Characterization of Nanoparticle Tracking Analysis for Quantification and Sizing of Submicron Particles of Therapeutic Proteins

A Comprehensive Evaluation of Nanoparticle Tracking Analysis (NanoSight) for Characterization of Proteinaceous Submicron Particles

“…The nature of protein aggregates is relevant for their stability upon sample dilution. That we observe a linear relationship using a protein-free diluent could reflect differences in the nature of the aggregates compared to the study by Zhou et al 25 or the minimal time delay between sample dilution and sample analysis used in the present study. Larger dilution factors were not assessed in this study because the particle concentration would be too low for standard NTA analysis, and the measurement time would have to be extended to get adequate statistics.…”

“…At more than 10 9 particles/mL, the close proximity of particles can make it impossible for the software to perform the so-called point tracking. Zhou et al 25 have previously observed that a linear relationship between particle count and dilution factor depended on the protein concentration of the diluent. We assessed 2 dilution factors (2-and 5-fold) using protein-free diluent for the stressed mAb (filtered through 5-mm PVDF syringe filters).…”

“…The size distribution presented a bell-shape profile as normally seen in other studies. 13,20,25 As discussed previously, NTA analysis of a polydisperse sample could compromise the sensitivity for smaller particles, and the number of protein aggregates with sizes <200 nm might be underestimated or completely undetected. After filtration of the samples through 1.2-and 0.22-mm syringe filters, hardly any submicron particles were detected (Fig.…”

“…The NTA technique has been evaluated for characterization of proteinaceous particles in several studies. 13,20,25 The main drawbacks of NTA are the relatively narrow concentration range (approximately 10 7 -10 9 particles/mL) and the scattering noise in solutions at high protein concentrations; thus, sample dilution is required in some cases. However, the particle count and dilution factor may not be in a linear relationship which depends on the diluent used 25 and the reversibility of aggregate formation.…”

“…26 For the purpose of routine use in biopharmaceutical development, the efforts should be made to assess the potential impact of parameter settings during data collection and processing. Recently, Zhou et al 25 investigated the effects of minimum expected particle size, complete tracking numbers, and sample flow rate, which revealed that these parameters should be properly optimized to obtain accurate and reproducible results. Gross et al 27 further evaluated the importance of the detector threshold, camera level, and capture time settings for NTA analysis of monodisperse and polydisperse polystyrene beads and the chosen capture mode for protein samples.…”