Boronate affinity/hydrophobic charge induction synergistic adsorption for improving purification of antibody

Adsorbents modified with small molecular ligands have shown great advantages in the downstream purification of antibody, but they still face some challenges caused by single-site adsorption mechanism. Taking the advantage of hydrophobic binding site and sugar chain in Fc region of antibody molecular, a Y-type ligand-modified adsorbent with two-site synergistic adsorption towards antibody was created by integrating 5-aminobenzimidazole and 3-aminophenylboronic acid on the molecular backbone of triazine bonded on the surface of agarose gel. Using dissociation constant and adsorption capacity as indicators, the interaction between immunoglobulin G (IgG) and the adsorbent was probed by comparison with three control adsorbents and by changing pH, salt concentration, and competitive agents in buffer. All the obtained indicators imply a boronate affinity and hydrophobic charge induction synergistic effect imposed on IgG by the benzimidazole and boric acid group in the Y-type ligand, enabling the adsorbent to provide a stronger affinity (Kd = 4.31x10(-6) M), larger adsorption capacity (Qm = 187 mg/g) and more excellent selectivity for IgG over the adsorbents with single-site adsorption mechanism. This is further demonstrated by selectively isolating IgG from protein mixture and serum. Also, pH is confirmed as an important factor for improving the synergistic effect and adsorption selectivity of the adsorbent. Under optimal condition, IgG was successfully separated from human serum and monoclonal antibody (mAb) from cell culture supernatant, with purities of 94.9 % and 97.5 % and recoveries of 94.3 % and 96.8 %, respectively. The results are comparable to those with protein A adsorbent, suggesting a good application potential of the adsorbent in the separation of antibody drugs.