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

Messenger RNA (mRNA)-based vaccines and therapeutics are rapidly emerging as a new class of medicines. Capillary gel electrophoresis (CGE) is considered a promising analytical technology for characterizing critical quality attributes of mRNA, including its structural integrity and poly(A) tail length. However, limitations in method development, particularly in sample preparation methods for mRNA-loaded nanoparticle systems, throughput, and data processing, have hindered its broader implementation in high-throughput (HT) settings. In this study, we present a comprehensive parallel CGE HT analytical workflow, using the Agilent 5300 Fragment Analyzer with a 48-capillary array, to assess mRNA structural integrity and poly(A) tail length. We first developed an mRNA structural integrity workflow by thoroughly understanding instrument variability and systematically optimizing sample preparation procedures for both naked mRNA (70 °C, 5 min) and mRNA encapsulated in lipid nanoparticles (2 % Triton-X100, 70 °C, 5 min). We then further established a workflow to determine average poly(A) tail length by analyzing the apex of the peak through RNase T1 digestion, magnetic oligo(dT) beads purification, and a custom-designed size calibration ladder. Finally, we created an open-source-compatible informatics workflow that enables flexible peak integration and scalable batch analysis, significantly improving workflow efficiency and consistency. Together, this HT workflow offers a rapid and scalable approach for mRNA characterization, enabling efficient support of both early development and quality control of mRNA therapeutics.