Sediq Ahmad S, van Duijvenvoorde R B, Jiskoot Wim, Nejadnik M Reza
Division of Drug Delivery Technology, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, 2300 RA, The Netherlands.
Division of Drug Delivery Technology, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, 2300 RA, The Netherlands.
J Pharm Sci. 2016 Feb;105(2):519-529. doi: 10.1016/j.xphs.2015.10.003. Epub 2016 Jan 12.
This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution on protein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring set-up was designed in which the presence and magnitude of the contact between the stir bar and the container could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphate buffered saline was stirred at 300 rpm at room temperature. At different time points, samples were taken and analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography. In contrast to non-contact-stirred and unstirred samples, the contact-stirred sample contained several-fold more particles and showed a significant loss of monomer. No increase in oligomer content was detected. The number of particles formed was proportional to the contact area and the magnitude of the normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidine fluorescence indicated a conformational change for contact-stirred protein samples. Presence of polysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in which abrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surface for adsorption of a fresh protein layer.
本研究探讨了单克隆抗体溶液搅拌过程中的接触滑动对蛋白质聚集的影响，特别是在纳米和微米尺寸范围内。设计了一种顶置搅拌装置，其中搅拌棒与容器之间接触的存在和大小可以被控制。将0.1mg/mL单克隆抗体（IgG）的磷酸盐缓冲盐水溶液在室温下以300rpm搅拌。在不同时间点取样，并通过纳米颗粒跟踪分析、流动成像显微镜和尺寸排阻色谱进行分析。与非接触搅拌和未搅拌的样品相比，接触搅拌的样品含有多几倍的颗粒，并且单体显著损失。未检测到低聚物含量增加。形成的颗粒数量与接触面积以及搅拌棒与玻璃容器之间的法向压力大小成正比。外在的9-(2,2-二氰基乙烯基) 朱利啶荧光表明接触搅拌的蛋白质样品发生了构象变化。聚山梨酯20的存在抑制了微米级聚集体的形成。我们提出了一个模型，其中潜在不稳定的吸附蛋白质的磨损导致聚集，并为新鲜蛋白质层的吸附更新表面。
