An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Theoretical and Experimental Gas Volume Quantification of Micro- and Nanobubble Ultrasound Contrast Agents.

The amount of gas in ultrasound contrast agents is related to their acoustic activity. Because of this relationship, gas volume has been used as a key variable in normalizing the in vitro and in vivo acoustic behavior of lipid shell-stabilized bubbles with different sizes and shell components. Despite its importance, bubble gas volume has typically only been theoretically calculated based on bubble size and concentration that is typically measured using the Coulter counter for microbubbles and nanoparticle tracking analysis (NTA) for nanoscale bubbles. However, while these methods have been validated for the analysis of liquid or solid particles, their application in bubble analysis has not been rigorously studied. We have previously shown that resonant mass measurement (RMM) may be a better-suited technique for sub-micron bubble analysis, as it can measure both buoyant and non-buoyant particle size and concentration. Here, we provide validation of RMM bubble analysis by using headspace gas chromatography/mass spectrometry (GC/MS) to experimentally measure the gas volume of the bubble samples. This measurement was then used as ground truth to test the accuracy of theoretical gas volume predictions based on RMM, NTA (for nanobubbles), and Coulter counter (for microbubbles) measurements. The results show that the headspace GC/MS gas volume measurements agreed well with the theoretical predictions for the RMM of nanobubbles but not NTA. For nanobubbles , the theoretical gas volume using RMM was 10% lower than the experimental GC/MS measurements; meanwhile, using NTA resulted in an 82% lower predicted gas volume. For microbubbles, the experimental gas volume from the GC/MS measurements was 27% lower compared to RMM and 72% less compared to the Coulter counter results. This study demonstrates that the gas volume of nanobubbles and microbubbles can be reliably measured using headspace GC/MS to validate bubble size measurement techniques. We also conclude that the accuracy of theoretical predictions is highly dependent on proper size and concentration measurements.

Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization.

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.

Sink or float? Characterization of shell-stabilized bulk nanobubbles using a resonant mass measurement technique.

Nano-sized shell-stabilized gas bubbles have applications in various fields ranging from environmental science to biomedical engineering. A resonant mass measurement (RMM) technique is demonstrated here as a new and only method capable of simultaneously measuring the size and concentration of buoyant and non-buoyant particles in a nanobubble sample used as a next-generation ultrasound contrast agent.