title: Toward improving selectivity in affinity chromatography with PEGylated affinity ligands: the performance of PEGylated protein A.
abstract: Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or "PEGylation" can potentially improve selectivity by sterically suppressing non-specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media-associated non-specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono-PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media-associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two- to three-fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass-transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands.
authors.affiliation: Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Monterrey, NL, 64849, México.
authors.forename: José
authors.initials: J
authors.lastname: González-Valdez
authors.forename: Alex
authors.initials: A
authors.lastname: Yoshikawa
authors.forename: Justin
authors.initials: J
authors.lastname: Weinberg
authors.forename: Jorge
authors.initials: J
authors.lastname: Benavides
authors.forename: Marco
authors.initials: M
authors.lastname: Rito-Palomares
authors.forename: Todd M
authors.initials: TM
authors.lastname: Przybycien
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000906:Antibodies; D055767:Immobilized Proteins; D013205:Staphylococcal Protein A; D011092:Polyethylene Glycols
keywords: PEGylation; affinity chromatography; antibody purification; bioseparations; protein A