Differential binding of heavy chain variable domain 3 antigen binding fragments to protein a chromatography resins

•We characterized the binding of 15 different VH3 mAb and F(ab’)2 fragments to two protein A resins.•A recombinant protein A ligand and an engineered ligand made of four Z domain repeats was studied.•Binding behaviour differs depending on the VH3 F(ab’)2 sequence and the protein A ligand.•The differential binding behaviour is localized to the CDR2 domain of the VH3 Fab.•Protein A binding can be gained or lost by mutation of a single amino acid in the IgG VH3 CDR2 domain.

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.