In situ neutron scattering of antibody adsorption during protein A chromatography.
In situ neutron scattering of antibody adsorption during protein A chromatography.
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Protein A 层析过程中抗体吸附的原位中子散射。
DOI：
10.1016/j.chroma.2019.460842
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Papachristodoulou M
中科院分区：
文献类型：
--
作者：
Papachristodoulou M
更深入地了解色谱吸附剂的纳米级和中尺度结构以及介质中蛋白质的分布，对于使用这些材料的分离过程的机理理解至关重要。在这种规模上表征介质的结构和蛋白质的吸附，使用传统技术是具有挑战性的。在这项研究中，我们提出了一种新的树脂表征技术，可以在典型的色谱条件下测量树脂内吸附蛋白层的结构。设计并制作了一种石英流式细胞，用于小角中子散射（SANS），以测量单克隆抗体吸附过程中二氧化硅基蛋白A色谱树脂的纳米级到中尺度结构。我们能够使用对比匹配方法实时检测树脂在不同蛋白质负载和洗涤缓冲液下的孔间（≈133 nm）和孔径（≈63 nm）相关性和平面内吸附抗体分子（≈4.2 nm）相关性。当将0.03 M磷酸钠、1 M尿素和10%异丙醇缓冲液（pH 8）作为洗涤缓冲液引入该体系时，通过引起吸附抗体的部分展开，破坏了体系的秩序，证明了平面内蛋白相关性的丧失。该方法为研究层析树脂的纳米结构和配体固定化提供了新的途径；也许最重要的是了解在不同的流动相条件下，在复制色谱柱的样品环境中，吸附蛋白质在介质中的处境行为。
A deeper understanding of the nanoscale and mesoscale structure of chromatographic adsorbents and the distribution of proteins within the media, is critical to a mechanistic understanding of separation processes using these materials. Characterisation of the media's architecture at this scale and protein adsorption within, is challenging using conventional techniques. In this study, we propose a novel resin characterisation technique that enablesin-situmeasurement of the structure of the adsorbed protein layer within the resin, under typical chromatographic conditions. A quartz flow-through cell was designed and fabricated for use with Small Angle Neutron Scattering (SANS), in order to measure the nanoscale to mesoscale structures of a silica based protein A chromatography resin during the monoclonal antibody sorption process. We were able to examine the pore-to-pore (˜133 nm) and pore size (˜63 nm) correlations of the resin and the in-plane adsorbed antibody molecules (˜ 4.2 nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. When 0.03 M sodium phosphate with 1 M urea and 10 % isopropanol buffer, pH 8, was introduced into the system as a wash buffer, it disrupted the system's order by causing partial unfolding of the adsorbed antibody, as evidenced by a loss of the in-plane protein correlation. This method offers new ways to investigate the nanoscale structure and ligand immobilisation within chromatography resins; and perhaps most importantly understand thein-situbehaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.
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影响因子：
3.8
作者：
Goncalo L Silva;Jacek Plewka;H. Lichtenegger;A. C. Dias;A. Jungbauer;R. Tscheliessnig
通讯作者：
R. Tscheliessnig
DOI：
10.1515/bchm2.1978.359.2.975
发表时间：
1978-01-01
影响因子：
--
作者：
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通讯作者：
SJOQUIST, J
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影响因子：
7.4
作者：
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通讯作者：
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DOI：
--
发表时间：
2016
期刊：
In Analysis
影响因子：
--
作者：
Chadin Kulsing;András Komáromy;R. Boysen;M. Hearn
通讯作者：
M. Hearn
影响因子：
6.1
作者：
D. Mettus;A. Michels
通讯作者：
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