High Resolution Characterization of Lipid Nanoparticles Using the Xevo™ Charge Detection Mass Spectrometry (CDMS) Instrument - Single Particle Mass Analysis of Intact LNP-mRNA Formulations

Significance of the topic

Lipid nanoparticles (LNPs) are central to the delivery of mRNA therapeutics because they protect RNA cargo, modulate cellular uptake, and influence pharmacokinetics. However, LNP formulations are intrinsically heterogeneous in size, composition and payload loading. Conventional ensemble biophysical methods (DLS, SEC‑MALS, FFF‑MALS, cryo‑EM, AUC, CE) provide complementary but averaged or component‑specific information and cannot directly measure the intact mass of individual particles. Charge detection mass spectrometry (CDMS) fills this analytical gap by reporting per‑particle mass and charge, enabling direct resolution of subpopulations and low‑abundance species that are hidden in ensemble measurements.

Objectives and study overview

This application note demonstrates the use of the commercial Xevo CDMS Instrument for high‑resolution, single‑particle mass analysis of intact LNP‑mRNA vaccine formulations. Using a marketed LNP system (COMIRNATY®, Pfizer‑BioNTech) as a case study, the work aims to (i) show that intact LNPs can be detected and measured on a commercial CDMS platform, (ii) reveal multimodal mass distributions and particle‑to‑particle variability, and (iii) illustrate how CDMS data can support formulation development, process control and lot comparability.

Methodology

Sample preparation and acquisition workflow summarized:

• Sample: COMIRNATY® (2025–2026 formula) handled per manufacturer guidance.
• Buffer exchange: into 20 mM ammonium acetate, pH 7.4, using small‑volume dialysis for 15 min at 4 °C.
• Ionization: nano‑electrospray ionization (nESI) in positive mode to generate intact LNP ions.
• Acquisition mode and parameters: full‑scan CDMS; capillary voltage 1.4–1.8 kV; cone voltage 40; trapping duration 100 ms; total acquisition 60 minutes.
• Data capture and processing: waters_connect Informatics Platform (v4.2.0) with the CDMS Toolkit for single‑particle extraction and visualization.

Key data representations reported:

• 2D plots of mass versus charge and charge versus m/z to separate populations with overlapping m/z but different charges.
• Univariate histograms for mass, charge and m/z (reported bin widths: 1 MDa, 2 e, 1000 m/z) to visualize modal structure and distribution width.

Instrumentation used

• Xevo CDMS Instrument (ELIT‑based commercial CDMS platform).
• Nano‑electrospray ion source for intact particle introduction.
• waters_connect Informatics Platform (software) and CDMS Toolkit for data acquisition and processing.

Results and discussion

Main findings:

• Direct single‑particle mass measurement: The Xevo CDMS produced true per‑particle masses in the megadalton range without charge‑state deconvolution or a priori assumptions.
• Multimodal mass distributions: COMIRNATY LNPs exhibited peak‑structured mass profiles indicating multiple coexisting particle subpopulations with different degrees of payload loading and structural packing.
• Charge and m/z separation: 2D mass vs charge and charge vs m/z plots dissociated ions that overlap in m/z by charge state, clarifying features that ensemble spectra would conflate.
• Retention of low‑abundance features: CDMS preserved shoulders and minor populations across the distribution, enabling unbiased detection of rare heavy or light particles.
• Interpretation of heterogeneity: Observed distribution breadth reflects combined variability in lipid composition, associated solvent, structural packing and mRNA copy number per particle (reported qualitatively up to ~100+ copies per particle in the note), consistent with prior microscopy and ensemble studies.

Figures and visual data (described):

• Figure content included a schematic 3D liposome depiction to contextualize LNP structure.
• 2D density plots showed continuous charge ranges and distinct mass populations; axis truncation was used in display figures for clarity but full distributions were analyzed.
• Mass, charge and m/z histograms captured the complete distributions and highlighted modal peaks and distribution widths.

Benefits and practical applications

Practical advantages of CDMS for LNP analytics:

• Single‑particle resolution removes reliance on deconvolution and charge‑state assignment, improving confidence in intact mass values.
• Detection of subpopulations and low‑abundance species supports formulation optimization by revealing heterogeneity in payload encapsulation and particle composition.
• Direct mass readouts facilitate lot‑to‑lot comparisons and process monitoring without model‑dependent corrections used in ensemble methods.
• Rapid, label‑free measurements complement microscopy and light‑scattering techniques by adding intact mass as an orthogonal CQA (critical quality attribute).

Use cases where CDMS data are actionable:

• Early formulation screening to compare loading distributions across candidate lipid compositions.
• Process development and scale‑up monitoring to detect shifts in mass distributions indicative of altered encapsulation efficiency or particle aggregation.
• Comparability and stability studies to track emergence or disappearance of subpopulations over time or between lots.

Future trends and potential applications

Predicted developments and opportunities for CDMS in LNP and bioparticle characterization include:

• Integration with orthogonal separations (e.g., FFF or SEC) prior to CDMS to add retention‑time resolution and reduce complexity for highly heterogeneous samples.
• Higher throughput acquisition and automatisation to support routine QC and larger‑scale comparability studies.
• Standardization of reporting metrics (mass‑based CQAs, distribution descriptors) to facilitate regulatory acceptance and cross‑laboratory comparisons.
• Improved informatics and statistical frameworks for automated subpopulation identification, annotation and quantitation.
• Extension to absolute quantitation of payload (RNA copy number) through calibrated mass differences and correlation with orthogonal biochemical assays.

Conclusion

The Xevo CDMS Instrument provides a commercial, ELIT‑based CDMS solution capable of measuring intact LNP masses at single‑particle resolution. Applied to a marketed mRNA vaccine formulation, CDMS resolved multimodal mass profiles, detected minor subpopulations, and offered direct mass evidence of LNP heterogeneity that ensemble methods cannot provide. These capabilities support formulation optimization, process control and comparability testing by supplying an orthogonal, high‑resolution measurement of intact particle mass and composition.

References

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