Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis‐diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation‐exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge‐transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins. Phenylboronate chromatography has been applied for several years, however, the detailed mechanisms of interactions between the ligand and complex biomolecules are not well studied. In this work, authors show that phenylboronate can adsorb not only glycoproteins but also other protein classes under selected conditions. These findings can enhance the phenylboronate chromatography performance regarding its selectivity towards glycoproteins separation, and also provide a broader applicability for chromatography.