Ludwig-Maximilians Universität München, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, 81377 Munich, Germany.
Westflische Wilhelms-Universität Münster, Institute of Physical Chemistry, 48149 Münster, Germany.
Int J Pharm. 2018 Apr 25;541(1-2):234-245. doi: 10.1016/j.ijpharm.2018.02.009. Epub 2018 Feb 24.
Both, formulation parameters and the presence of liquid-air interfaces are known to affect the aggregation of protein drugs. In this study, the impact of pH on the liquid-air interfacial behavior of three proteins, a polyclonal and two monoclonal antibodies (IgG, mAB and mAB) was investigated using different surface sensitive methods. Equilibrium surface pressure values revealed only a minor impact of pH. Infrared Reflectance Absorbance Spectroscopy (IRRAS) proved not only the presence of the proteins at the interface but also showed that the secondary structure was not considerably affected by the adsorption to the interface independent of pH between pH 3 and 9. Additionally, the physical resistance of the film as determined by the interfacial compressibility in a Langmuir trough was not affected by pH. Compression of the interfacial film caused the formation of telescoped areas which were no longer present after decompression at all pH values as investigated by underwater Atomic Force Microscopy (AFM). Brewster Angle Microscopy (BAM) showed some slight changes in the film reflectivity depending on pH, indicating changes in the interfacial film thickness. IRRAS experiments at different angles of incidence as well as section analysis of AFM images proved not only that the film thickness increased upon compression, but also that the interfacial film is thinner at pH 4 than at pH 9. Continuous compression and decompression of the protein film resulted in particle formation with increasing numbers of particles at higher pH value as detected by Light Obscuration (LO) and Micro-Flow Imaging (MFI). The use of different surface sensitive methods provides expedient information on how liquid-air interfacial events are affected by formulation pH. These findings enable a better understanding of not only the events and processes happening at the interface but can also be directly linked to the interface-related formation of particles.
配方参数和液-气界面的存在都已知会影响蛋白质药物的聚集。在这项研究中，使用不同的表面敏感方法研究了 pH 对三种蛋白质（多克隆和两种单克隆抗体（IgG、mAB 和 mAB）的液-气界面行为的影响。平衡表面压力值仅显示 pH 有轻微影响。红外反射吸收光谱（IRRAS）不仅证明了蛋白质在界面上的存在，还表明二级结构不受 pH 影响，在 pH 3 至 9 之间，吸附到界面上不会对其产生显著影响。此外，通过 Langmuir 槽中的界面可压缩性确定的界面膜物理阻力不受 pH 影响。界面膜的压缩导致形成了伸缩区，在所有 pH 值下进行减压后，这些伸缩区都不再存在，这可以通过水下原子力显微镜（AFM）进行研究。偏振光显微镜（BAM）显示出界面膜反射率取决于 pH 的一些轻微变化，表明界面膜厚度发生变化。不同入射角的 IRRAS 实验以及 AFM 图像的截面分析不仅证明了膜厚度随压缩而增加，还证明了 pH 4 时的界面膜比 pH 9 时更薄。通过光阻塞（LO）和微流成像（MFI）检测到，蛋白质膜的连续压缩和解压缩导致颗粒形成，在较高 pH 值下颗粒数量增加。使用不同的表面敏感方法可以快速获得有关配方 pH 如何影响液-气界面事件的信息。这些发现不仅使我们更好地了解界面上发生的事件和过程，而且可以直接与与界面相关的颗粒形成联系起来。
