Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, Maryland 20899; XSOLIS, Nashville, Tennessee 37217.
J Pharm Sci. 2018 May;107(5):1304-1312. doi: 10.1016/j.xphs.2018.01.013. Epub 2018 Feb 1.
The spatially averaged density of protein aggregates is an important parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of protein in aggregate form in a sample. We obtained a series of images of protein aggregates exhibiting Brownian diffusion while settling under the influence of gravity in a sealed capillary. The aggregates were formed by stir-stressing a monoclonal antibody (NISTmAb). Image processing yielded particle tracks, which were then examined to determine settling velocity and hydrodynamic diameter down to 1 μm based on mean square displacement analysis. Measurements on polystyrene calibration microspheres ranging in size from 1 to 5 μm showed that the mean square displacement diameter had improved accuracy over the diameter derived from imaged particle area, suggesting a future method for correcting size distributions based on imaging. Stokes' law was used to estimate the density of each particle. It was found that the aggregates were highly porous with density decreasing from 1.080 to 1.028 g/cm as the size increased from 1.37 to 4.9 μm.
蛋白质聚集体的空间平均密度是一个重要的参数，可用于将正交方法测量的大小分布相关联，用于描述蛋白质颗粒，并可能用于估计样品中以聚集形式存在的蛋白质的量。我们获得了一系列在密封毛细管中受重力影响沉降时表现出布朗扩散的蛋白质聚集体的图像。这些聚集体是通过搅拌一种单克隆抗体（NISTmAb）形成的。图像处理得到了粒子轨迹，然后根据均方根位移分析来检查这些轨迹，以确定沉降速度和水动力直径低至 1 μm。对尺寸范围为 1 至 5 μm 的聚苯乙烯校准微球的测量表明，均方根位移直径比基于成像的粒子面积得出的直径具有更高的准确性，这表明未来可能会有一种基于成像的方法来校正尺寸分布。斯托克斯定律用于估计每个粒子的密度。结果发现，聚集体具有很高的多孔性，随着尺寸从 1.37 增加到 4.9 μm，密度从 1.080 降低到 1.028 g/cm。
