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

抗体药物在癌症治疗和免疫诊断中起着重要作用,但抗体的分离纯化通常采用酸性洗脱,易导致抗体聚集失活等问题。本研究以硅胶为基质,以温敏嵌段聚合物聚[(N-异丙基丙烯酰胺)-b-4-乙烯基吡啶](P[NIPAM-b-4VP])为间隔臂,以4-巯基乙基吡啶(MEP)为配基,制备了一种嵌段共聚温敏亲和色谱固定相SiO_(2)-P[NIPAM-b-4VP]-MEP,并以牛血清白蛋白(BSA)和γ-球蛋白为模型蛋白,对制备的温敏亲和色谱固定相的色谱性能进行了表征。分别考察了流动相盐浓度和温度对二者分离性能的影响。结果表明,在40℃时该固定相只选择性保留γ-球蛋白,而不保留BSA;在5℃时采用含0.6 mol/L NaCl的Tris-HCl(pH 8.0)缓冲溶液进行洗脱,γ-球蛋白的质量回收率为92.7%。该固定相对γ-球蛋白的吸附量为(71.5±2.1)mg/g(n=3),是传统温敏亲和色谱固定相SiO_(2)-PNIPAM-MEP的2倍。将该固定相用于人血清中IgG的分离纯化,仅通过改变流动相温度和盐浓度即可一步实现对抗体的分离纯化,纯度大于97.4%±0.7%。上述结果表明该温敏亲和色谱固定相不仅对抗体具有特异的选择性,而且洗脱条件温和,绿色环保,从根本上解决了传统亲和色谱采用酸性洗脱易使抗体蛋白变性失活的难题。该技术在抗体药物的分离纯化和工业生产中具有重要的应用价值。

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 aqueous 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,SiO_(2)-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopropylacrylamide-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)andγ-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 retainedγ-globulin due to the specific affinity interaction between antibodies and the ligand MEP.At 5℃,γ-globulin can be eluted 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 ofγ-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 SiO_(2)-PNIPAM-MEP.The stationary phase was also used to separate and purify immunoglobulin(IgG)in human serum in one step by altering the temperature 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 potential for the industrial production of antibody drugs.