Characterizing Nucleic Acid Payloads in Lipid Nanoparticles and Viral Vectors via Charge Detection Mass Spectrometry (CDMS)

Significance of the topic

The accurate characterization of nucleic acid payloads in delivery vehicles such as lipid nanoparticles (LNPs) and adeno-associated viruses (AAVs) is central to the development, quality control and regulatory compliance of nucleic acid therapeutics. Payload integrity, length distribution and impurity content directly affect potency, safety and stability. Conventional ensemble measurements provide limited resolution for high-molecular-weight, heterogeneous genomes; single-particle techniques that deliver direct mass and charge information are therefore strategically important for both R&D and QC applications.

Objectives and study overview

This work demonstrates a unified analytical workflow based on charge detection mass spectrometry (CDMS) to:

• Directly measure intact AAV capsids and quantify packaging efficiency at single-particle level.
• Isolate and determine intact masses of nucleic acid payloads released from AAV and LNP formulations while minimizing fragmentation.
• Resolve full-length genomes, truncated species and other impurities to better define payload heterogeneity across viral and non-viral platforms.

Methods

Key experimental steps for both vector classes were tailored to maximize recovery of intact nucleic acids and to preserve their mass signatures for CDMS analysis:

• AAV processing: USP AAV8 full reference capsids were heat-digested at 56°C for 40 min with agitation (1000 rpm). Genomic payloads were isolated using a spin-column DNA cleanup kit, followed by buffer exchange to 20 mM ammonium acetate via P6 gel columns prior to analysis.
• LNP deformulation: Ionizable LNP samples were diluted into a solution of 60 mM ammonium acetate in isopropanol (100% IPA), vortexed for 30 s to release payloads and impurities. Intact LNP samples intended for whole-particle measurement were buffer-exchanged (P6 columns or dialysis using Slide-A-Lyzer devices) at 4°C.
• Introduction and detection: Samples were introduced by nano-electrospray ionization into a benchtop Xevo CDMS instrument. CDMS provides simultaneous single-particle mass and charge measurements. Data processing and quantitative evaluation used the CDMS Toolkit within waters_connect software.

Instrumentation used

• Xevo CDMS benchtop charge-detection mass spectrometer (Waters Corporation).
• Monarch Spin PCR and DNA Cleanup kit (New England Biolabs) for AAV genome isolation.
• Micro Bio-Spin P6 Gel Columns (Bio-Rad) for buffer exchange; Slide-A-Lyzer MINI Dialysis Devices (Thermo Scientific) for dialysis.

Main results and discussion

• AAV capsid populations: Single-particle CDMS resolved distinct capsid classes and enabled quantification of packaging states: Empty ~4%, Partial ~16%, Full ~77%, Overfull ~3%. This demonstrates robust assessment of packaging efficiency at the particle level rather than ensemble averages.
• Isolated AAV genomes: The applied AAV workflow preserved intact genomes and revealed populations corresponding to expected full-length genomes along with truncated and impurity species. Observed centroid masses matched expected genome masses within a few percent for most species, with some deviations indicating truncated species or co-isolated contaminants.
• LNP and mRNA payloads: Intact LNPs produced broad mass distributions consistent with variable payload loading per particle. Deformulation combined with CDMS detected full-length modified mRNA (observed mass ~1.46 MDa for a 4101-nt payload) alongside lower-mass impurities originating from truncated nucleic acids or LNP-derived components. Charge versus mass 2D plots and charge histograms provided orthogonal information on particle ionization behavior and helped distinguish nucleic acid species from non-nucleic acid debris.
• Sensitivity to heterogeneity: CDMS resolved reproducible mass intervals corresponding to full-length payloads, truncated genomes and other low-mass species, demonstrating its utility to dissect complex, polydisperse samples that challenge conventional mass analyzers.

Practical benefits and applications

• Direct single-particle mass and charge readout enables accurate quantitation of packaging efficiencies and payload heterogeneity relevant for potency and safety assessments.
• The ability to measure intact released genomes avoids reliance on indirect surrogate assays and provides direct evidence for truncations and degradation products.
• Applicable to both viral (AAV) and non-viral (LNP) platforms with vector-specific sample preparation workflows, making CDMS a cross-platform analytical approach for next-generation nucleic acid therapeutics.
• Useful across development stages—formulation optimization, stability testing, degradation pathway elucidation and QC release testing—where knowledge of intact payload mass distributions is required.

Future trends and possibilities for use

• Identification of impurities: Follow-up studies combining CDMS with orthogonal identity methods (next-generation sequencing, capillary electrophoresis, enzymatic digestion or LC-MS of degraded fragments) will clarify the molecular origin of lower-mass species detected by CDMS.
• Correlation workflows: Integrating intact particle measurements with extracted payload profiles will enable mechanistic insight into loading efficiency, capsid-payload interactions and degradation routes during storage or processing.
• Method standardization: Development of standardized sample preparation and data-processing protocols for CDMS will support broader adoption in regulated environments.
• Higher-throughput implementations and improved mass/charge resolving power could expand CDMS applicability to routine QC screening and in-process monitoring for biologics manufacturing.

Conclusion

Charge detection mass spectrometry provides a powerful single-particle approach to directly quantify and characterize nucleic acid payloads from both AAV and LNP delivery systems. Tailored sample preparation workflows preserve payload integrity and allow resolution of full-length genomes, truncated species and impurities. CDMS bridges analytical gaps left by ensemble techniques and offers a robust platform for payload integrity assessment that can inform formulation, development and quality control of nucleic acid therapeutics.

Reference

1. Keifer DZ, Pierson EE, Jarrold MF. Charge detection mass spectrometry: weighing heavily charged ions. Anal Chem. 2017;89:826–833.
2. Wörner TP, Snijder J, Bennett A, et al. Resolving heterogeneous macromolecular assemblies by charge detection mass spectrometry. Nat Methods. 2020;17:395–398.
3. Hartmann MD, et al. Characterization of adeno-associated virus capsids by charge detection mass spectrometry. Mol Ther Methods Clin Dev. 2021;21:231–239.
4. Kulkarni JA, Cullis PR, van der Meel R. Lipid nanoparticles enabling gene therapies: from concepts to clinical utility. Nat Nanotechnol. 2018;13:112–121.