Determination of mRNA Encapsulation Efficiency with the Agilent 1290 Infinity II Bio LC System

Significance of the Topic

Efficient encapsulation of mRNA into lipid nanoparticles (LNPs) is a critical quality attribute for RNA-based therapeutics. Reliable quantification of encapsulation efficiency ensures proper dosing, stability and safety of mRNA vaccines and other gene-based medicines. Traditional fluorescence assays like RiboGreen cannot distinguish intact mRNA from truncated fragments, highlighting the need for orthogonal analytical methods with higher specificity.

Objectives and Study Overview

This study aimed to develop and validate an ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) method using the Agilent 1290 Infinity II Bio LC system to quantify encapsulation efficiency of F-luciferase mRNA loaded into LNPs. The method provides an alternative to fluorescence assays by separating full-length mRNA from impurities and quantifying extracted mRNA against a calibration curve.

Methodology

mRNA Synthesis and Quantification:

• In vitro transcription of F-luciferase mRNA from a PCR-amplified dsDNA template using a commercial T7 kit.
• Purification by spin columns and concentration determination by UV-Vis spectrophotometry (Agilent Cary 60), with calibration standards at 4.35, 8.7, 13, 34.8 and 43.5 µg/mL.

mRNA-LNP Preparation:

• LNPs formulated with a lipid composition matching Moderna’s Spikevax (SM-102, DSPC, DMG-PEG2K, cholesterol) via microfluidic mixing (aqueous : organic = 3 : 1, total flow 12 mL/min, N : P = 5.67 : 1).
• Buffer exchange into Tris pH 7.4 and concentration by ultracentrifugation (30 kDa cutoff).
• Samples either lyophilized or stored without freeze-drying, with or without cryoprotectants (mannitol, trehalose).

mRNA Extraction:

• Isopropanol precipitation: sample diluted in ammonium acetate/isopropanol, centrifuged, pellet washed and resuspended in nuclease-free water.

Chromatographic Conditions:

• Column: Agilent PLRP-S, 2.1×150 mm, 8 µm, 4000 Å, 50 °C.
• Mobile phase A: 100 mM triethylammonium acetate (TEAA), pH 7; B: 100 mM TEAA in acetonitrile.
• Gradient from 10 % to 90 % B over 27 min; flow rate 0.3 mL/min.
• Detection: diode array detector at 260 nm (4 nm bandwidth), reference 360 nm.

Instrumentation

• Agilent 1290 Infinity II bio LC system: high-speed pump, multisampler, multicolumn thermostat, DAD detector.
• Agilent OpenLab CDS v2.7 or later.
• Agilent Cary 60 UV-Vis spectrophotometer for concentration measurements.

Main Results and Discussion

• Chromatographic Separation: F-luciferase mRNA eluted at 13.2 min, with a minor fragment peak at 10.6 min, demonstrating clear resolution of intact mRNA and impurities.
• Calibration Performance: linear response over 4.35–43.5 µg/mL (R² > 0.997), LOQ < 5 µg/mL; retention time %RSD < 1 %, area %RSD < 10 % (n = 6).
• Extracted mRNA Analysis: LNP-extracted mRNA co-eluted with the standard, confirming method suitability for quantification of recovered mRNA.
• Encapsulation Efficiency: lyophilized LNPs with mannitol or trehalose showed ~108–110 % recovery (apparent over-recovery due to analytical variation), while non-lyophilized samples without protectant yielded lower efficiencies (~70 %). Cryoprotectants improved mRNA stability during storage.

Benefits and Practical Applications

• Direct separation of intact mRNA and fragments enhances specificity over fluorescence assays.
• High precision and linearity support routine QC and batch release testing of mRNA-LNP therapeutics.
• Method can be integrated into development workflows for formulation optimization and stability studies.

Future Trends and Opportunities

• Extending IP-RP-HPLC to other mRNA constructs and chemical modifications.
• Coupling with mass spectrometry for sequence-level confirmation of mRNA integrity.
• Automation of extraction and chromatographic workflows for higher throughput.
• Development of on-line sample preparation to reduce manual handling and increase reproducibility.

Conclusion

An IP-RP-HPLC method on the Agilent 1290 Infinity II Bio LC system offers a robust alternative to fluorescence assays for quantifying mRNA encapsulation efficiency in LNP formulations. The technique reliably separates full-length mRNA from fragments, delivers high precision and linearity, and reveals the beneficial effect of cryoprotectants on mRNA recovery.

References

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