title: Antibody purification by affinity chromatography based on small molecule affinity ligands identified by SPR-based screening of chemical microarrays.
abstract: Libraries of small molecules were searched for Fc-fragment selective binders to a recombinant human antibody ("MDJ8″, IgG(1)-subtype, κ-light chain) via SPR-based screening of chemical microarrays. Identified hit structures were immobilised on NHS-activated Sepharose for the determination of MDJ8 binding and selectivity versus typical proteineous impurities represented by the spend cell culture supernatant. Columns were packed and the most promising ligands further characterized in terms of binding constants, binding kinetics, as well as dynamic and equilibrium binding capacities. The performance of the best ligand, 2A10, was compared to standard Protein A chromatography. Using ligand 2A10 antibody capture from unprocessed cell culture supernatants was possible at similar recovery yield (>90%), purity (>80%), and eluting concentration (approximately 1 g/L) as with Protein A. Affinity constants (K(d)) of 2A10 were an order of magnitude higher than for the Protein A material, but still in the nM-range, while maximum binding capacities and binding kinetics were in the same order of magnitude. Ligand 2A10 was also able to capture a murine monoclonal antibody, again with similar efficiency as Protein A, as well as a number of humanised therapeutic antibodies. Antibody elution from the 2A10 column was possible using the Protein A standard protocol, i.e. 100mM glycine HCl pH 3.0, but also at near physiological pH, when some organic solvent or modifiers were present. Ligand 2A10 thus constitutes a cheaper, more robust alternative to Protein A as possible generic antibody binder. Moreover, the outlined approach to ligand selection could in principle by used to create suitable affinity ligands for other high value biotech products.
authors.affiliation: Graffinity Pharmaceuticals GmbH, Heidelberg, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Arnold
authors.forename: Holger
authors.initials: H
authors.lastname: Bittermann
authors.forename: Bernd
authors.initials: B
authors.lastname: Kalbfuss-Zimmermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Neumann
authors.forename: Kristina
authors.initials: K
authors.lastname: Schmidt
authors.forename: Renate
authors.initials: R
authors.lastname: Sekul
authors.forename: Frank
authors.initials: F
authors.lastname: Hilbrig
authors.forename: Heiko
authors.initials: H
authors.lastname: Ludolph
authors.forename: Ruth
authors.initials: R
authors.lastname: Freitag
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046228:Microarray Analysis / Q000379:methods*; D009994:Osmolar Concentration; D011485:Protein Binding; D055550:Protein Stability; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D054852:Small Molecule Libraries; D013205:Staphylococcal Protein A
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D012685:Sepharose; D054852:Small Molecule Libraries; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A