Practical Considerations for Detection and Characterization of Sub-Micron Particles in Protein Solutions by...

Nanoparticle Tracking Analysis (NTA) is an emerging analytical technique developed for detection, sizing, and counting of sub-micron particles in liquid media. Its feasibility for use in biopharmaceutical development was evaluated with particle standards and recombinant protein solutions. Measurements of aqueous suspensions of NIST-traceable polystyrene particle standards showed accurate particle concentration detection between 2 × 10(7) and 5 × 10(9) particles/mL. Sizing was accurate for particle standards up to 200 nm. Smaller than nominal value sizes were detected by NTA for the 300-900 nm particles. Measurements of protein solutions showed that NTA performance is solution-specific. Reduced sensitivity, especially in opalescent solutions, was observed. Measurements in such solutions may require sample dilution; however, common sample manipulations, such as dilution and filtration, may result in particle formation. Dilution and filtration case studies are presented to further illustrate such behavior. To benchmark general performance, NTA was compared against asymmetric flow field flow fractionation coupled with multi-angle light scattering (aF4-MALS) and dynamic light scattering, which are other techniques for sub-micron particles. Data shows that all three methods have limitations and may not work equally well under certain conditions. Nevertheless, the ability of NTA to directly detect and count sub-micron particles is a feature not matched by aF4-MALS or dynamic light scattering.

Thorough characterization of particulate matter present in protein therapeutics is limited by the lack of analytical methods for particles in the sub-micron size range. Emerging techniques are being developed to bridge this analytical gap. In this study, Nanoparticle Tracking Analysis is evaluated as a potential tool for biologics development. Our results indicate that method performance is molecule-specific and may not work as well under all solution conditions, especially when testing opalescent solutions. Advantages and disadvantages of Nanoparticle Tracking Analysis are discussed in comparison to other analytical techniques for particles in the sub-micron size range.