Characterization and quantitation of aggregates and particles in interferon-ß products: potential links between product quality attributes and immunogenicity.

Interferon-ß (IFN-ß) products have been used for many years in the treatment of multiple sclerosis and include recombinant IFN-ß-1b (Betaseron®) and IFN-ß-1a (Avonex® and Rebif®). All three products lead to the formation of neutralizing antibodies (NAbs) and resulting loss of efficacy in patients but to different extents. Across several clinical trials, the reported rates of neutralizing-antibody formation were 22%-47% (Betaseron®), 5%-35% (Rebif®), and 2%-13% (Avonex®). In the current study, all products were purchased from the pharmacy and aggregates were characterized and/or quantified using size-exclusion chromatography (SEC), analytical ultracentrifugation, gel electrophoresis, and dot-blotting immunoassays. Particle characterization and counting were performed using microflow imaging, particle tracking analysis, and resonant mass measurement. Betaseron® and Rebif®, which are formulated with human serum albumin, had the greatest amount of aggregated protein and particles (e.g., 9%-15% high molecular weight species by SEC and >100,000 particles/mL by flow imaging). Avonex® was found to have the least amount of aggregated protein, with >95% monomer content by both SEC and analytical ultracentrifugation, and the particles detected in Avonex® were determined to be primarily silicone oil droplets. These results strongly suggest that protein aggregate and particle contents are key product quality attributes in a given product's propensity to elicit the production of NAbs in patients.

Weighing of biomolecules, single cells and single nanoparticles in fluid.

Nanomechanical resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10(-21) g) have been weighed in vacuum, and proton-level resolution seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone-that is, with a high quality factor. In solution, viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnology and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resolution is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the solution inside a hollow resonator that is surrounded by vacuum. Here we demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resolution (1 Hz bandwidth). Central to these results is our observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of our silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resolution of six orders of magnitude over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the non-optical sizing and mass density measurement of colloidal particles.

Simultaneous nitrification-denitrification and clarification in a pseudoliquified activated sludge system.

This paper describes results from a pilot study of a novel wastewater treatment technology, which incorporates nutrient removal and solids separation to a single step. The pseudoliquified activated sludge process pilot system was tested on grit removal effluent at flowrates of 29.4 to 54.7 m3/d, three different solid residence times (SRT) (15, 37, and 57 days), and over a temperature range of 12 to 28 degrees C. Despite wide fluctuations in the influent characteristics, the system performed reliably and consistently with respect to organics and total suspended solids (TSS) removals, achieving biochemical oxygen demand (BOD) and TSS reductions of > 96% and approximately 90%, respectively, with BOD5 and TSS concentrations as low as 3 mg/L. Although the system achieved average effluent ammonia concentrations of 2.7 to 3.2 mg/L, nitrification efficiency appeared to be hampered at low temperatures (< 15 degrees C). The system achieved tertiary effluent quality with denitrification efficiencies of 90 and 91% total nitrogen removal efficiency at a total hydraulic retention time of 4.8 hours and an SRT of 12 to 17 days. With ferric chloride addition, effluent phosphorous concentrations of 0.5 to 0.8 mg/L were achieved. Furthermore, because of operation at high biomass concentrations and relatively long biological SRTs, sludge yields were over 50% below typical values for activated sludge plants. The process was modeled using activated sludge model No. 2, as a two-stage system comprised an aerobic activated sludge system followed by an anoxic system. Model predictions for soluble BOD, ammonia, nitrates, and orthophosphates agreed well with experimental data.

Studies of tip wear processes in tapping mode atomic force microscopy.

Tip integrity is crucial to atomic force microscope image quality. Tip wear not only compromises image resolution but also introduces artifacts. However, the factors that govern wearing have not been systematically studied. The results presented here of tip wearing on a rough titanium surface were determined by monitoring changes in tip shape and the evolution of histograms of complex surface curvatures under different control parameters. In contrast with the common assumption that operating at a low set point (the ratio of tapping amplitude to free oscillation amplitude) wears the tip quickly, we observed that a low set point actually minimizes tip wear on a hard surface regardless of the free amplitude. The results can be interpreted qualitatively with theoretical calculations based on momentum exchange at tapping impact. Operating at a low set point allows more robust scanning than with a high set point (tapping near free amplitude), providing a method to slow down tip wear. Another advantage of a low set point is that amplitude error grows faster than with a high set point by nearly an order of magnitude, permitting an increase in scanning speed.