Using a 2.1 mm ID Narrow Bore GTxResolve™ Premier™ SEC Column to Reduce Sample Consumption During Size Exclusion Analyses

Importance of the Topic

The accurate separation and quantitation of biotherapeutic molecules and their impurities are critical during the development and quality control of cell and gene therapy products.
Size exclusion chromatography (SEC) under native conditions is widely used to detect aggregates, degraded forms, and residual process-related impurities, ensuring product safety and efficacy.

Study Aims and Overview

This application note evaluates the performance of a narrow bore 2.1 × 150 mm GTxResolve™ Premier™ BEH SEC 450 Å 2.5 µm column for reducing sample consumption during SEC analyses of therapeutic proteins and viral vectors.
The column’s separation efficiency, salt dependence, and sensitivity are compared to a commercially available 2.1 × 150 mm titanium hardware SEC column (3 µm, 700 Å).

Methodology and Instrumentation

The following instrumentation and conditions were employed:

• Liquid chromatograph: ACQUITY Premier Quaternary LC System with high-pH flowpath kit
• Column heater: CH-A 15 cm
• Detector: TUV detector set at 280 nm
• Test columns: GTxResolve Premier BEH SEC 450 Å 2.5 µm, 2.1 × 150 mm; comparator column: 700 Å 3 µm titanium, 2.1 × 150 mm
• Mobile phase: 20 mM phosphate, 276 mM NaCl, 5.4 mM KCl at pH 7.4 (2× dPBS) or varied NaCl concentrations for salt‐dependence studies
• Flow rate: 0.052 mL/min (SEC) and 0.073 mL/min (protein chemistry tests)
• Column temperature: 30 °C
• Analytes: BEH 450 Protein Mix Standard, NISTmAb RM 8671, empty AAV2 viral vector

Prior to analysis, pre- and post-column tubing was minimized to reduce extra-column dispersion.

Main Results and Discussion

Comparison of protein mix separations demonstrated that the GTxResolve narrow bore column achieved full USP half-height resolution for all test proteins, whereas the comparator column failed to resolve several peak pairs.
Salt‐dependence experiments with NISTmAb showed negligible changes in peak tailing or recovery on the GTxResolve column over 0–200 mM NaCl, indicating minimal electrostatic interactions.
In contrast, the titanium column exhibited decreased peak tailing only at higher salt, revealing metal‐induced adsorption.
Analysis of empty AAV2 revealed that the GTxResolve column provided consistent recovery and sharp peak shapes in both 1× and 2× dPBS mobile phases, while the comparator column showed poor recovery at 1× dPBS.

Benefits and Practical Applications

Use of the 2.1 mm ID GTxResolve Premier SEC column offers:

• Significant reduction in precious sample consumption
• Improved sensitivity due to reduced dispersion
• High separation efficiency comparable to wider bore columns
• Minimal method development for mobile phase optimization

Its performance makes it suitable for routine purity testing and biophysical characterization in research, process development, and QA/QC laboratories.

Future Trends and Opportunities

The adoption of narrow bore SEC columns is expected to accelerate cell and gene therapy development by preserving lead material and enabling more extensive analytical campaigns.
Integration with multi-detector systems (e.g., SEC-MALS) and further column hardware innovations will expand applications to other complex biotherapeutics, including antibody-drug conjugates and virus-like particles.

Conclusion

The 2.1 × 150 mm GTxResolve Premier BEH SEC 450 Å 2.5 µm narrow bore column demonstrated high resolution, low salt sensitivity, and consistent recovery for proteins and viral vectors while halving sample consumption.
This custom column outperformed a commercially available titanium column, offering an effective solution for limited-sample SEC analyses.

References

1. Kizekai L., Addepalli B., Lauber M. A. Heightened Characterization of AAVs by SEC-MALS with XBridge™ Premier GTx BEH™ SEC 450 Å 2.5 µm Columns. Waters Application Note 720007969, June 2023.
2. Imiołek M., Fekete S., Kizekai L., Addepalli B., Lauber M. Fast and Efficient Size Exclusion Chromatography of Adeno Associated Viral Vectors With 2.5 Micrometer Particle Low Adsorption Columns. Journal of Chromatography A, 1714 (2024) 464587.