A Novel Separation Medium For Rapid Purification Of Antibodies Based On Zwitterionic Polymer

Affinity ligand is the foundation of affinity chromatography in the application of protein separation and purification.Protein A is the most widely used affinity ligand for antibody purification,yet with some disadvantages,e.g.,poor stability and high cost.Applications of synthetic ligands are limited by their poor specificity.It has been a hot topic to synthesize ligands with high selectivity and acid/base tolerance.In this thesis,we reported a zwitterionic polymer-based affinity ligand for rapid antibody purification with high selectivity.Main work of this thesis is as follows.Firstly,poly（sulfobetaine methacrylate）（pSBMA）and derivatives were prepared by reversible addition-fragmentation chain transfer（RAFT）,reduction and thiol-conjugation reactions.Two types of zwitterionic polymer-modified NPs were fabricated by conjugating pSBMA via either one-step method（1S NPs）or two-step method（2S NPs）.Then,protein adsorption on 1S NPs and 2S NPs was studied by static adsorption experiment of protein,using human immunoglobulin G（hIgG）and bovine serum albumin（BSA）as model proteins.The results showed that 1S NPs are capable of resisting both hIgG and BSA,while 2S NPs exhibit specificity toward hIgG adsorption.The effects of divinylsulfone（DVS）treatment time,ligand structure,pSBMA chain length and NPs size as well as solution factors on protein adsorption behaviors for 2S NPs were investigated.2S NPs exhibit high specificity towards hIgG adsorption in both protein solutions and complex biological samples,and could be used at least five times.Secondly,the real-time monitoring of protein adsorption on pSBMA-based sensor surface was conducted on a biofilm interference（BLI）.The dissociation constant（K_D）was calculated to be 0.18 mg/m L(1.2×10^-6 M).The functionalized sensor surface exhibits hIgG adsorption with high specificity in simulated complex biological fluids.Finally,the interaction sites of hIgG with the ligand were estimated to be disulfide bonds through protein binding experiments using Fc-fusion protein.