Chromatography Column Frits with MaxPeak High Performance Surfaces Prevent Nonspecific Binding of Nucleic Acids

Importance of the Topic

Chromatographic analysis of biotherapeutics requires high recovery and consistent performance.
Nonspecific adsorption of charged macromolecules on column frits leads to sample loss, distorted peaks, and compromised quantification.
Addressing frit-sample interactions is crucial for reliable nucleic acid separations.

Objectives and Study Overview

This study evaluates the performance of traditional stainless steel frits versus hybrid organic/inorganic MaxPeak High Performance Surfaces (HPS) frits for preventing nonspecific binding of a 23 kbp dsDNA ladder.
It examines recovery under varying pressure (low ~790 psi and high ~9,000–10,000 psi) and porosity (0.2 μm and 0.5 μm).

Methodology

Flow injection experiments used a two-frit assembly without stationary phase to isolate frit effects.
A 23 kbp dsDNA ladder was prepared at 5 μg/mL in aqueous buffer and injected through assemblies containing either stainless steel or MaxPeak HPS frits.
Tests were performed at low and high back pressures, and recovery was determined by comparing chromatographic signal to a frit-free system.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System
• ACQUITY TUV Detector (260 nm)
• PEEK and PEEKsil tubing assemblies
• Empower 3 chromatography software

Main Results and Discussion

At low pressure, stainless steel frits achieved 69% DNA recovery, while MaxPeak HPS frits reached 100%.
Under high pressure, recovery on stainless steel frits dropped to 20%, reflecting increased nonspecific adsorption under shear stress.
MaxPeak HPS frits maintained near-complete recovery under both pressure regimes.
Porosity studies showed 0.2 μm and 0.5 μm MaxPeak HPS frits provided comparable performance, indicating channel size did not influence adsorption for 23 kbp DNA.
Thyroglobulin passivation of stainless steel frits improved recovery to 69% but suffered from poor reproducibility.

Benefits and Practical Applications

• Complete preservation of large nucleic acids during flow injections
• Elimination of sample-based passivation steps
• Compatibility with high-pressure slalom chromatography
• Simplified method development for nucleic acid quantification and separations

Future Trends and Potential Applications

Advancements in surface engineering may extend to other chromatographic hardware to further minimize biomolecule adsorption.
Integration of HPS frits with emerging slalom and size-exclusion platforms could enhance workflows for gene therapies, vaccines, and high-molecular-weight protein complexes.
Custom pore architectures may optimize separations across diverse biomolecules.

Conclusion

MaxPeak HPS frits effectively prevent nonspecific binding of large nucleic acids under both normal and elevated pressures, outperforming traditional stainless steel frits.
Their consistent performance and compatibility with varied porosities make them well suited for sensitive bioseparation applications.

References

1. J. Simeone et al., J Chromatogr A 1666 (2022) 462855.
2. G. J. Guimaraes and M. G. Bartlett, Anal Chim Acta 1250 (2023) 340994.
3. F. Gritti, J Chromatogr A 1738 (2024) 465487.