Preparation Of Hydrophobic Charge-induction Adsorbent And Its Application In Antibody Separation

As one of the most important biopharmaceutical products,antibody-based pharmaceuticals have a broad application prospect due to high specificity and affinity.Antibody drugs already accounted for about 20% of the biopharmaceutical market,and the utility ratio of antibody increases year by year.With the increase of market requirement and production scale,it has become an inevitable problem that how to fast separate and purity antibodies from complex feedstock,and it puts forward more and more high demands to separation techniques.Although a series of chromatographic methods have been applied to purified antibody drugs,there are still many problems have not been overcome,such as low binding capacity and high cost.This paper proposed a novel hydrophobic charge-induction chromatography(HCIC)based on triazole group to solve the impracticable problems which arisen in conventional hydrophobic interaction chromatography(HIC).Antibody can be adsorbed to or detached from HCIC resins easily by using of pH-dependent property of designed ligands.This method can overcome the problems that tedious loading processes and severe eluting conditions existed in HIC.The main research contents of this paper include the following aspects:(1)The first part of this paper focused on synthesizing the triazole derivative through chick reaction and investigating adsorption behaviors of triazole for several model proteins in various experimental conditions.The research shows that the triazole derivative has typical characteristics of HCIC during the static adsorption in protein solutions,such as pH-dependent and salt-independent properties.Static adsorption capacity of human IgG can reach to 15 mg/m L gel at the neutral conditions,whereas other proteins have very low binding capacity.Those properties can provide a basis for preparing high-capacity HCIC adsorbent.(2)The second part focused on preparing the triazole-functionalized polystyrene microspheres(triazole-PS)based on atom transfer radical polymerization technique(ATRP)and Cu-catalyzed click reaction.The experiments examined effects of solution conditions and properties of adsorbent for IgG adsorption.The static saturated adsorption capacity and dynamic binding capacity(DBC)are 240 and 88 mg/g,respectively.The elution efficiency over 80% by usage of citrate buffer at p H 3.0.The triazole-PS can effective separate human IgG from human serum and purity over 95% by gray scale scanning.(3)The last part of this paper focused on designing and screening a novel HCIC ligand to avoid the problems of complicated reactions processed,dispose of copper ion,and so on.A novel HCIC adsorbent was prepared with benzotriazole-5-carboxylic acid as a functionalized ligand.The DBC of IgG is up to 57.7 mg/m L gel and enhances more than 60% compared to traditional HIC under physiological conditions.IgG was separated from human plasma and eluted with citrate buffer(pH 4.0)with the purity of over 95% and recovery ratio of over 70%.Besides,binding capacity of IgG can be efficiently enhanced through decreasing the eluent pH.Finally,according related zoological experiments,the benzotriazole possesses very low toxicity for human and no carcinogenic potential.Therefore,benzotriazole adsorbent has a good sepaparation ability for bivine and horse serum,the purity of IgG mare than 80% and recovery rate is reach to 80% when the ratio of gel and serum is 1:1.The results of this paper indicated HCIC adsorbent based on triazole group allowed the direct purification of human IgG from complex antibody samples with a satisfactory purity.Meanwhile,triazole group could provide new direction for designing and screening of ligands and power for purification of antibody drugs.