SEC-MALS for mRNA Characterization with the Agilent 1260 Infinity II Multi-Angle Light Scattering Detector

Importance of the Topic

The emergence of mRNA therapeutics, accelerated by COVID-19 vaccine development, has underscored the need for reliable analytical methods to characterize large and complex RNA molecules. Accurate measurement of molecular weight and aggregation is essential for ensuring product quality, safety, and efficacy in biopharmaceutical applications.

Objectives and Study Overview

This application note evaluates the combination of size exclusion chromatography with multi-angle light scattering detection (SEC-MALS) using the Agilent 1260 Infinity II MALS detector coupled to the Agilent 1290 Infinity II Bio LC System. The primary goals are to determine the molecular weight of mRNA monomers and aggregates, assess aggregate percentage, and demonstrate method precision and resolution.

Methodology

The SEC-MALS workflow employs an Agilent AdvanceBio SEC 1000Å, 7.8 × 300 mm, 2.7 µm column with a mobile phase of 150 mM phosphate buffer (pH 7). Key chromatographic conditions include a 0.6 mL/min flow rate, 30 min runtime, and injection volumes of 5–20 µL. Concentration detection is achieved via UV absorbance at 260 nm and refractive index (RI), with dn/dc values of 0.172 mL/g for mRNA. Calibration for interdetector delay and detector constant is performed with rituximab (MW 145,000 Da).

Used Instrumentation

• Agilent 1290 Infinity II Bio LC System (bio-inert flow path)
• Agilent 1260 Infinity II Multi-Angle Light Scattering Detector
• Agilent 1290 Infinity II Variable Wavelength Detector (260 nm)
• Agilent 1290 Infinity II Refractive Index Detector
• Agilent AdvanceBio SEC 1000Å Column, 7.8 × 300 mm, 2.7 µm
• Milli-Q ultrapure water system and 0.2 µm filters for buffer preparation

Key Findings and Discussion

• The monomeric mRNA peak (4,546 nucleotides) exhibited a measured MW of 1,453,000 Da, matching the theoretical value with ~2 % RSD precision.
• Aggregate analysis by UV and RI signals indicated 74 % monomer, 20 % dimer (3,205,000 Da), and 6 % higher-order species (~7,150,000 Da).
• Wide pore SEC columns enabled clear separation of mRNA monomer and dimer; calibration with rituximab ensured accurate light scattering calibration.

Benefits and Practical Applications

SEC-MALS provides direct determination of molecular weight and aggregation profiles without reliance on molecular standards. This approach enhances quality control for mRNA-based therapeutics, supports formulation development, and aids in bioprocess monitoring by delivering robust, biocompatible analysis under physiological buffer conditions.

Future Trends and Potential Applications

• Integration of higher throughput SEC-MALS platforms for screening large mRNA libraries.
• Combination with orthogonal detectors (e.g., mass photometry) for advanced biophysical characterization.
• Application to diverse RNA constructs, including modified nucleotides and lipid nanoparticle formulations.
• Automated workflows for in-process control and real-time monitoring in mRNA manufacturing.

Conclusion

The coupling of Agilent SEC and MALS technologies delivers precise molecular weight determination and aggregate quantification for large mRNA molecules. This method offers high resolution, reproducibility, and compatibility with biopharmaceutical buffer conditions, making it a valuable tool for mRNA therapeutic development and quality assurance.

Reference

1. Sahin U., Karikó K., Türeci Ö. mRNA-Based Therapeutics—Developing a New Class of Drugs. Nat. Rev. Drug Discov. 2014;13(10):759–780.
2. United States Pharmacopeia–National Formulary. Analytical Procedures for mRNA Vaccine Quality (Draft Guidelines), 2nd Edition; 2023.
3. De Vos J., Morreel K., Alvarez P., Vanluchene H., Vankeirsbilck R., Sandra P., Sandra K. Evaluation of Size-Exclusion Chromatography, Multi-Angle Light Scattering Detection and Mass Photometry for the Characterization of mRNA. J. Chromatogr. A. 2024;1719:464756.
4. Held D. Tips & Tricks GPC/SEC: How to Determine dn/dc Values. The Column. 2015;10(7):10–15.
5. Radke W. Tips & Tricks: New and Old Approaches to Handle Unknown dn/dc in GPC/SEC-LS. The Column. 2017;13(14):19–23.
6. Agilent Technologies. Advanced SEC-MALS Analysis of Monoclonal Antibodies with the Agilent 1260 Infinity II Multi-Angle Light Scattering Detector. Application Note. 2024;5994-7664EN.