Neutron reflectivity measurement of protein A-antibody complex at the solid-liquid interface.
Neutron reflectivity measurement of protein A-antibody complex at the solid-liquid interface.
复制标题
DOI：
10.1016/j.chroma.2017.03.084
复制
发表时间：
2017-05-26
期刊：
影响因子：
--
通讯作者：
Bracewell DG
中科院分区：
文献类型：
--
作者：
Mazzer AR;Clifton LA;Perevozchikova T;Butler PD;Roberts CJ;Bracewell DG
探讨了吸附在固液界面的IgG 4的取向。通过将蛋白A附着到二氧化硅表面来模拟层析树脂。中子反射率用于测量蛋白A和吸附的IgG结构。在IgG吸附之前，用BSA或PEG封闭蛋白A修饰的二氧化硅。吸附的IgG从表面延伸至230 μ m，这取决于阻断策略。色谱法是生物药物纯化中普遍存在的单元操作，但很少有研究涉及溶液-树脂界面处蛋白质的生物物理表征。层析和其它吸附和解吸过程已显示诱导蛋白质聚集，这在生物制药产品中是不期望的。为了进一步了解吸附过程如何影响蛋白质的稳定性，使用中子反射率来表征模型表面上吸附的免疫球蛋白G（IgG）的结构。在第一个模型系统中，IgG被直接吸附到二氧化硅上，并表现出具有高表面接触的侧面取向。在pH 4.1的条件下，观察到在表面法线方向上的最大尺寸为60 μ m和高密度的表面覆盖度。在色谱缓冲液中，发现pH影响IgG在固液界面处的堆积密度和取向。在第二个模型系统中，其被设计为模拟亲和层析表面，蛋白A被连接到二氧化硅表面，以产生代表多孔玻璃层析树脂的配置。在从配体附着到IgG结合和洗脱的连续阶段期间探测界面结构。吸附的IgG结构从表面延伸至250 μ m，并表现出对表面阻断策略的依赖性。该数据提示两个IgG分子以稍微偏斜的取向结合至蛋白A，并且非常接近二氧化硅表面。这些发现提供了深入了解吸附的抗体结构的条件下，色谱分离过程中遇到的方向。
The orientation of IgG4 adsorbed at the solid-liquid interface was probed. A chromatography resin was mimicked by attaching protein A to a silica surface. Neutron reflectivity was used to measure protein A and adsorbed IgG structures. Protein A-modified silica was blocked with either BSA or PEG before IgG adsorption. Adsorbed IgG extended up to 230 Å from the surface, depending on blocking strategy. Chromatography is a ubiquitous unit operation in the purification of biopharmaceuticals yet few studies have addressed the biophysical characterisation of proteins at the solution-resin interface. Chromatography and other adsorption and desorption processes have been shown to induce protein aggregation which is undesirable in biopharmaceutical products. In order to advance understanding of how adsorption processes might impact protein stability, neutron reflectivity was used to characterise the structure of adsorbed immunoglobulin G (IgG) on model surfaces. In the first model system, IgG was adsorbed directly to silica and demonstrated a side-on orientation with high surface contact. A maximum dimension of 60 Å in the surface normal direction and high density surface coverage were observed under pH 4.1 conditions. In chromatography buffers, pH was found to influence IgG packing density and orientation at the solid-liquid interface. In the second model system, which was designed to mimic an affinity chromatography surface, protein A was attached to a silica surface to produce a configuration representative of a porous glass chromatography resin. Interfacial structure was probed during sequential stages from ligand attachment, through to IgG binding and elution. Adsorbed IgG structures extended up to 250 Å away from the surface and showed dependence on surface blocking strategies. The data was suggestive of two IgG molecules bound to protein A with a somewhat skewed orientation and close proximity to the silica surface. The findings provide insight into the orientation of adsorbed antibody structures under conditions encountered during chromatographic separations.