Studying the Absorption and Escape Kinetics of Biomolecules

The study of large biomolecules, such as monoclonal antibodies, double-stranded deoxyribonucleic acid (dsDNA), and virus-like particles (VLPs) has led to questions about how these molecules absorb and escape from fully porous particles. One study, published in the Journal of Chromatography A, used size exclusion chromatography (SEC) columns to analyze the concentration profiles of these large biomolecules (1).

There is uniform spatial distribution of analytes in a particle’s volume; however, this is not the case with large biomolecules. The scientists determined that non-porous particles or monolithic structures are the most effective stationary phases for the separation and purification of the biggest biomolecules. The researchers determined the concentration profiles by looking at time and radial position across a single sub-3 μm Bridge-Ethylene-Hybrid (BEH) Particle present in SEC columns.

Fabrice Gritti | Image Credit: © Fabrice Gritti

Fabrice Gritti, principal consulting scientist at Waters Corporation, told LCGC in an email that BEH Particles can be used “over a wider range of pH (examples: SEC separation of biomolecules such as oligonucleotide or DNA are operated at basic pH > 7) than that for pure silica particles and they are prepared fully inert (no undesirable and residual adsorption of large biomolecules) unlike classical pure silica-based SEC materials. This improves SEC resolution and increases the accuracy of the determination of the desired analyte size.”

All BEH particles present in the column reached near-instantaneous thermodynamic equilibrium with the bulk mobile phase during the passage of the chromatographic band. Larger biomolecules, including dsDNA and VLPs, are exceptions to this, partially due to SEC particles that are located near the column inlet and designed for high velocities. Biomolecule egress has slower kinetics than ingress, which causes pronounced peak tailing. Additionally, the mean concentration of the SEC particles’ largest biomolecules was found to be typically smaller than the maximum bulk concentration. These results all have implications on how the observed retention factors and plate heights were expressed. But more work still needs to be done, Gritti said.

“They can be tested experimentally by recording the peak shapes of small molecules, mAbs, DNAs, and viruses on these different SEC particles,” Gritti said. “In the end, most importantly, the results strongly suggest using non-porous separation systems to handle the separation of the largest biomolecules such as viruses because it would take too long for these analytes to escape the inner volume of the existing porous SEC particles.”

Reference

Gritti, F. Absorption and escape kinetics of spherical biomolecules from fully porous particles utilized in size exclusion chromatography. J. Chromatogr. A. 2023,1701, 464050. DOI: https://doi.org/10.1016/j.chroma.2023.464050