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 enables in-situ measurement 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 the in-situ behaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.

中文翻译：

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蛋白A层析过程中抗体吸附的原位中子散射。

对色谱吸附剂的纳米级和中级结构以及蛋白质在介质中的分布的更深入了解，对于机械理解使用这些材料的分离过程至关重要。使用常规技术来表征这种规模的介质结构及其内部的蛋白质吸附是具有挑战性的。在这项研究中，我们提出了一种新颖的树脂表征技术，该技术能够在典型的色谱条件下原位测量树脂中吸附的蛋白质层的结构。设计并制造了石英流通池，用于小角度中子散射（SANS），以便在单克隆抗体吸附过程中测量二氧化硅基蛋白A色谱树脂的纳米级到中尺度结构。我们能够检查在不同蛋白质上样量和洗涤缓冲液下树脂与孔内吸附抗体分子（约4.2 nm）的相关性，其中孔与孔（约133 nm）和孔径（约63 nm）的相关性，使用对比匹配方法实时进行。当将0.03 M磷酸钠与1 M尿素和10％的异丙醇缓冲液（pH 8）作为洗涤缓冲液引入系统时，它会通过引起吸附的抗体的部分展开而破坏系统的顺序，这可通过减少in平面蛋白的相关性。该方法提供了研究色谱树脂内纳米级结构和配体固定化的新方法。