20 mM HEPES, 0.15 M NaCl
Variants were purified on an Äkta Express chromatography system using affinity purification using Mabselect Sure Protein A resin followed by a gel filtration column based on the Superdex200 resin (GE Healthcare). The
MabSelect Sure Protein A column was washed with 0.1 M Hepes, 150 mM NaCl, pH 7.4 and antibodies were eluted from the affinity column with 0.1 M sodium formate, pH 3.5 onto the pre-equilibrated gel filtration column via an interconnected loop. The gel filtration column was operated using a running buffer based on 20 mM HEPES, 0.15 M NaCl, pH 7.4. Eluted peaks were fractionated in 96-well plates and fractions were pooled to obtain high purities with minimum of high-molecular weight protein. Antibody concentrations were determined by 280 nm absorbance with Dropsense 96 (Trinean).