Totoki Shinichiro, Yamamoto Gaku, Tsumoto Kouhei, Uchiyama Susumu, Fukui Kiichi
Shimadzu Corporation, Kyoto, 604-8511, Japan.
J Pharm Sci. 2015 Feb;104(2):618-26. doi: 10.1002/jps.24288. Epub 2014 Dec 1.
Laser diffraction (LD) has been recognized as a method for estimating particle size distribution. Here, a recently developed quantitative LD (qLD) system, which is an LD method with extensive deconvolution analysis, was employed for the quantitative assessment of protein particles sizes, especially aimed at the quantification of 0.2-10 μm diameter subvisible particles (SVPs). The qLD accurately estimated concentration distributions for silica beads with diameters ranging from 0.2 to 10 μm that have refractive indices similar to that of protein particles. The linearity of concentration for micrometer-diameter silica beads was confirmed in the presence of a fixed concentration of submicrometer diameter beads. Similarly, submicrometer-diameter silica beads could be quantified in the presence of micrometer-diameter beads. Subsequently, stir- and heat-stressed intravenous immunoglobulins were evaluated by using the qLD, in which the refractive index of protein particles that was determined experimentally was used in the deconvolution analysis. The results showed that the concentration distributions of protein particles in SVP size range differ for the two stresses. The number concentration of the protein particles estimated using the qLD agreed well with that obtained using flow microscopy. This work demonstrates that qLD can be used for quantitative estimation of protein aggregates in SVP size range.
激光衍射（LD）已被公认为一种估算颗粒尺寸分布的方法。在此，采用了一种最近开发的定量激光衍射（qLD）系统，它是一种具有广泛去卷积分析的激光衍射方法，用于对蛋白质颗粒大小进行定量评估，尤其旨在对直径为0.2 - 10μm的亚可见颗粒（SVP）进行定量。qLD准确估算了直径范围为0.2至10μm且折射率与蛋白质颗粒相似的二氧化硅珠的浓度分布。在存在固定浓度的亚微米级直径珠子的情况下，确认了微米级直径二氧化硅珠浓度的线性关系。同样，在存在微米级直径珠子的情况下，可以对亚微米级直径的二氧化硅珠进行定量。随后，使用qLD对搅拌和热应激的静脉注射免疫球蛋白进行了评估，其中在去卷积分析中使用了通过实验测定的蛋白质颗粒的折射率。结果表明，两种应激下SVP尺寸范围内蛋白质颗粒的浓度分布有所不同。使用qLD估算的蛋白质颗粒数量浓度与使用流式显微镜获得的结果吻合良好。这项工作表明，qLD可用于定量估算SVP尺寸范围内的蛋白质聚集体。
