The binding mechanism between the engineered C domain of the Amsphereâ„¢ A3 protein A (PrA) ligand and a VHH single domain antibody (sdAb) was revealed. Binding sites in the PrA ligand in helices 2 and 3 and in framework regions 1 and 3 of the VHH were confirmed. Identified VHH residues are not involved in antigen recognition. Overlap with a human VH showed the same interaction sites. These results provide insight on why Amsphere A3 is a suitable tool for antibody fragment purification, with the known benefits of high process robustness, selectivity and caustic stability. Amsphere A3 has the same capacity for sdAbs as the current non-PrA affinity ligand resins (20-30 g/L).

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