Ludwig-Maximilians-Universität Munich, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, 81377 Munich, Germany.
AbbVie Deutschland GmbH & Co. KG, 67061 Ludwigshafen am Rhein, Germany.
Eur J Pharm Biopharm. 2017 Oct;119:396-407. doi: 10.1016/j.ejpb.2017.07.006. Epub 2017 Jul 22.
The presence of liquid-air interfaces in protein pharmaceuticals is known to negatively impact product stability. Nevertheless, the mechanisms behind interface-related protein aggregation are not yet fully understood. Little is known about the physical-chemical behavior of proteins adsorbed to the interface. Therefore, the combinatorial use of appropriate surface-sensitive analytical methods such as Langmuir trough experiments, Infrared Reflection-Absorption Spectroscopy (IRRAS), Brewster Angle Microscopy (BAM), and Atomic Force Microscopy (AFM) is highly expedient to uncover structures and events at the liquid-air interface directly. Concentration-dependent adsorption of a human immunoglobulin G (IgG) and characteristic surface-pressure/area isotherms substantiated the amphiphilic nature of the protein molecules as well as the formation of a compressible protein film at the liquid-air interface. Upon compression, the IgG molecules do not readily desorb but form a highly compressible interfacial film. IRRA spectra proved not only the presence of the protein at the interface, but also showed that the secondary structure does not change considerably during adsorption or compression. IRRAS experiments at different angles of incidence indicated that the film thickness and/or packing density increases upon compression. Furthermore, BAM images exposed the presence of a coherent but heterogeneous distribution of the protein at the interface. Topographical differences within the protein film after adsorption, compression and decompression were revealed using underwater AFM. The combinatorial use of physical-chemical, spectroscopic and microscopic methods provided useful insights into the liquid-air interfacial protein behavior and revealed the formation of a continuous but inhomogeneous film of native-like protein molecules whose topographical appearance is affected by compressive forces.
在蛋白质药物中存在气-液界面会对产品稳定性产生负面影响。然而，界面相关的蛋白质聚集的机制尚未完全理解。对于吸附在界面上的蛋白质的物理化学行为知之甚少。因此，组合使用适当的表面敏感分析方法，如 Langmuir 槽实验、红外反射吸收光谱（IRRAS）、布鲁斯特角显微镜（BAM）和原子力显微镜（AFM），非常有助于直接揭示气-液界面上的结构和事件。人免疫球蛋白 G（IgG）的浓度依赖性吸附和特征表面压/面积等温线证实了蛋白质分子的两亲性质以及在气-液界面形成可压缩的蛋白质膜。在压缩时，IgG 分子不易解吸，而是形成高度可压缩的界面膜。IRRA 谱不仅证明了蛋白质在界面上的存在，还表明在吸附或压缩过程中二级结构没有明显变化。不同入射角的 IRRAS 实验表明，在压缩时膜厚和/或堆积密度增加。此外，BAM 图像暴露了蛋白质在界面上存在一致但不均匀的分布。吸附、压缩和解压缩后使用水下 AFM 揭示了蛋白质膜内的形貌差异。物理化学、光谱和显微镜方法的组合使用为研究气-液界面蛋白质行为提供了有用的见解，并揭示了形成连续但不均匀的天然样蛋白质分子膜，其形貌受压缩力的影响。
