Characterization of submicron (0.1-1 μm) particles in therapeutic proteins by nanoparticle tracking analysis.

The importance of 0.1-1 μm submicron particles characterization in therapeutic proteins, which was limited because of a lack of suitable methods, has been recognized recently. An application of nanoparticle tracking analysis (NTA) for characterization of 18 lots of recombinant fusion protein (rP1) drug product presentation along with stressed samples of this material exposed to heat at 50°C, agitation, and UV light was studied. In addition, monodisperse polystyrene standards with nominal sizes of 60-800 nm and rP1 samples spiked with 100-400 nm polystyrene standards were analyzed. The NTA technique was capable of demonstrating good sizing of monodisperse polystyrene standards, detect small particle size population in 800 nm standard, and resolve three size populations in the mixture of four standards (60-400 nm). The NTA was also capable of resolving 400 nm polystyrene standard from the main rP1 peak, but was not able to resolve 100 and 200 um standards because of the particle distribution profiles overlap. A characterization of 0.1-1 μm submicron particles in rP1 showed a relatively diverse range of mean particle diameters, D90, and size distributions, which was not linked to the lots storage duration prior to analysis. The size distribution profile of rP1was specific for a single lot and did not show significant variability, which allowed detection of larger particle population in stressed samples compared with a control. Overall, the NTA technique is suitable for characterization of submicron particles in a studied therapeutic protein. However, the NTA can only be used as a semiquantitative methodology, because frequent sample dilution is required to achieve optimal particle concentration.