Abstract：
Staphylococcal protein-A affinity chromatography has been optimized for antibody purification, achieving a current capacity of up to 90 mg/ml in packed bed. The morphology of the particles, the number of antibodies bound per ligand and the spatial arrangement of the ligands were assessed by in-situ Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) combined with measurement of adsorption isotherms. We employed SAXS measurements to probe the nanoscale structure of the chromatographic resin. From scanning electron microcopy, the morphology and area of the beads were obtained. The adsorption isotherm revealed a bi-Langmuirian behavior where the association constant varied with the critical bulk concentration, indicating multilayer adsorption. Determining the antibody-ligand stoichiometry was crucial for understanding the adsorption mechanism, which was estimated to be 4 at lower concentrations and 4.5 at higher concentrations, suggestive of reversible protein-protein interactions. The same results were reached from the in-situ small angle X-ray scattering measurements. A stoichiometry of 6 cannot be achieved since the two protein A monomers are anchored to the stationary phase and thus sterically hindered. Normalization through ellipsoids facilitated SAXS analysis, enabling the determination of distances between ligands and antibody-ligand complexes. Density fluctuations were examined by subtracting the elliptical fit, providing insights into ligand density distribution. The dense ligand packing of TOYOPEARL® AF-rProtein A HC was confirmed, making further increases in ligand density impractical. Additionally, SAXS analysis revealed structural rearrangements of the antibody-ligand complex with increasing antibody surface load, suggesting reversible association of antibodies.
摘要：
葡萄球菌蛋白-A亲和色谱已针对抗体纯化进行了优化，在填充床中实现高达90mg/ml的电流容量。颗粒的形态，通过原位小角度X射线散射（SAXS）和扫描电子显微镜（SEM）结合吸附等温线测量来评估每个配体结合的抗体数量和配体的空间排列。我们使用SAXS测量来探测色谱树脂的纳米级结构。从扫描电子显微镜来看，获得珠子的形态和面积。吸附等温线揭示了双Langmuirian行为，其中缔合常数随临界体积浓度而变化，表明多层吸附。确定抗体-配体化学计量对于理解吸附机理至关重要，在较低浓度下估计为4，在较高浓度下估计为4.5，提示可逆的蛋白质-蛋白质相互作用。从原位小角度X射线散射测量得到相同的结果。不能实现6的化学计量,因为两个蛋白A单体锚定到固定相并因此空间受阻。通过椭圆体的归一化促进了SAXS分析，能够确定配体和抗体-配体复合物之间的距离。通过减去椭圆拟合来检查密度波动，提供对配体密度分布的见解。确认了TOYOPEARL®AF-r蛋白AHC的致密配体包装,进一步增加配体密度是不切实际的。此外,SAXS分析显示，随着抗体表面负载的增加，抗体-配体复合物的结构重排，提示抗体的可逆关联。
