Rapid and Simple Analysis of LNP-Encapsulated mRNA Using a Microchip Electrophoresis System

Significance of the topic

mRNA therapeutics and vaccines rely on robust delivery systems such as lipid nanoparticles (LNPs) to protect nucleic acids and enable cellular uptake. Quality control of mRNA-LNP formulations (size, integrity, concentration and purity) is critical for safety, efficacy and regulatory compliance. Rapid, automated analytical methods that can assess encapsulated mRNA without time‑consuming purification steps are therefore valuable for development, manufacturing and stability testing of mRNA-based products.

Objectives and study overview

This application note demonstrates a simple, rapid workflow for analysis of LNP‑encapsulated mRNA (mRNA‑LNP) using the Microchip Electrophoresis System MultiNA II (MCE‑301). The goals were to show that the system can (1) detect and size mRNA inside LNPs without lipid removal, (2) provide sensitive concentration estimates, and (3) operate with a fully automated sample‑to‑result procedure suitable for high‑throughput screening in a QC/development context.

Methodology

Key methodological points summarized from the study:

• Fluorescent dye: SYBR Green II was used after dilution (1:100) to label RNA.
• Size standard: RNA 6000 Ladder served as a size marker (diluted prior to use).
• Sample preparation: equal volumes of sample and RNA marker solution were mixed; the mixture was heat‑denatured at 70 °C for 5 minutes and subsequently cooled at 4 °C for 5 minutes in accordance with draft USP guidance for mRNA analysis.
• LNP lysis: heat treatment alone was insufficient to liberate mRNA from LNPs; the nonionic surfactant Triton X‑100 (final 0.1%) was added prior to heat denaturation to release encapsulated RNA.
• Analysis: the RNA kit for MultiNA II was used; the platform automates gel preparation, sample application and data acquisition and yields rapid runs (~100 seconds per sample).

Used instrumentation

Instrumentation and reagents explicitly reported:

• Microchip Electrophoresis System MultiNA II (MCE‑301).
• RNA Kit for MultiNA II (P/N: 292‑27913‑91).
• SYBR Green II fluorescent dye (P/N: S‑7564), diluted 1:100 for use.
• RNA 6000 Ladder size standard (P/N: AM7152), diluted in THE RNA Storage Solution (P/N: AM7001).
• Formamide and TE buffer components as part of RNA separation buffer formulation.

Main results and discussion

Summary of analytical findings:

• MultiNA II generated clear electropherograms with identifiable mRNA peaks for prepared samples and for mRNA released from LNPs after surfactant treatment.
• Heat treatment alone produced only minimal detectable signal from mRNA‑LNP samples, indicating that thermal denaturation without lipid disruption is insufficient for quantitative release of encapsulated mRNA.
• Addition of 0.1% Triton X‑100 followed by heat denaturation produced a clear RNA peak whose estimated size matched a model mRNA control, indicating full‑length mRNA recovery rather than fragmented degradation products.
• No significant short RNA fragments (indicative of degradation) were observed under the tested conditions, supporting integrity of the detected mRNA.
• Analysis time per sample was approximately 100 seconds and the MultiNA II workflow automated gel preparation, loading and data acquisition, reducing hands‑on time compared with agarose gels or manual capillary methods.

Benefits and practical applications

Practical advantages highlighted by this work include:

• Rapid throughput: short migration times (~100 s/sample) enable many samples to be processed per day.
• Automation: automated gel preparation, sample application and data capture reduces operator variability and labor.
• No lipid removal required: surfactant‑assisted release (Triton X‑100) permits direct analysis of mRNA‑LNP formulations without prior lipid extraction or purification.
• Sensitivity and sizing: fluorescent detection with SYBR Green II yields sensitive detection and reliable sizing against an RNA ladder, useful for assessing integrity and presence of full‑length mRNA.
• Compliance alignment: the sample handling (heat denaturation and use of surfactant) follows draft USP guidance for mRNA quality assessment, facilitating method adoption in regulated environments.

Future trends and potential applications

Possible developments and uses building on this approach:

• Method validation and qualification for GMP/QC release testing of mRNA therapeutics and vaccines, including robustness, linearity and limit-of‑detection studies.
• Integration with orthogonal techniques (e.g., capillary electrophoresis with laser‑induced fluorescence, HPLC, nanoparticle tracking analysis) for comprehensive LNP characterization (encapsulation efficiency, particle size, polydispersity, lipid composition).
• Extension to different LNP formulations, payload types (siRNA, saRNA, oligonucleotides) and varied surfactant or enzymatic release strategies to broaden applicability.
• High‑throughput screening during formulation development, stability monitoring and batch release workflows thanks to the rapid automated cycle time.
• Potential for regulatory acceptance as standard QC tooling as industry and agencies converge on common analytical procedures for mRNA products.

Conclusion

The MultiNA II microchip electrophoresis platform enables a fast, automated and sensitive workflow for evaluating mRNA encapsulated in LNPs. The study shows that combining a mild surfactant (0.1% Triton X‑100) with heat denaturation releases intact mRNA for direct electrophoretic analysis without lipid removal. This approach shortens analysis time, reduces hands‑on sample preparation and aligns with draft regulatory guidance, making it attractive for development and quality control of mRNA therapeutics and vaccines.

References

• Analytical Procedures for Quality of mRNA Vaccines and Therapeutics, Draft Guidelines: 3rd Edition.