Advanced SEC with Online LS/DLS Detection for the Analysis of Lipid Nanoparticles CQAs

Significance of the Topic

Precise measurement of lipid nanoparticle size is critical for their performance as delivery vehicles in biopharmaceuticals. Size influences biodistribution, stability, aggregation, circulation half-life and cellular uptake. Combining size exclusion chromatography with advanced light scattering detection provides a comprehensive profiling of lipid nanoparticle critical quality attributes and ensures reproducible analytical control in drug development and quality assurance.

Objectives and Study Overview

This study evaluates an online SEC-LS/DLS setup for simultaneous separation and sizing of loaded and empty lipid nanoparticles. Key aims include comparison with batch dynamic light scattering, assessment of column performance across different pore sizes, investigation of aggregation and stability under freeze-thaw stress, and demonstration of co-analysis of nanoparticles and targeting antibodies.

Methodology and Instrumentation

Equipment and software

• Agilent 1290 Infinity III Bio LC System with flexible pump, multisampler and column thermostat
• Agilent 1260 Infinity II Bio-SEC Multidetector System featuring dual-angle static light scattering and dynamic light scattering detection
• Agilent GPC/SEC Software version 2.2 or later

Columns

• AdvanceBio SEC, 300 Å, 7.8 × 300 mm, 2.7 µm
• Bio SEC-5, 2000 Å, 7.8 × 300 mm, 5 µm
• PL aquagel-OH MIXED-H, 7.5 × 300 mm, 8 µm

Chemicals and samples

• Milli-Q water and phosphate buffered saline filtered to 0.2 µm
• Bovine serum albumin (20 mg/mL in PBS) for calibration and column evaluation
• Loaded and empty lipid nanoparticles with different lipid compositions
• Monoclonal antibody spiked for co-analysis

Chromatographic conditions were isocratic PBS at 0.6 mL/min and 30 °C, with detector settings optimized for combined RI, LS and DLS signals.

Main Results and Discussion

Column comparison

• AdvanceBio SEC 300 Å provided the best resolution of BSA monomer, oligomers and lipid nanoparticle peaks, with clean DLS signals and strong correlation to batch DLS
• Wide-pore columns (2000 Å, 8 µm) showed broader peaks, lower resolution and required higher sample loading to detect RI signals
• Hydrodynamic chromatography effects on small-pore columns enabled partial separation of large aggregates

Size accuracy and precision

• Online SEC-DLS Rh values deviated less than 15 % from batch DLS for all loaded and empty LNPs using the 300 Å column
• Precision of Rh measurements reached RSD ≤ 5 % (n = 5), demonstrating excellent reproducibility
• Calculated shape factors Rg/Rh aligned with dense sphere theory, except for an empty LNP sample showing evidence of aggregation

Stability under freeze-thaw stress

• Polydispersity index (PDI) from batch DLS increased over three cycles for empty nanoparticles, mirrored by rising Rh RSD in online SEC-DLS
• Loaded LNP B maintained monodispersity and low PDI throughout stress tests, confirming robust formulation

Antibody co-analysis

• Purified LNPs and spiked monoclonal antibody were baseline separated, enabling independent detection of free antibody
• Static light scattering identified the antibody mass at 166 kDa, illustrating method utility for detecting unbound targeting moieties

Benefits and Practical Applications

The optimized SEC-LS/DLS method offers

• High-resolution separation of nanoparticles and aggregates
• Accurate and precise sizing directly linked to chromatography retention
• Simultaneous assessment of aggregation, size distribution, stability and co-formulated biomolecules
• Realistic dynamic range for combined RI and light scattering detection in a metal-free flow path

This approach streamlines critical quality attribute monitoring in LNP development, formulation screening and quality control workflows.

Future Trends and Potential Applications

Advances may include

• Integration with multi-attribute detection for lipid composition and RNA content profiling
• Real-time process monitoring in continuous manufacturing
• Automation of data analysis and reporting for regulatory compliance
• Extension to other nanoparticle platforms such as exosomes and polymeric carriers

Conclusion

The combination of Agilent 1290 Infinity III Bio LC with the 1260 Infinity II Bio-SEC Multidetector System and the AdvanceBio SEC 300 Å column forms a robust platform for comprehensive characterization of lipid nanoparticle critical quality attributes. High correlation with batch DLS, precise reproducibility, and the ability to monitor aggregation, stability and co-formulated antibodies make this method ideal for biopharmaceutical development and quality control.

Reference

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