The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography
The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography
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DOI：
10.1016/j.pep.2007.02.010
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发表时间：
2007-07-01
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;Tsurnoto, Kouhei;Ejima, Daisuke
精氨酸在抑制蛋白质聚集方面是有效的，在纯化过程中加入精氨酸可能是有益的。我们已经证明，精氨酸在凝胶渗透色谱中减少非特异性蛋白质结合，并有助于从蛋白质- a柱中洗脱抗体。本研究利用重组单克隆抗体（mab）和人白细胞介素-6检测了精氨酸在疏水相互作用（HIC）和离子交换色谱（IEC）中对蛋白质结合和洗脱的影响。在HIC的情况下，蛋白质在有或没有精氨酸的硫酸铵（AS）存在的情况下与苯基sepharose柱结合，并用浓度下降的AS洗脱。虽然在加载缓冲液中使用1 M的AS可使单抗完全结合，但在加载和平衡缓冲液中加入1 M的精氨酸，仅当使用低取代的苯基- sepharose时，导致蛋白质的结合较弱。虽然将AS浓度降低到0.75 M可以部分洗脱mAB，但加入0.5-1 M精氨酸有助于洗脱。在IEC的情况下，精氨酸包含在加载样品中。即使在没有精氨酸的情况下进行洗脱，在与IEC柱结合过程中包含精氨酸也会产生更大的回收率和更少的聚集。这些结果表明精氨酸增强了与树脂结合的蛋白质的洗脱，表明其作为HIC和IEC洗脱溶剂的有效性。(c) 2007爱思唯尔公司版权所有。
Arginine is effective in suppressing aggregation of proteins and may be beneficial to be included during purification processes. We have shown that arginine reduces non-specific protein binding in gel permeation chromatography and facilitates elution of antibodies from Protein-A columns. Here we have examined the effects of arginine on binding and elution of the proteins during hydrophobic interaction (HIC) and ion-exchange chromatographies (IEC) using recombinant monoclonal antibodies (mAbs) and human interleukin-6. In the case of HIC, the proteins were bound to a phenyl-Sepharose column in the presence of ammonium sulfate (AS) with or without arginine and eluted with a descending concentration of AS. While use of 1 M AS in the loading buffer resulted in complete binding of the mAb, inclusion of 1 M arginine in loading and equilibration buffer, only when using low-substituted phenyl-Sepharose, resulted in weaker binding of the proteins. While decreasing AS concentration to 0.75 M resulted in partial elution of the mAB, elution was facilitated with inclusion of 0.5-1 M arginine. In the case of IEC, arginine was included in the loading samples. Inclusion of arginine during binding to the IEC columns resulted in a greater recovery and less aggregation even when elution was done in the absence of arginine. These results indicate that arginine enhances elution of proteins bound to the resin, suggesting its effectiveness as a solvent for elution in HIC and IEC. (c) 2007 Elsevier Inc. All rights reserved.