MULTIMODAL CHARACTERIZATION OF TARGETED LIPID NANOPARTICLES USING CHARGE DETECTION MS AND ORTHOGONAL SEPARATION TECHNIQUES

Significance of the topic

Targeted lipid nanoparticles (tLNPs) are a rapidly maturing delivery platform for nucleic acids with direct relevance to in vivo cell therapies and programmable immunology. Accurate physicochemical characterization of tLNPs is essential for linking formulation variables to biological performance, ensuring batch-to-batch consistency, and meeting regulatory expectations. Traditional bulk assays and ensemble sizing methods (e.g., encapsulation assays, DLS) provide fast screening but fail to resolve particle-level heterogeneity and complex structure–function relationships inherent to targeted LNPs. Single-particle mass analysis by charge detection mass spectrometry (CDMS), combined with orthogonal separation and light-scattering approaches, addresses this gap by directly measuring intact particle mass distributions and revealing subpopulations masked in ensemble averages.

Objectives and study overview

This study aims to apply a multimodal analytical workflow to characterize tLNPs differing in antibody (mAb) surface conjugation (0, 1, 2.5, 5% DBCO-lipids). Specific objectives were to: provide direct whole-particle mass distributions using CDMS; compare and complement CDMS findings with field-flow fractionation–multi-angle light scattering (FFF-MALS), DLS/ELS and chromatographic/optical detectors; evaluate the impact of progressive mAb incorporation on particle heterogeneity and structural complexity; and demonstrate the value of combining single-particle and ensemble techniques for LNP development and quality assessment.

Methods

tLNP production and sample preparation:

• tLNPs were prepared by conjugating azide-modified monoclonal antibodies to DBCO-functionalized lipids via copper-free click chemistry at nominal surface incorporation levels of 0, 1, 2.5, and 5% DBCO.
• Samples were buffer-exchanged into 20 mM ammonium acetate, pH 7.4, prior to analysis.
• tLNP material was sourced from Phosphorex.

CDMS analysis:

• Samples were introduced by nano-electrospray ionization (nESI) and measured on a benchtop Xevo CDMS instrument under native-like conditions.
• CDMS data were processed with the CDMS Toolkit in waters_connect software to obtain single-particle mass distributions and two-dimensional m/z vs. charge density plots.

Orthogonal techniques:

• Field-flow fractionation with a short, fixed-height channel (Eclipse FFF) using PBS as mobile phase, coupled online to DAWN MALS, Optilab differential refractometer (dRI), and UV detection at 260 nm. Data acquisition and analysis used VISION and ASTRA software.
• Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) were employed as complementary ensemble methods for size and surface charge/mobility.

Instrumentation used

• Xevo CDMS benchtop instrument (Waters) for charge detection mass spectrometry.
• Eclipse Field-Flow Fractionation (FFF) system with 350 µm fixed-height short channel and HPLC pump/autosampler.
• DAWN Multi-Angle Light Scattering (MALS) detector and Optilab differential refractometer (dRI) for on-line molar mass and size determination.
• UV detector at 260 nm; VISION, ASTRA, and waters_connect software suites for control and analysis.

Main results and discussion

CDMS findings:

• Even unconjugated LNPs (0% DBCO) exhibited substantial intrinsic mass heterogeneity, illustrating limits of ensemble-only characterization.
• Low-level mAb conjugation (1% DBCO) broadened the mass distribution, indicating increased particle-to-particle variability without a simple uniform mass shift—consistent with nonuniform antibody loading across particles.
• Mid-level conjugation (2.5% DBCO) produced more complex, multimodal distributions suggestive of distinct subpopulations with varying degrees of antibody incorporation and potentially altered internal structure or aggregation states.
• High-level conjugation (5% DBCO) yielded the broadest and most heterogeneous mass distribution, implying that increasing ligand density can amplify structural variability rather than produce uniform surface decoration.
• Two-dimensional m/z versus charge density plots provided visual resolution of subpopulations and charge-state distributions that are not accessible through ensemble techniques.

FFF-MALS and orthogonal data:

• FFF-MALS provided size-resolved molar mass profiles and enabled estimation of average antibodies per particle across elution time ranges, supporting the CDMS observations of distribution broadening with increased conjugation.
• DLS/ELS delivered ensemble-average size and mobility metrics but masked the heterogeneity revealed by CDMS.
• Combined outputs showed concordant trends: increasing mAb loading correlates with greater heterogeneity in mass and size distributions, though the relationship is non-linear and composition-dependent.

Interpretation and implications:

• CDMS uniquely resolves intact-particle mass heterogeneity and subpopulations, providing direct evidence that surface conjugation strategies may produce highly variable particle-level compositions.
• The lack of a single mass shift with increasing mAb indicates heterogeneous ligand incorporation mechanisms (partial surface coverage, variable stoichiometry, or selective incorporation into subpopulations).
• These structural heterogeneities can influence delivery performance, biodistribution, and immunogenicity, underscoring the need to include single-particle mass analysis in tLNP development workflows.

Benefits and practical applications of the method

• Direct, label-free whole-particle mass measurement enables quantitative assessment of population heterogeneity, complementing ensemble physicochemical assays used in formulation screening and QC.
• CDMS combined with FFF-MALS allows correlation of mass, size, and composition across resolved subpopulations—useful for optimizing conjugation chemistry, lipid composition, and payload loading strategies.
• The workflow can support critical quality attribute (CQA) definition, release testing, and troubleshooting of manufacturing variability by detecting subpopulations and nonuniform modifications that would be invisible to bulk methods.

Future trends and potential applications

• Integration of CDMS with on-line separations (e.g., FFF-CDMS hyphenation) to directly link fraction-resolved mass spectra with light-scattering and refractive index data for richer characterization.
• Improvements in CDMS throughput, mass resolution, and quantitation to enable routine ligand counting per particle and estimation of payload stoichiometry at scale.
• Development of standardized workflows and reference materials for regulatory adoption and interlaboratory comparability of LNP mass-distribution metrics.
• Combination with orthogonal single-particle tools (e.g., cryo-EM, nanoparticle tracking analysis with fluorescent labeling) to correlate structural, compositional, and functional readouts such as encapsulation efficiency and cellular delivery efficacy.
• Application of multimodal characterization during formulation optimization, stability studies, and process development to reduce batch heterogeneity and improve in vivo performance predictability.

Conclusion

Multimodal characterization anchored by charge detection mass spectrometry reveals substantial and composition-dependent particle heterogeneity in targeted lipid nanoparticles that is not captured by conventional ensemble methods. Progressive antibody conjugation increases distribution breadth and generates complex subpopulations rather than inducing uniform surface modification. Integrating CDMS with orthogonal separation and light-scattering techniques yields complementary size, composition, and mass information critical for establishing structure–function relationships, guiding formulation design, and strengthening quality control strategies for advanced LNP therapeutics.

References

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