MEP HyperCel chromatography II: Binding, washing and elution
MEP HyperCel chromatography II: Binding, washing and elution
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DOI：
10.1016/j.pep.2009.11.004
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发表时间：
2010-06-01
影响因子：
1.6
通讯作者：
Ejima, Daisuke
中科院分区：
文献类型：
--
作者：
Arakawa, Tsutomu;Futatsumori-Sugai, Mutsumi;Ejima, Daisuke
用含有人源化IgG1和牛血清白蛋白(BSA)的两种条件培养液(CM)对MEP HyperCel层析的结合、洗脱和洗脱条件进行了考察。来自哺乳动物表达系统的单抗无需预处理就能与柱结合，尽管CM中存在的大多数污染蛋白也表现出结合。在所考察的条件下，无机盐、乙醇和甘油不能有效洗脱蛋白质，这表明静电或疏水相互作用不是抗体与MEP树脂结合的主要因素。乙二醇、异丙醇、尿素和精氨酸对结合蛋白有不同程度的洗脱作用。结合的污染蛋白和BSA用乙二醇洗脱，结合的抗体在中性pH下用精氨酸水溶液洗脱。MEP对BSA表现出选择性，因此可用于从样品中去除BSA。有趣的是。来自家蚕幼虫的Fc融合蛋白没有检测到结合。家蚕幼虫的血清蛋白与Fc融合蛋白和单抗强烈竞争与MEP树脂结合，而同样的Fc融合蛋白可以很容易地被Protein-A树脂一步纯化，再次证实了MEP树脂的弱选择性。(C)2009 Elsevier Inc.保留所有权利。
Binding, washing and elution conditions for MEP HyperCel chromatography were examined using two conditioned media (CM) containing monoclonal antibodies (humanized IgG1) and bovine serum albumin (BSA). Monoclonal antibodies derived from mammalian expression system bound to the column without pretreatment, although a majority of contaminating proteins present in the CM also showed binding. Inorganic salts, ethanol and glycerol were ineffective in eluting proteins under the conditions examined, suggesting that electrostatic or hydrophobic interactions are not a major factor for antibody binding to the MEP resin. Ethylene glycol, 2-propanol, urea and arginine were effective, to varying degrees, in elution of the bound proteins. The bound contaminating proteins and BSA were effectively eluted with ethylene glycol and the bound antibodies were finally eluted with aqueous arginine solutions at neutral pH. MEP showed selectivity toward BSA and hence utility for removing BSA from the samples. Interestingly. Fc-fusion proteins derived from silkworm larvae showed no detectable binding. Serum proteins present in silkworm larvae strongly competed with the Fc-fusion proteins and monoclonal antibody for binding to MEP resin, while the same Fc-fusion proteins can be readily purified in one-step by Protein-A resin, again confirming weak selectivity of the MEP resin. (C) 2009 Elsevier Inc. All rights reserved.