Characterization of methacrylate chromatographic monoliths bearing affinity ligands
| Autor: | Urška Vidic, Urh Černigoj, Jernej Gašperšič, Blaž Nemec, Nika Lendero Krajnc, Aleš Štrancar, Aleš Podgornik, Jana Vidič |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
02 engineering and technology
Ligands 01 natural sciences Biochemistry Chromatography Affinity Analytical Chemistry symbols.namesake Adsorption Affinity chromatography Monolayer Monolith Staphylococcal Protein A geography geography.geographical_feature_category Chromatography Chemistry Ligand 010401 analytical chemistry Organic Chemistry dBc Langmuir adsorption model General Medicine 021001 nanoscience & nanotechnology 0104 chemical sciences IgG binding Hydrodynamics symbols Methacrylates 0210 nano-technology Protein Binding |
| Zdroj: | Journal of Chromatography A. 1464:72-78 |
| ISSN: | 0021-9673 |
| Popis: | We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12 mg/mL exceeding theoretical monolayer adsorption value of 7.8 mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11 nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4 nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher. |
| Databáze: | OpenAIRE |
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