Modeling of the transient diffusion regime in fully porous particles-Application to the analysis of large biomolecules by ultra-high pressure liquid chromatography

The transient diffusion regime of large biomolecules such as monoclonal antibodies, double-stranded (ds) DNA (base pair number similar to 100), and virus-like particles is modeled in a single fully porous particle utilized as size exclusion chromatography (SEC) packing materials in ultra-high pressure liquid chromatography (UHPLC). The expression of the time and space dependent concentration profiles is derived for a step concentration change. Four different UHPLC particles were considered in the calculations depending on the size of the analyte : 2.0 mu m 125 angstrom Bridge-Ethylene-Hybrid (BEH) XBridge (TM) particles for small molecules (size: 4 angstrom), 2.0 mu m 125 angstrom and 250 angstrom BEH particles for monoclonal antibodies (size: 55 angstrom), 2.0 mu m 250 angstrom and 2.5 mu m 450 angstrom BEH particles for dsDNA (size: 160 angstrom), and 2.5 mu m 900 angstrom BEH particles for virus-like particles (size: 500 angstrom). The accessible porosities and the hindrance diffusion factors of these analytes were determined from the physical reconstruction of the internal structure of a 2.0 mu m 130 angstrom BEH particle and from the simulation of the analyte mobility in the reconstructed mesopores. The analysis of the transient diffusion profiles reveals that there is insufficient time for the largest analytes to fully equilibrate the BEH particles in UHPLC. The dynamic capacity of the BEH particles is estimated to decrease from more than 99% for small molecules to 96% for monoclonal antibodies, 74% for 100 base pair dsDNA, and to less than 2% for virus-like particles. A full but reduced column capacity relative to fully porous particles can be recovered for monoclonal antibodies by considering superficially porous particles with a core-to-particle diameter ratio of about 0.8. In contrast, this is not possible for either large dsDNA (base pair number >= 100) or virus-like particles because the shell thickness would become smaller than the mesopore size required. The presented results open up two research avenues in order to analyze such large biomolecules by UHPLC: prepare ultra-high pressure-resistant sub-3 mu m silica-based particles with pore sizes much larger than 1000 angstrom and design proper nonporous macrostructures to load, trap, separate, and elute rapidly by gradient UHPLC. (C) 2022 Elsevier B.V. All rights reserved.

Automated summary

This study models the transient diffusion of large biomolecules in ultra-high pressure liquid chromatography using a single fully porous particle. The research reveals that different sized analytes have varying dynamic capacities and equilibrium issues in the chromatography column.