色谱2023，Vol.41Issue(12)：1045-1051,7.DOI:10.3724/SP.J.1123.2023.09028
嵌段共聚温敏亲和色谱固定相的制备及其对抗体的分离纯化
Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification
摘要
Abstract
Antibodies play an essential role in cancer diagnosis and treatment because of the specificity for target biomolecules and reduction of side effects.However,antibodies separation and purification still face some challenges.Antibody elution from columns using a low-pH aque-ous solution leads to aggregation or loss of activity of the antibody drugs.In this paper,a block copolymer-based temperature-responsive affinity chromatography(TRAC)stationary phase,SiO2-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopro-pylacrylamide-b-4-vinylpyridine)(P[NIPAM-b-4VP])as the space arms and 4-mercaptoethyl pyridine(MEP)as the ligand was prepared for antibody separation.The TRAC column was tested using bovine serum albumin(BSA)and γ-globulin as model proteins,and the effects of salt concentration in the mobile phase and temperature on their separation were studied in detail.At 40℃,the TRAC stationary phase only selectively retained γ-globulin due to the spe-cific affinity interaction between antibodies and the ligand MEP.At 5℃,γ-globulin can be elu-ted from the column with a mass recovery of 92.7%using a Tris-HCl buffer(pH 8.0)solution containing 0.6 mol/L NaCl.The adsorption capacity of γ-globulin on this stationary phase was(71.5±2.1)mg/g(n=3),which was twice that of a traditional temperature-sensitive affinity chromatography stationary phase SiO2-PNIPAM-MEP.The stationary phase was also used to separate and purify immunoglobulin(IgG)in human serum in one step by altering the tempera-ture and ion strength of the mobile phase,resulting in a purity of 97.4%±0.7%.Thus,this new technology has specific selectivity for antibodies,as well as mild and green elution conditions,ultimately resolving the problem of traditional affinity chromatography using acid elution,which can lead to the antibodies aggregation/inactivation.This technology has great application poten-tial for the industrial production of antibody drugs.关键词
温敏色谱/亲和色谱/抗体分离/聚(N-异丙基丙烯酰胺)/4-乙烯基吡啶Key words
temperature-responsive chromatography/affinity chromatography/antibody sepa-ration/poly(N-isopropylacrylamide)/4-vinylpyridine分类
化学化工引用本文复制引用
郭冬梅,夏一然,Mujeeb ur RAHMAN,王建忠,刘家玮,白泉..嵌段共聚温敏亲和色谱固定相的制备及其对抗体的分离纯化[J].色谱,2023,41(12):1045-1051,7.基金项目
国家自然科学基金(22174108,21874106) （22174108,21874106）
陕西省自然科学基金(2021JM-310). National Natural Science Foundation of China(Nos.22174108,21874106) （2021JM-310）
Natural Science Foundation of Shaanxi Province(No.2021JM-310). （No.2021JM-310）
