Developing a high-throughput capillary gel electrophoresis workflow for mRNA integrity and poly(A) tail analysis.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Messenger RNA (mRNA) therapeutics represent a significant advancement in medicine.
• Capillary gel electrophoresis (CGE) is a key technology for mRNA quality assessment.
• Current CGE methods face challenges in high-throughput (HT) settings due to sample preparation and data processing limitations.

Purpose of the Study:

• To develop a comprehensive, parallel CGE HT analytical workflow for mRNA characterization.
• To assess mRNA structural integrity and poly(A) tail length efficiently.
• To enable scalable batch analysis for mRNA therapeutics development and quality control.

Main Methods:

• Utilized a 48-capillary array Agilent 5300 Fragment Analyzer.
• Optimized sample preparation for naked mRNA and lipid nanoparticle-encapsulated mRNA.
• Developed poly(A) tail length determination using RNase T1 digestion and magnetic oligo(dT) beads.
• Created an open-source-compatible informatics workflow for data analysis.

Main Results:

• Established optimized sample preparation protocols for both naked and encapsulated mRNA.
• Successfully determined mRNA structural integrity and average poly(A) tail length.
• Implemented a scalable informatics workflow improving analysis efficiency and consistency.
• Demonstrated a rapid and robust HT CGE approach for mRNA characterization.

Conclusions:

• The developed HT CGE workflow provides a rapid and scalable solution for mRNA characterization.
• This analytical approach supports efficient quality control in mRNA therapeutic development.
• The workflow enhances the broader implementation of CGE in high-throughput settings.