Nanoparticle tracking analysis of particle size and concentration detection in suspensions of polymer and protein samples: Influence of experimental and data evaluation parameters.

Nanoparticle Tracking Analysis (NTA) is an emerging technique for detecting simultaneously sub-micron particle size distributions and particle concentrations of a sample. This study deals with the performance evaluation for the detection and characterisation of various particles by NTA. Our investigation focusses on the NTA measurement parameter set-ups, as will be shown in this study, are very crucial parameters to correctly analyse and interpret the data. In order to achieve this, we used (i) polystyrene standard particles as well as (ii) protein particles. We show the highly precise and reproducible detection of particle size and concentration in monodisperse polystyrene particle systems, under specified and constant parameter settings. On the other hand, our results exemplify potential risks and errors while setting inadequate parameters with regards to the results and thus interpretation thereof. In particular changes of the parameters, camera level (CL) and detection threshold (DT), led to significant changes in the determined particle concentration. We propose defined and specified "optimal" camera levels for monodisperse particle suspension characterisations in the size range of 20-1000nm. We illustrate that the results of polydisperse polystyrene standard particle solution measurements, highly depend on the used parameter settings, which are rarely published with the data. Changes in these settings led to the "appearance" or "disappearance" of particle populations ("peaks") for polydisperse systems. Thus, a correct evaluation of the particle size populations in the sample becomes very challenging. For the use of NTA in biopharmaceutical analysis, proteinaceous samples were investigated. We analysed protein particle suspensions and compared unstressed and stressed (formation of aggregates) protein samples similar to polystyrene particle analysis. We also studied these samples in two different measuring modes (general capture mode and live monitoring mode) that the commercially available analysis software is offering. Our results stated the live monitoring mode as more suitable for protein samples, as the results were more reproducible and less operator-depending. In conclusion, NTA is a potential technique and unique in quantitative evaluation of particle suspensions in the subvisible size range, especially for monodisperse suspensions. We strongly urge on not underestimating the influence of the measuring parameters on the obtained results, which should be presented with the data in order to better judge and interpret the NTA results.