title: Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.
abstract: This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding.
authors.affiliation: Drug Substance Development, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, United States.
authors.forename: Julia
authors.initials: J
authors.lastname: Bach
authors.affiliation: Drug Substance Development, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, United States.
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Lewis
authors.affiliation: Drug Substance Development, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, United States.
authors.forename: Kathy
authors.initials: K
authors.lastname: Maggiora
authors.affiliation: Drug Substance Development, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, United States.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Gillespie
authors.affiliation: Drug Substance Development, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, United States. Electronic address: connelll@amgen.com.
authors.forename: Lisa
authors.initials: L
authors.lastname: Connell-Crowley
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
keywords: F(ab’)(2) fragment; IgG; Protein A chromatography; Protein A variant; V(H)3 heavy chain; Variable affinity