated the impact of mobile phase composition, including magnesium chloride (MgCl2) concentration.
• Evaluated the effects of column length and flow rate on mRNA separation and impurity detection.

Main Results:

• A 1000 Å pore size column was optimal for analyzing mRNA products across a wide size range (500–5000 nucleotides).
• Addition of 10 mM MgCl2 improved resolution and recovery of large mRNA variants in specific samples.
• Increased column length or decreased flow rate led to apparent increases in low-molecular-weight species and peak tailing.

Conclusions:

• Optimized SEC methods provide effective characterization of mRNA products, crucial for therapeutic development.
• The 1000 Å SEC method is suitable for analyzing mRNA products from various suppliers and conditions.
• Careful consideration of chromatographic parameters is necessary to avoid artifacts and ensure accurate mRNA impurity profiling.