WHAT’S INSIDE A VACCINE? A CLOSER LOOK USING CHARGE DETECTION MASS SPECTROMETRY

Significance of the topic

Vaccines are moving beyond simple subunit formats toward large, heterogeneous assemblies such as virus‑like particles (VLPs), recombinant capsids and nanoparticle platforms. Accurate mass characterization of these macromolecular assemblies is essential for structure confirmation, quality control, batch comparability, detection of defective or incomplete particles, and development of next‑generation vaccine modalities. Charge detection mass spectrometry (CDMS) provides single‑particle mass measurements that bypass limitations of conventional native MS for very large, polydisperse systems, making it highly relevant for modern vaccine analytics.

Objectives and study overview

This study demonstrates the application of an ELIT‑based CDMS system (Waters Xevo CDMS) to characterize a range of vaccine materials and recombinant VLPs. Primary aims were to: (1) obtain direct single‑particle masses for vaccine antigens across commercial formulations, (2) resolve intact capsids versus subassemblies and impurities, and (3) evaluate the method’s throughput and mass accuracy for particles spanning several MDa to >60 MDa.

Methods and experimental workflow

Samples: Commercial vaccines analyzed included Engerix‑B (HBsAg VLPs), Rotateq (rotavirus), and IPOL (inactivated poliovirus). Recombinant VLPs (Dengue serotypes 1–4, Norovirus genotypes, Chikungunya) were also examined.

Sample preparation: Buffer exchange into 200 mM ammonium acetate using Micro Bio‑Spin P‑6 desalting columns or Amicon 100 kDa centrifugal filters to maintain near‑native conditions and remove nonvolatile salts.

Ionization and acquisition: Static nanoelectrospray ionization in positive mode. Ions trapped in an electrostatic linear ion trap (ELIT) detection cylinder with ion mirrors; trap times reported ~100 ms. Time‑domain signals were recorded for individual ions; FFT provided oscillation frequency (m/z) while signal amplitude and calibration gave charge (z), enabling direct mass calculation for each particle.

Data processing: Single‑ion m/z and charge pairs were combined into histograms and 2D charge vs. mass plots. Typical acquisitions collected thousands of ions per sample over 10–30 minute runs. Reported charge measurement precision (RMSD) was ~0.9 elementary charges.

Instrumentation used

- Waters Xevo CDMS instrument configured with an electrostatic linear ion trap (ELIT).
- Static nanoESI emitters for gentle ionization of large particles.
- Conductive detection cylinder with ion mirrors to induce charge signal; low‑noise charge‑sensitive amplifier and FFT‑based signal processing for independent determination of m/z and z.

Main results and discussion

General performance:

• CDMS produced direct single‑particle masses across a wide mass range (several MDa up to ~61 MDa reported), with acquisition times suitable for routine laboratory workflows (10–30 minutes).
• Measured mass centroids were broadly consistent with expected capsid masses when accounting for adducts, counterions, trapped solvent, and chemical crosslinking; measured peaks showed narrow-to-moderate FWHM depending on sample heterogeneity.
• The method resolved intact capsids, empty versus full particles, subassemblies and impurities (e.g., defective C‑antigen particles in IPOL).

Selected sample findings:

• Engerix‑B (HBsAg VLPs): Observed oligomeric subassemblies and T=1 particles; well‑defined T=3 particles (higher‑order assembly) were not clearly detected in the vaccine formulation, although a small population in the expected mass range appeared — suggesting formulation or preparation effects that limit detection of larger assembled species.
• Rotateq (rotavirus): Detected rotavirus double layered particle (DLP) at 61.07 MDa with a mass error of −1.75% and FWHM ≈ 0.95 MDa. Extracted m/z distributions spanned wide m/z ranges (80,000–200,000), reflecting large particle heterogeneity.
• IPOL (poliovirus): Resolved both empty (C‑antigen) and full (D‑antigen) particles with measured masses ≈5.98 MDa (+1.53%, FWHM 0.12 MDa) and ≈8.62 MDa (+2.25%, FWHM 0.18 MDa). Observed mass excesses attributed to noncovalent adducts, counterions, trapped solvent and formaldehyde crosslinking from vaccine formulation.
• Recombinant VLPs: Norovirus and Chikungunya VLPs displayed classical capsid mass signatures consistent with expected architectures. Dengue serotypes largely produced subviral assemblies rather than intact canonical capsids under the conditions analyzed.

Interpretation and limitations:

• Measured masses frequently exceed theoretical protein‑only masses due to adducts, bound salts and residual solvent—an expected phenomenon in native and gas‑phase measurements of high‑mass assemblies.
• Formulation components and chemical treatments used in commercial vaccines (e.g., formaldehyde) can alter apparent mass distributions and assembly stability, complicating direct comparisons to recombinant or theoretical masses.
• Sensitivity to detect low‑abundance populations is demonstrated but depends on acquisition time and ion throughput; very large or highly heterogeneous assemblies may require optimization of desolvation and sample handling to enhance detection of intact higher‑order capsids.

Benefits and practical applications

CDMS with ELIT detection provides several practical advantages for vaccine analytics:

• Direct single‑particle mass measurement without charge state deconvolution simplifies interpretation of highly charged, high‑mass ions.
• Ability to distinguish empty, full and defective particles enables orthogonal characterization for potency and quality control workflows.
• Detection of subassemblies and minor populations aids in formulation assessment, process development, and comparability studies.
• Reasonable throughput (minutes per sample) and robustness for commercial vaccine formulations suggest applicability in R&D and specialized QC environments.

Future trends and potential applications

Advances and likely directions include:

• Improved resolution and charge precision through electronics and trap optimisation to better separate closely spaced mass populations and reduce charge uncertainty.
• Integration with orthogonal native‑MS, ion mobility and top‑down approaches to provide complementary structural and compositional information (e.g., protein composition, PTMs, crosslinks).
• Method standardization and sample preparation protocols tailored for complex vaccine matrices to reduce adduct contributions and improve accuracy of mass assignment.
• Expanded use in development of nanoparticle vaccines, gene therapy vectors and heterogeneous biologics where intact mass and particle composition are critical quality attributes.

Conclusions

ELIT‑based CDMS (Waters Xevo) enables reliable single‑particle mass measurements for a broad range of vaccine antigens and VLPs, resolving intact capsids, empty versus full particles and subassemblies across masses from a few MDa to tens of MDa. While observed masses include contributions from nonvolatile adducts and formulation chemistry, the method offers direct, interpretable mass data that support vaccine characterization, impurity profiling and development of next‑generation modalities.

Reference

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