Toward Improving Selectivity in Affinity Chromatography with PEGylated Affinity Ligands: The Performance of PEGylated Protein A
Toward Improving Selectivity in Affinity Chromatography with PEGylated Affinity Ligands: The Performance of PEGylated Protein A
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DOI：
10.1002/btpr.1994
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发表时间：
2014-11-01
影响因子：
2.9
通讯作者：
Przybycien, Todd M.
中科院分区：
文献类型：
--
作者：
Gonzalez-Valdez, Jose;Yoshikawa, Alex;Przybycien, Todd M.
用聚乙二醇链或PEG化对大分子亲和色谱配体进行化学修饰可以通过空间抑制非特异性结合相互作用而不牺牲结合能力来潜在地提高选择性。对于商业蛋白A亲和介质和酵母提取物（YE）和胎牛血清（FBS）作为模拟污染物，我们发现，配体占90%以上的介质相关的非特异性结合，证明了改进的机会。发现使用生物分子相互作用筛选平台保留了用5.0和20.7 kDa聚（乙二醇）链单聚乙二醇化的蛋白A的IgG静态结合亲和力。对商业蛋白A培养基进行类似的原位聚乙二醇化，并用掺入YE和FBS的IgG溶液对改性培养基进行功能表征。配体PEG化将介质相关污染物的质量降低了2至3倍或更多。奇怪的是，我们还发现PEG化后洗脱时IgG的平均回收率增加了高达15%。结合起来，当加标YE时，这些效应平均使IgG选择性增加一个数量级，当加标FBS时，相对于商业培养基，IgG选择性增加2 - 3倍。动态结合能力和传质阻力的测量结果表明，动态容量的减少归因于IgG有效孔扩散率的降低和可能较慢的IgG缔合动力学的聚乙二醇化蛋白A配体。配体聚乙二醇化是一种提高大分子配体亲和色谱选择性的可行方法。(c)2014年美国化学工程师学会生物技术。程序，30：1364-1379，2014
Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or PEGylation can potentially improve selectivity by sterically suppressing non-specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media-associated non-specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono-PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media-associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two- to three-fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass-transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1364-1379, 2014