title: Characterization of methacrylate chromatographic monoliths bearing affinity ligands.
abstract: 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 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm 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.
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Urh
authors.initials: U
authors.lastname: Černigoj
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Urška
authors.initials: U
authors.lastname: Vidic
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Blaž
authors.initials: B
authors.lastname: Nemec
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Jernej
authors.initials: J
authors.lastname: Gašperšič
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Vidič
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia.
authors.forename: Nika
authors.initials: N
authors.lastname: Lendero Krajnc
authors.affiliation: BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia; COBIK, Tovarniška 26, 5270 Ajdovščina, Slovenia.
authors.forename: Aleš
authors.initials: A
authors.lastname: Štrancar
authors.affiliation: COBIK, Tovarniška 26, 5270 Ajdovščina, Slovenia; Faculty for Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia. Electronic address: ales.podgornik@fkkt.uni-lj.si.
authors.forename: Aleš
authors.initials: A
authors.lastname: Podgornik
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D057446:Hydrodynamics; D008024:Ligands; D008689:Methacrylates / Q000737:chemistry*; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000737:chemistry
chemical_list: D008024:Ligands; D008689:Methacrylates; D013205:Staphylococcal Protein A
keywords: Affinity chromatography; Antibodies; Immobilization; Immunoaffinity; Monoliths; Protein A; Protein A - IgG interaction