Aggregate Analysis of mRNA Using the Agilent Infinity II Bio LC System and Bio-SEC Columns

Significance of the Topic

The reliable analysis of mRNA aggregates is critical for ensuring the safety and efficacy of mRNA-based biopharmaceuticals. Aggregation can impair translation, affect lipid nanoparticle formulation, and alter vaccine stability during storage, especially under low-temperature or acidic conditions. Robust methods for aggregate detection support quality control in both drug substance and drug product stages.

Objectives and Study Overview

This application note evaluates how column pore size and mobile phase composition influence size-exclusion HPLC analysis of mRNA aggregates in accordance with the United States Pharmacopeia (USP) Analytical Procedures for mRNA Vaccine Quality draft guidelines. Key aims include:

• Determining optimal SEC column dimensions for mRNA of varying lengths.
• Comparing phosphate buffer and Tris acetate/EDTA mobile phases for aggregate resolution.
• Quantifying aggregate levels in representative mRNA samples.

Methodology and Instrumentation

Standards and Reagents:

• Poly(A) RNA (4,831 nt average), DNA ladders (100 bp, 1 kbp), mRNA (CleanCap FLuc, CleanCap β-gal).
• Buffers: 150 mM phosphate (pH 7.0); 100 mM Tris acetate/2.5 mM EDTA.

Instruments and Software:

• Agilent 1290 Infinity II Bio LC System (high-speed pump, multisampler, column thermostat, diode array detector with bio-inert flow path).
• Columns: Agilent Bio SEC-5 with pore sizes 500 Å, 1000 Å, and 2000 Å (7.8 × 300 mm, 5 μm).
• Data processing: Agilent OpenLab CDS v2.7 with GPC add-on for molecular weight calibration.

Analytical Conditions:

• Flow rate: 0.6 mL/min; injection volume: 5 μL; UV detection at 260 nm; analysis time: 25 min.
• Column temperature: 25 °C (phosphate buffer) or 40 °C (Tris/EDTA buffer).

Main Results and Discussion

Column Screening with Ladder Standards:

• 500 Å column separates species <300 bp effectively; 1000 Å suits up to ~1000 bp; 2000 Å accommodates ~2000 bp and larger.
• Tris/EDTA mobile phase yielded slightly shorter retention times and higher apparent molecular weights than phosphate buffer but similar resolution patterns.

Aggregate Analysis of CleanCap FLuc (1,929 nt):

• Under 1000 Å column with Tris/EDTA, target mRNA and fronting aggregates eluted within the column range; aggregate content ~8.5%.
• 2000 Å column offered less clear separation between monomer and aggregates.

Aggregate Analysis of CleanCap β-gal (3,420 nt):

• 1000 Å column with Tris/EDTA showed ~24.9% aggregates but limited size range coverage.
• Screening recommended 2000 Å column for comprehensive aggregate detection: sample-dependent aggregates ~12.7% and ~9.6%.

Structural Considerations:
mRNA’s complex secondary structures and branching increase hydrodynamic radius compared to linear dsDNA, leading to broader peaks in SEC.

Benefits and Practical Applications

• Demonstrates a workflow for selecting column and buffer conditions matching mRNA size and structure.
• Enables reliable quantification of monomer versus aggregate fractions to support USP guideline compliance.
• Offers a biocompatible, iron-free flow path to prevent metal-induced RNA degradation.

Future Trends and Opportunities

Next-generation approaches may include:

• High-throughput or miniaturized SEC formats for rapid screening.
• Integration with mass spectrometry for direct mass measurements of aggregates.
• Advanced software and machine-learning tools for automated peak deconvolution and aggregate characterization.
• Development of novel column chemistries tailored to RNA secondary-structure analysis.

Conclusion

This study confirms that careful selection of SEC column pore size and mobile phase composition is essential for accurate mRNA aggregate analysis. The Agilent 1290 Infinity II Bio LC System, equipped with Bio-SEC columns and bio-inert components, provides a robust platform for characterizing mRNA quality attributes per USP guidelines. Column screening tailored to mRNA length ensures optimal monomer-aggregate resolution across diverse vaccine candidates.

References

1. USP. Analytical Procedures for mRNA Vaccine Quality Draft Guidelines: 2nd Edition. 2023.
2. Park C.; Chen X.; Yang J. R.; Zhang J. Differential Requirements for mRNA Folding Partially Explain Why Highly Expressed Proteins Evolve Slowly. PNAS 2013, 110(8), E678–E686.
3. Borodavka A.; Singaram S. W.; Stockley P. G.; Gelbart W. M.; Ben-Shaul A.; Tuma R. Sizes of Long RNA Molecules Are Determined by the Branching Patterns of Their Secondary Structures. Biophys. J. 2016, 111(10), 2077–2085.
4. Cheng F.; Wang Y.; Bai Y.; Liang Z.; Mao Q.; Liu D.; Wu X.; Xu M. Research Advances on the Stability of mRNA Vaccines. Viruses 2023, 15(3), 668.