Preparation Of Hydrophobic Charge-inducing Membrane Adsorbents And Their Studies On The Purification Performance Of IgG

Monoclonal antibodies are the most important bioproducts which are in great demand in the biomedical field due to their excellent clinlcal features and specificity.However,for the antibody purification,the cost accounts for 50-80%of the whole preparation process via the traditional separation methods.The most popular purification method based on the Protein A affinity chromatography has several disadvantages,such as high pressure drop,limited mass transfer and falling off the ligands.Therefore,it is impulsory for developing new type of chromatographic media and antibody purification methods for industrial applications.Hydrophobic charge induction chromatography(HCIC)has emerged as a novel and efficient method for antibody purification.It can combine low cost advantage with fair selectivity.On the other hand,commercial macroporous membranes have high transport efficiency.In this context,we use a commercial regenerated cellulose(RC)membrane as a starting material for the synthesis of chromatographic media considering its high content of hydroxyl groups,large pore diameter,strong hydrophilicity and ease of modification.The RC membrane was modified to prepare a series of novel HCIC membrane adsorbers and their application in IgG purification was investigated.(Ⅰ)Two different polymers-diethylene glycol diglycidyl ether(DEGDE)and trimethyl-propane triglycidyl ether(TMPEG)were grafted on RC memrbane by cationic ring-opening polymerization(CROP)to prepare RC-g-DEGDE membrane and RC-g-TMPEG membrane,respectively.The effects on grafting yield,pore diameter and morphology of membranes with two different polymers and reaction time were studied.Meanwhile,the grafting conditions were optimized.The results demonstrated that only 0.5 h was needed to prepare the RC-g-DEGDE membrane.(Ⅱ)Four different ligands-2-mercaptobenzimidazole(MBI),2-mercapto-l-methylimidazole(MMI),2-mercaptopyridine(2MP)and 4-mercaptopyridine(4MP)were chosen to further graft on the RC-g-DEGDE membrane to prepare a series of HCIC membrane adsorbers.The influence factors of ligands grafting density,the influence of different ligands on hydrophilic and zeta potential of membrane surface were investigated.The results confirmed that the graft density of the ligands was closely related to the pKa value.The higher ligands density can be achieved with smaller pKa value with the same type of ligands.The isoelectric point(pI)value of the membrane adsorber corresponded to the ligand pKa value.The reason is that aromatic thiol with low pKa value can be easier to react with epoxy group.(Ⅲ)Bovine serum albumin(BSA)and human Immunoglobulin G(IgG)were used as model contaminant and antibody to evaluate the HCIC performance via the static and dynamic adsorption study.The RC-g-MBI membrane exhibited the highest ligand density of 370 mmol/L.The static and dynamic adsorption capacity of IgG reached 195 mg/mL and 65 mg/mL,respectively.The high dynamic capacity and high IgG recovery(96%)can be ascribed to the branched structure of DEGDE and the convective transport in membrane pores.In addition,for the IgG adsorption,the modified membrane displayed excellent tolerance to salt even in the different salt species and salt concentrations.(Ⅳ)The SiO2 nanoparticles with two different thiol groups(SiO2-W-SH and SiO2-SH)were introduced into the RC-g-DEGDE membrane through the ring opening reaction of epoxy and thiol.RC-SiW-4VP and RC-SiS-4VP were obtained via the thiol-ene reaction between thiol on sulphur nano silicon dioxide and the double bond on 4-vinylpyridine(4VP)ligand.The results show that the introduction of SiO2 nanoparticles can increase the ligand density by about five times.However,the static adsorption capacity of IgG improved only about 30%.It’s due to steric hindrance and the reduction of ligand utilization efficiency.