Introduction: With the expansion of industrial production of various recombinant pharmaceuticals, it is essential to improve the purification efficiencies. developing boronic acid substrates as affinity adsorbents with appropriate spacer arms is essential to improve adsorption capacity of glycoproteins such as recombinant human erythropoietin (rhEPO). Therefore, this study compares the use of two amine activating agents (ammonia and ethylene diamine) with different chemical structure lengths as the spacer arms for covalent binding with boronate (CPBA) ligand to study the effect of ligand length and presence of amine functional groups in the boronate structure on affinity adsorption of a model glycoprotein (i.e. rhEPO) on agarose microparticles.
Methods: Immobilization of spacer arms: Agarose microparticles were activated with highly reactive epoxid groups according to Porath et al [18]. Then, the activated microparticles were aminated following Matsumoto et al [19]. For this purpose, NH3 and EDA respectively as two proportionally short and long length spacer arms were employed.
Functionalization of the substrate with boronate affinity ligands: In order to functionalize the bed with boronate affinity ligands, 1.25 mL of activated and dried microparticles was placed in the reaction buffer of phosphate-buffered saline (PBS) solution of 0.1 molar (pH 7.4), 0.15 molar NaCl, 100 mM carbodiimide (EDC), 100 mM N- hydroxysuccinimide (NHS), and 50 mM 3-carboxyphenylboronic acid (CPBA) as affinity ligand. The reaction was carried out at ambient temperature for 2 hours. On completion of the reaction, the functionalized microparticles were washed with 0.01 M sodium phosphate buffer. The microparticles were stored in 20% ethanol at 4℃. In brief, two types of agarose microparticles namely EDA-CPBA and NH3-CPBA were prepared. By comparing the adsorption performance of NH3-CPBA and EDA-CPBA resins, the role of the spacer arm length and structure on glycoprotein adsorption was compared
. In order to confirm the formation of favorable chemical bonds on agarose microparticles, Fourier-transform infrared spectroscopy (FTIR) was performed using PerkinElmer Spectrum Gx, Perkin-Elmer. Finally, the immobilized CPBA density was measured by elemental analysis (Thermo Finnegan).
Affinity capture of glycoprotein: To evaluate the effect of ligand structure (spacer arms) affinity capturing of rhEPO by two boronate functionalized microparticleswas conducted.
To compare the ligands' performance, affinity capture efficiencieswere calculatedas ratio of the amount of captured protein (Mcaptured) to the amount of protein loaded (Mloaded) in the feeding stage [20] according to equation (1)
Affinity capture efficiency (%) = (Mcaptured/Mloaded) × 100 equation (1)
Results: The results show that the adsorbent aminated with type 2 amine had a better performance in protein adsorption than the adsorbent aminated with type 1 amine. This can be due to two reasons. One is that the presence of two amine groups behind the CPBA ligand can improve the tendency of the ligand to capture proteins due to more significant electron withdrawal. Second, the employed type 2 amine holds four atoms thus can reduce the steric hindrance by providing a longer amine spacer arm which makes the ligands more accessible for the protein molecules to bind to. In conclusion, desiging the structure of boronate affinity ligand and the vicinal functional groups based on target biomolecules enhances the capturing capacity of boronate functionalized materials for further applications in diagnosis, separation and sensing of glycoproteins.
Conclusion: The adsorbent aminated with type 2 amine had a better performance in protein adsorption than the adsorbent aminated with type 1 amine.This can be due to two reasons. One is that the presence of two amine groups behind the CPBA ligand can improve the tendency of the ligand to capture proteins due to more significant electron withdrawal. Second, the use of type 2 amine can reduce the steric hindrance by providing a longer amine spacer arm and leading to better binding of the protein to the ligand. In conclusion, desiging the structure of boronate affinity ligand and the vicinal functional groups based on target biomolecules enhances the capturing capacity of boronate functionalized materials for further applications in diagnosis, separation and sensing of glycoproteins.
Keywords: Boronate affinity materials, Protein Purification, Glycoprotein, Recombinant human Erythropoietin