Enhancing Size-Exclusion Chromatography of Large Biomolecular Analytes With Rigorously Designed GTxResolve™ Premier SEC 1000 Å 3 μm Columns

Significance of the Topic

Size‐exclusion chromatography (SEC) is a cornerstone technique for characterizing macromolecules by hydrodynamic volume. As cell and gene therapies, antibody‐drug conjugates, and nucleic acid therapeutics become more prevalent, demands on SEC increase for higher resolution, reproducibility, and minimal sample loss. The development of columns tailored for large biomolecules is critical to ensure accurate impurity profiling, quality control, and regulatory compliance in biopharmaceutical research and manufacturing.

Study Objectives and Overview

This study evaluates the performance of Waters GTxResolve™ Premier SEC 1000 Å columns packed with novel 3 µm ethylene‐bridged PEO‐bonded particles and MaxPeak™ HPS surfaces. Key aims include demonstrating batch‐to‐batch and column‐to‐column reproducibility, enhanced resolution for proteins and nucleic acids, and reduced secondary interactions compared to conventional 5 µm 1000 Å silica SEC columns.

Methodology

Samples comprised a standard five‐component protein mix (thyroglobulin dimer/monomer, IgG, BSA, myoglobin, uracil), a dsDNA ladder (50–1350 bp), an antibody‐drug conjugate (Kadcyla™), and the NIST monoclonal antibody reference material. Separations employed phosphate buffers, PBS, NaCl, and acetonitrile titrations to probe electrostatic and hydrophobic interactions. Column dimensions of 4.6 × 150 mm and 4.6 × 300 mm were tested at flow rates of 0.1–0.25 mL/min, temperatures of 6–35 °C, and UV detection at 260/280 nm.

Instrumentation

• ACQUITY™ UPLC H-Class Bio System (Premier equivalent)
• ACQUITY™ Tunable UV Detector
• Polypropylene screw‐neck vials with septumless caps

Key Results and Discussion

Column architecture featuring MaxPeak HPS surfaces eliminated metal oxide interactions and reduced nonspecific adsorption. The 3 µm packing delivered significantly higher signal (0.12 AU vs. 0.09 AU), sharper peaks, and doubled plate counts (19 K vs. 9 K for 150 mm columns) compared to 5 µm silica counterparts. Nucleic acid separations resolved up to eight species in the 400–766 bp range with an average of 20 K plates versus 11 K on 5 µm columns. Electrostatic and hydrophobic interaction tests showed minimal changes in tailing factors (<6% and <11%, respectively), whereas 5 µm columns exhibited 21–31% variation.

Benefits and Practical Applications

• Improved resolution and peak capacity for high‐molecular‐weight proteins and nucleic acids
• Robust batch‐to‐batch and column‐to‐column reproducibility
• Minimal sample adsorption and secondary interactions
• Faster and more reliable impurity profiling for advanced biotherapeutics

Future Trends and Potential Applications

Advancements in SEC column technology will drive higher‐throughput analyses of mRNA, viral vectors, and lipid nanoparticles. Integration with mass spectrometry, expanded pore size distributions, and further miniaturization of particle diameters may enable real‐time process monitoring and ultra‐high sensitivity characterization.

Conclusion

GTxResolve™ Premier SEC 1000 Å 3 µm columns represent a step change in SEC performance for large biomolecules, delivering superior resolution, reproducibility, and inertness that meet the stringent needs of modern biopharmaceutical development.

References

1. Barth HG, Boyes BE. Size Exclusion Chromatography. Anal Chem. 1990;62(12):268–303.
2. Hong P, Koza S, Bouvier ESP. A review of size‐exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chromatogr Relat Technol. 2012;35(20):2923–2950.
3. Fekete S, Kizekai L, Sarisozen YT, et al. Investigating secondary interactions of packing materials for size‐exclusion chromatography of therapeutic proteins. J Chromatogr A. 2022;1676:463262.