Shinichiro Totoki

Nanoparticle size analysis with relaxation of induced grating by dielectrophoresis

We propose an alternative approach to the use of dynamic light scattering (DLS) for the analysis of particle sizes ranging from 5 nm to 100 nm. This approach employs a combination of 1) diffusion, 2) density grating, and 3) dielectrophoresis (DEP), and measures the diffusion coefficient from the decay rate of the diffracted light intensity in the relaxation process of particle density modulation generated by DEP. Both the experiments and the theoretical analysis confirm the reliable determination of particle size independently of the refractive index. The new method records a decay signal directly without an autocorrelator and is expected to have a less extreme sensitivity dependence on particle size than DLS.

Quantitative laser diffraction method for the assessment of protein subvisible particles.

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.

Quantitative Laser Diffraction for Quantification of Protein Aggregates: Comparison With Resonant Mass Measurement, Nanoparticle Tracking Analysis, Flow Imaging, and Light Obscuration

In the past, analysis of micron-sized (>1.0 μm) aggregates of therapeutic proteins has been limited to light obscuration (LO), and appropriate quantitative methods of evaluating protein aggregates need to be developed. Recently, novel methods with enhanced reliability and sensitivity, such as nanoparticle tracking analysis (NTA), resonant mass measurement (RMM), and flow imaging (FI), have emerged. We have found that quantitative laser diffraction (qLD) is also effective for quantitative evaluation of protein aggregates over a wide size range. However, the different detection principles of the methods potentially lead to inconsistencies in results. This study aimed to compare particle size distributions and concentrations of protein aggregates using the orthogonal methods. Protein aggregates were generated by stirring an immunoglobulin solution. Serial dilutions of the aggregates stock were analyzed by RMM, FI, and qLD to obtain the particle size distribution and concentration using each method. In addition, size distribution of a protein aggregates solution was compared by RMM, NTA, FI, LO, and qLD. Both particle size distribution and concentration were in good agreement between RMM and qLD (0.3-2 μm) and between FI and qLD (2-20 μm). Thus, we concluded that qLD enables covering of the overlapping particle size range between RMM and FI.