Efficient Profiling of Lipid Nanoparticle Formulations Using Waters GTxResolve 2000 Å SEC Column, MaxPeak Premier 3 μm

Importance of the Topic

Lipid nanoparticles (LNPs) have become essential carriers for RNA-based therapeutics, offering protection and targeted delivery of fragile nucleic acids. Precise control of LNP size and polydispersity is critical for biodistribution, efficacy, and tissue targeting. Size exclusion chromatography (SEC) is widely used for biologics and is now adapted for megadalton-size nanoparticles. The introduction of the Waters GTxResolve 2000 Å SEC Column with MaxPeak Premier 3 µm particles addresses challenges in resolving and quantifying LNPs under gentle, low-ionic-strength conditions.

Objectives and Study Overview

This study evaluates the fit-for-purpose performance of the Waters GTxResolve 2000 Å SEC Column in profiling two commercial COVID-19 vaccine LNP formulations (Comirnaty™ and Spikevax™). Key aims include demonstrating resolution and reproducibility under a low ionic strength mobile phase (0.1× DPBS), comparing recoveries and size profiles, assessing column lifetime, and correlating SEC data with dynamic light scattering (DLS) and field-flow fractionation (FFF) results.

Methodology and Instrumentation

Mobile Phase and Sample Preparation:
0.1× Dulbecco’s Phosphate-Buffered Saline (DPBS) in Milli-Q water was used to preserve native LNP integrity and minimize salt-mediated aggregation. Vaccine samples were diluted 1:1 in mobile phase and injected at low volumes.

Chromatographic System:

• ACQUITY™ Premier System with Binary Solvent Manager, Flow-Through Needle Sample Manager, Column Manager-A
• Waters GTxResolve 2000 Å SEC Column, MaxPeak Premier 3 µm (4.6 × 150 mm)
• ACQUITY Premier eLambda PDA detector with 5 mm titanium flow cell, 260 nm detection
• Column temperature 35 °C, flow rate 0.1 mL/min, sample temperature 5 °C

Column and Particle Design:
The column hardware features MaxPeak High-Performance Surface (HPS) to prevent metal-mediated adsorption. The 3 µm hybrid silica particles have a 2000 Å average pore size and hydrophilic PEO bonding to minimize secondary interactions.

Results and Discussion

Resolution and Recovery:
Comirnaty and Spikevax produced distinct SEC-UV profiles consistent with their DLS-measured hydrodynamic diameters (≈94 nm vs. 156 nm). Waters GTxResolve columns outperformed comparable 5 µm columns in total peak area recovery and reproducibility (%RSD < 6 for retention time, < 6 for area).

Column Lifetime:
Fifty consecutive injections showed stable peak shape, retention, and area (area %RSD 0.9) with no passivation steps. A periodic 6 M guanidine HCl wash is recommended to sustain performance.

Orthogonal Correlation:
DLS confirmed size and zeta potential differences. FFF-MALS literature data corroborate SEC findings, demonstrating SEC-UV as a rapid screening complement to high-resolution orthogonal techniques.

Additive-Enhanced Recovery:
Using a larger 7.8 mm column format, inclusion of 0.006% polysorbate 80 and 10 mM arginine increased Spikevax recovery from 60% to 88%, illustrating the role of mobile phase additives and flow rate optimization.

Benefits and Practical Applications

• Superior resolution of large LNP species via optimized 2000 Å pores and 3 µm particles
• Enhanced recovery under low-ionic conditions using hydrophilic, non-ionic surfaces
• Fast, robust SEC method compatible with UV, MALS, dRI, and fluorescence detection
• High reproducibility supports batch QC, release testing, and stability studies

Future Trends and Applications

Emerging efforts will focus on:

• Integrating SEC with multi-angle light scattering and mass spectrometry for absolute quantification
• Optimizing flow rates and column formats for diverse LNP formulations
• Assessing the impact of additional mobile phase excipients on LNP integrity
• Developing standardized QC workflows combining SEC, DLS, ELS, and FFF

Conclusion

The Waters GTxResolve 2000 Å SEC Column, MaxPeak Premier 3 µm offers a fit-for-purpose SEC solution for LNP characterization under native, low-salt conditions. Its advanced hardware and packing material deliver high resolution, minimal analyte interaction, and exceptional reproducibility, making it a powerful orthogonal tool alongside DLS, ELS, and FFF in therapeutic vector development and quality control.

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