SEC-CDMS ENABLES ONLINE BUFFER EXCHANGE AND CHARACTERIZATION OF PROTEIN-BASED THERAPEUTICS

Significance of the topic

Size-exclusion chromatography (SEC) coupled to mass spectrometry (MS) is an increasingly important approach for characterizing large biomolecules under near-native conditions. For very high-mass assemblies (megadalton scale), Charge Detection Mass Spectrometry (CDMS) provides direct mass determination but historically suffered from low throughput that limited integration with chromatography. Demonstrating a practical SEC–CDMS workflow that preserves chromatographic timescales and consumes minimal sample addresses a critical need for routine, reliable characterization of protein therapeutics, viral vectors, virus-like particles (VLPs) and other heterogeneous assemblies in pharmaceutical and research settings.

Goals and overview of the study

The study aims to establish and demonstrate a simple, robust online SEC–CDMS method capable of: 1) performing buffer exchange inline during SEC separation, 2) transferring eluate into MS-compatible conditions for direct mass analysis, and 3) delivering throughput and sensitivity compatible with chromatographic peak widths. The work evaluates the approach on representative high-mass analytes including AAV8 capsids (empty and full), dengue VLPs, keyhole limpet hemocyanin (KLH), and the monoclonal antibody adalimumab to illustrate mass-range coverage and quantitative performance.

Methods and used instrumentation

Workflow and chromatography:

• SEC was performed with low-flow, ultra-wide-pore (1000 Å) size-exclusion columns operated at 30 °C and a flow rate of 50 µL/min using 50 mM ammonium acetate (isocratic) as mobile phase to maintain native-like conditions and enable online buffer exchange.
• Narrow-bore columns and a 10:1 post-column flow split were used to direct approximately 5 µL/min to a nano-electrospray ionization (nano-ESI) source adapted with a silica-fused tip for continuous spray, minimizing sample consumption (injection volumes: 1–10 µL depending on sample).

Mass spectrometry and acquisition:

• Measurements were acquired on a Waters Xevo CDMS system operated in continuous trapping mode (trapping time ~100 ms).
• High-throughput data acquisition was achieved with the Multiple Ion Charge Extraction (MICE) algorithm to recover overlapping ion signals and support ion fluxes compatible with SEC peak widths.
• Electrospray parameters: nebulizer gas ~1.8 L/min (N2), source temperature 150–250 °C (reduced settings for labile species), spray voltage ~3.65–3.9 kV; inlet heating and reduced voltages were applied to minimize ion activation where required.

Samples tested:

• AAV8 capsids (empty and full) in defined volumetric mixtures (0–100% empty in 25% increments).
• Dengue virus serotype 1 VLP (~0.32 mg/mL).
• Keyhole limpet hemocyanin (KLH) at 5 mg/mL.
• Adalimumab (Humira) at 1 mg/mL.

Data handling:

• Processing and visualization were performed using waters_connect software. MICE was essential to extract charge states and mass distributions from overlapping ion populations.

Main results and discussion

Feasibility and throughput:

• The SEC–CDMS configuration delivered sufficient ion flux and time resolution to analyze chromatographic peaks (typical elution times reported between ~10–26 min for different analytes) using small injection volumes (1–10 µL), demonstrating practical compatibility with standard chromatographic workflows.

Quantitative performance and examples:

• AAV8 empty/full capsid mixtures were quantified by CDMS after SEC separation, producing a linear correlation between measured and expected empty-capsid percentages (R² = 0.989), indicating accurate relative quantification across defined mixtures.
• Dengue VLPs were detected as intact assemblies with masses around ~6.95 MDa and charge distributions near ~188 e, consistent with preservation of high-order structure through SEC and MS transfer.
• KLH showed multiple distinct oligomeric species with resolved mass peaks at approximately 3.13, 3.88, 6.93, 7.67 and 11.24 MDa and a dominant charge population near 185 e, illustrating the method’s ability to deconvolute heterogeneous megadalton oligomers.
• Adalimumab (monoclonal antibody) was observed at ~147 kDa with an average charge around 22 e and well-resolved m/z charge-state features, showing applicability across the kilo- to megadalton range.

Method considerations:

• Continuous trapping CDMS combined with MICE enabled recovery of overlapping ion signals, overcoming a primary throughput limitation of traditional single-ion CDMS and allowing analysis on chromatographic timescales.
• Instrumental settings (source temperature, spray voltage) were tuned to balance desolvation efficiency and preservation of noncovalent assemblies; reduced activation conditions were important for fragile megadalton complexes.

Benefits and practical applications

The demonstrated SEC–CDMS workflow provides several practical advantages for laboratories handling protein therapeutics and viral products:

• Online buffer exchange: SEC replaces manual offline desalting, reducing sample handling variability and time.
• Low sample consumption: compatible with precious or limited samples due to small injection volumes and low effluent flow to the MS.
• Broad mass-range capability: enables direct mass characterization from antibodies (~100–200 kDa) up to intact VLPs and megadalton oligomers with single-platform analysis.
• Quantification and heterogeneity assessment: enables accurate relative quantification (example R² = 0.989 for AAV8 empty/full content) and resolves complex oligomeric distributions important for product quality assessment.

These attributes make SEC–CDMS attractive for applications in biologics development, viral vector QC, vaccine research, and structural characterization of large assemblies.

Future trends and potential uses

Prospective developments and broader impacts include:

• Scaling throughput and automation: further optimization of MICE and trapping strategies could increase duty cycle and enable routine high-throughput QC workflows.
• Integration with orthogonal separations: combining SEC–CDMS with additional separations (e.g., ion-exchange, affinity capture) or upstream sample handling could enhance specificity for complex formulations.
• Regulatory and QC adoption: reproducible inline buffer exchange and quantitation could support adoption of CDMS-based assays in regulated environments for lot release and stability testing of viral vectors and complex biologics.
• Advances in data analytics: improved algorithms for charge extraction and deconvolution will increase confidence in mass assignments for highly heterogeneous populations and enable automated reporting.

Conclusion

The presented preliminary work establishes a practical SEC–CDMS approach that couples low-flow, wide-pore SEC with high-throughput CDMS acquisition (MICE) to enable online buffer exchange and mass characterization of biomolecules across a broad mass range. The method is compatible with chromatographic timescales, consumes minimal sample, and demonstrates accurate relative quantification and resolution of complex assemblies, supporting its potential as a scalable tool for characterization of protein-based therapeutics, viral vectors and megadalton assemblies.

Reference

1. Parikh, R. A.; Miller, L. M.; Draper, B. E.; Lavelay Kizekai; Balasubrahmanyam Addepalli; Chen, M.; Lauber, M. A.; Jarrold, M. F. Coupling of Size Exclusion Chromatography to High Throughput Charge Detection Mass Spectrometry for the Analysis of Large Proteins and Virus-like Particles. Analytical Chemistry 2025, 97 (5).
2. Parikh, R. A.; Draper, B. E.; Jarrold, M. F. Multiple Ion Charge Extraction (MICE) for High-Throughput Charge Detection Mass Spectrometry. Analytical Chemistry 2024, 96.