Benzotriazole-5-carboxylic as a mixed-mode ligand for chromatographic separation of antibody with enhanced adsorption capacity.

Mixed-mode chromatography provides a promising strategy for industrial protein purification for its potential merit of balancing efficiency and cost-effectiveness. However, mixed-mode media with satisfactory selectivity and binding capacity towards antibody are still urgently needed. A new type of mixed-mode chromatography resin was prepared using benzotriazole-5-carboxylicas as ligand (BTA MM), and its application in antibody separation was explored. A typical pH-dependent protein binding was observed, and the neutral condition was favorable for antibody adsorption. Dynamic binding capacity of human immunoglobulin G (hIgG) was 57.7 mg/mL at pH 7.4 (10 mM phosphate buffer, containing 150 mM NaCl), while elution with acidic solutions (pH 3-4) could achieve a recovery of more than 85%. Protein adsorption on the resin showed a salt-independent manner, thus it could work under physiological solution conditions, with satisfied antibody selectivity. One-step purification of antibody components from human serum samples could obtain a product with the purity more than 84%. Satisfied performance was also observed when the adsorbent was used for purifying a IgG1-type monoclonal antibody (mAb) from cell culture supernatant. In addition, the benzotriazole adsorbent has been found stable enough to withstand autoclave sterilization and other harsh conditions, including 1 M NaOH, 1 M HCl, and 75% ethanol. The results proved the potential of this type of mixed-mode chromatography medium for industrial antibody purification.