Heparin Sepharose Binding Chromatographic Assay
The interaction of the antibodies with heparin was measured using a chromatographic method in which approximately 100 µg of each antibody variant in 1X PBS, pH 7.4 was injected onto a heparin sepharose fast flow column (Cytiva) which had been equilibrated in 20 mM potassium phosphate, pH 7.0. Bound protein was eluted using a linear gradient of 2-100% 20 mM potassium phosphate, 1 M sodium chloride, pH 7.0 and extent of heparan sulfate binding activity was assessed based on the observed column retention (measured in minutes) and converted to the corresponding concentration of sodium chloride required to elute the bound protein from the column and into a heparin interaction potential.11