Pore Size Distribution Study of GTxResolve™ SEC Columns for Analysis of Genetic Medicine Drug Substances and Products

Significance of the Topic

Size exclusion chromatography (SEC) has become a cornerstone technique for rapid quality control of genetic medicine drug substances and products. With the rise of large biomolecular modalities—such as lipid nanoparticles, plasmids, nucleic acids and viral vectors—robust and high-resolution methods are required to assess aggregate content, heterogeneity and purity. The pore size and pore size distribution (PSD) of SEC packing materials directly govern separation performance, making informed column selection critical to reliable size variant analysis.

Objectives and Overview of the Study

This study aims to characterize three wide-pore SEC columns (450 Å, 1000 Å, 2000 Å nominal pore sizes) from the GTxResolve family. By applying an inverse SEC approach—calibration with analytes of known hydrodynamic diameter (flexible dsDNA ladder and rigid gold nanoparticles, AuNPs)—the work seeks to:

• Derive effective median pore sizes (d50) and PSD curves under chromatographic conditions.
• Compare chromatographically derived PSDs against mercury intrusion porosimetry (MIP) data.
• Provide guidance on optimal column choice for genetic medicine analytes based on size accessibility.

Methodology and Instrumentation

A two-pronged calibration strategy was followed:

• Flexible analytes: A dsDNA ladder (50–1350 bp) was separated on each SEC column using 2× PBS mobile phase and UV detection (260 nm).
• Rigid analytes: Gold nanoparticles (5–60 nm), functionalized with bovine serum albumin or mercaptoundecanoic acid, were separated using 0.1× PBS/2 % isopropanol/0.02 % SDS mobile phase and monitored by multi-angle light scattering (MALS) and dynamic light scattering (DLS).

Elution times (te), interstitial volume markers (ti) and total void markers (tM) were used to calculate partition coefficients (KSEC). A modified Richard’s model, combined with Ogston theory, was fitted to KSEC versus log(analyte diameter) data in Excel™ Solver™ to extract PSD and median pore diameters.

Instrumentation

• LC System: ACQUITY UPLC H-Class Bio QSM.
• Detectors: ACQUITY UPLC Tunable UV (260 nm, 280 nm) and Wyatt DAWN™ MALS with QELS™ DLS module.
• Columns: GTxResolve Premier BEH SEC 450 Å (2.5 µm), GTxResolve Premier SEC 1000 Å (3 µm), GTxResolve 2000 Å SEC (3 µm, MaxPeak Premier).
• Temperatures: Column 25 °C; sample 6 °C.
• Flow rates: 0.05–0.6 mL/min; injection volumes: 1 µL (DNA), 10 µL (AuNPs).

Main Results and Discussion

Chromatograms revealed distinct elution windows for each column, confirming nominal pore size ranks: 2000 Å columns favored large analyte penetration (KSEC > 0.5 for species >20 nm), while 450 Å columns restricted large species (KSEC < 0.5). Calibration curves produced log-normal PSD profiles. Median accessible pore diameters matched nominal values within ~10 %:

• 450 Å column d50 ≈ 450 Å
• 1000 Å column d50 ≈ 1000 Å
• 2000 Å column d50 ≈ 2000 Å

Comparisons with MIP highlighted methodological differences: MIP focuses on geometric pore entries, while inverse SEC emphasizes hydrodynamic accessibility, pore connectivity and tortuosity under flow conditions.

Benefits and Practical Applications

By linking analyte size to elution behavior, this approach enables:

• Informed column selection for novel genetic medicine analytes.
• Prediction of optimal size ranges for highest resolution (KSEC ≈ 0.5 region).
• Reduction of trial-and-error method development time.

Future Trends and Opportunities

Extending inverse SEC characterization to emerging modalities—such as messenger RNA, viral capsids and exosomes—can further refine column design. Integration with online MALS/DLS and high-throughput workflows will accelerate quality assessment of gene therapies. Additionally, development of tailored wide-pore packing chemistries may improve selectivity for complex nanomedicines.

Conclusion

This work demonstrates that calibration with flexible and rigid standards provides direct access to functional PSD metrics of SEC columns. The GTxResolve 450 Å, 1000 Å and 2000 Å SEC columns exhibit median pore sizes consistent with their designations and serve distinct size-separation niches for genetic medicine analytics. Inverse SEC serves as a practical tool for method development and column selection in biopharmaceutical laboratories.

References

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