Facile Capture of Recombinant Human Erythropoietin on Mesoporous Affinity Hydrogel Matrix Functionalized with Azoboronate.

Boronate affinity ligands (BALs) have gained attention for glycoproteins capture and recognition due to their unique affinity interaction with glycans. In this paper, the effect of azo immobilization of phenylboronic acid on the reduction of adsorption pH of a recombinant glycoprotein (i.e., rhEPO) on hydrogel microparticles was investigated. To evaluate the influence of intraparticle porosity on protein adsorption, microporous (MicroBead) and mesoporous (MesoBead) agarose beads carrying two levels of amine densities were functionalized with azoboronate ligand. Affinity adsorption of the glycoprotein during static and dynamic adsorptions at relatively low pHs of 8 and 7 was studied. Results revealed successful adsorption of rhEPO at pH = 8 through affinity capture of glycans by azoboronate ligands. Increased amine density provided 1.1 and 1.5 times higher static adsorption capacities and dynamic performance efficiencies, respectively. In addition, adsorption capacities and initial adsorption rates of rhEPO on MesoBeads were respectively 1.4 and 2.5-2.8 times of MicroBeads. Also, at pH = 8, MesoBeads recorded higher dynamic recoveries (59 and 91%) compared with microporous ones (46 and 69%) since mesoporosity facilitates intraparticle mass transfer. Reduction of binding pH from 8 to 7 resulted in a sharp decrease in dynamic recovery (14%), indicating the appropriate binding pH of azoPBA to be above 7. The azoboronate affinity ligand is a leading candidate for capturing glycoproteins at relatively low pH. Also, mesoporous microparticles are appropriate tools in more efficient medium-sized protein binding applications.