Global Analysis of Aggregation Profiles of Three Kinds of Immuno-Oncology mAb Drug Products Using Flow Cytometry.

Accurately quantifying the protein particles in both subvisible (1-100 µm) and submicron (≤1 µm) ranges remains a prominent challenge in the development and manufacturing of protein drugs. Due to the limitation of the sensitivity, resolution, or quantification level of various measurement systems, some instruments may not provide count information, while others can only count particles in a limited size range. Moreover, the reported concentrations of protein particles commonly have significant discrepancies owing to different methodological dynamic ranges and the detection efficiency of these analytical tools. Therefore, it is extremely difficult to accurately and comparably quantify protein particles within the desired size range at one time. To develop an efficient protein aggregation measurement method that can span the entire range of interest, we established, in this study, a single particle-sizing/counting method based on our highly sensitive lab-built flow cytometry (FCM) system. The performance of this method was assessed, and its capability of identifying and counting microspheres between 0.2 and 25 µm was demonstrated. It was also used to characterize and quantify both subvisible and submicron particles in three kinds of top-selling immuno-oncology antibody drugs and their lab-produced counterparts. These assessment and measurement results suggest that there may be a role for an enhanced FCM system as an efficient investigative tool for characterizing and learning the molecular aggregation behavior, stability, or safety risk of protein products.

Rapid evaporation at the superheat limit of methanol, ethanol, butanol and n-heptane on platinum films supported by low-stress SiN membranes.

The bubble nucleation temperatures of several organic liquids (methanol, ethanol, butanol, n-heptane) on stress-minimized platinum (Pt) films supported by SiN membranes is examined by pulse-heating the membranes for times ranging from 1 µs to 10 µs. The results show that the nucleation temperatures increase as the heating rates of the Pt films increase. Measured nucleation temperatures approach predicted superheat limits for the smallest pulse times which correspond to heating rates over 108 K/s, while nucleation temperatures are significantly lower for the longest pulse times. The microheater membranes were found to be robust for millions of pulse cycles, which suggests their potential in applications for moving fluids on the microscale and for more fundamental studies of phase transitions of metastable liquids.

Distribution of Average Aggregate Density from Stir-Stressed NISTmAb Protein.

Quantifying the heterogeneous nature of protein aggregates is important to understanding the impact aggregates may have on the performance of antibody therapeutics. The spatially averaged density ρp of aggregates, defined as the total mass, including water, divided by the volume, is a parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of aggregated protein in a sample. We report measurements by two methods on the distribution of density values for different aggregate sizes, where the aggregates were produced by stir-stressing fluorescently labeled monoclonal antibody (NISTmAb). A fluorescence microscope was used to image particles. Each particle was analyzed for brightfield equivalent circular diameter (ECD) and fluorescence intensity and the results converted to average density. Measurements were also obtained using video holography. The aggregates were highly porous with median density decreasing from 1.07 g/cm3 to 1.02 g/cm3 as the size increased from 0.9 µm to 6 µm by fluorescence, and similar results by video holography. The distribution in density for a given particle size was asymmetrical and broad. For example, particles with an ECD of 2.5 µm ranged in density from 1.005 g/cm3 to 1.1 g/cm3.

Improving Diameter Accuracy for Dynamic Imaging Microscopy for Different Particle Types.

Dynamic imaging analysis instruments are used for sizing particles of different types that might appear in a biopharmaceutical. These instruments are calibrated using polystyrene latex microspheres in water, which is a significantly different system than the typical particles imaged in a formulation. We show how the instruments, when reporting an equivalent diameter, set a threshold for image processing and then apply a built-in correction to account for fuzzy boundary effects. We investigate the degree to which the threshold value and built-in correction influences the size, and ultimately particle size distribution, that the instrument reports on other particle types. Size corrections for a dynamic imaging system in a typical optical configuration were determined by comparison of equivalent image diameters with diameters from Brownian motion tracking of particles. A variety of particles were characterized: aggregates made from a monoclonal antibody available as reference material RM 8671 from the National Institute of Standards and Technology, bovine serum albumin aggregates, silicone oil droplets, polystyrene microspheres, and ethylene tetrafluoroethylene particles, a protein aggregate simulant (National Institute of Standards and Technology reference material RM 8634). The results show that the protein aggregates and ethylene tetrafluoroethylene are very similar to one another but quite different from the polystyrene calibration spheres. This points the way to developing new correction factors and calibration procedures based on particle type.

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells.

BACKGROUND: Nanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells. METHODS: The complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining. RESULTS: Using a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously. CONCLUSION: We have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Phase-Appropriate Application of Analytical Methods to Monitor Subvisible Particles Across the Biotherapeutic Drug Product Life Cycle.

The phase-appropriate application of analytical methods to characterize, monitor, and control particles is an important aspect of the development of safe and efficacious biotherapeutics. The AAPS Product Attribute and Biological Consequences (PABC) focus group (which has since transformed into an AAPS community) conducted a survey where participating labs rated their method of choice to analyze protein aggregation/particle formation during the different stages of the product life cycle. The survey confirmed that pharmacopeial methods and SEC are the primary methods currently applied in earlier phases of the development to ensure that a product entering clinical trials is safe and efficacious. In later phases, additional techniques are added including those for non-GMP extended characterization for product and process characterization. Finally, only robust, globally-accepted, and stability-indicating methods are used for GMP quality control purposes. This was also consistent with the feedback during a webinar hosted by the group to discuss the survey results. In this white paper, the team shares the results of the survey and provides guidance on selecting phase-appropriate analytical methods and developing a robust particle control strategy.

Measurement of Average Aggregate Density by Sedimentation and Brownian Motion Analysis.

The spatially averaged density of protein aggregates is an important parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of protein in aggregate form in a sample. We obtained a series of images of protein aggregates exhibiting Brownian diffusion while settling under the influence of gravity in a sealed capillary. The aggregates were formed by stir-stressing a monoclonal antibody (NISTmAb). Image processing yielded particle tracks, which were then examined to determine settling velocity and hydrodynamic diameter down to 1 µm based on mean square displacement analysis. Measurements on polystyrene calibration microspheres ranging in size from 1 to 5 µm showed that the mean square displacement diameter had improved accuracy over the diameter derived from imaged particle area, suggesting a future method for correcting size distributions based on imaging. Stokes' law was used to estimate the density of each particle. It was found that the aggregates were highly porous with density decreasing from 1.080 to 1.028 g/cm3 as the size increased from 1.37 to 4.9 µm.

Variable Threshold Method for Determining the Boundaries of Imaged Subvisible Particles.

An accurate assessment of particle characteristics and concentrations in pharmaceutical products by flow imaging requires accurate particle sizing and morphological analysis. Analysis of images begins with the definition of particle boundaries. Commonly a single threshold defines the level for a pixel in the image to be included in the detection of particles, but depending on the threshold level, this results in either missing translucent particles or oversizing of less transparent particles due to the halos and gradients in intensity near the particle boundaries. We have developed an imaging analysis algorithm that sets the threshold for a particle based on the maximum gray value of the particle. We show that this results in tighter boundaries for particles with high contrast, while conserving the number of highly translucent particles detected. The method is implemented as a plugin for FIJI, an open-source image analysis software. The method is tested for calibration beads in water and glycerol/water solutions, a suspension of microfabricated rods, and stir-stressed aggregates made from IgG. The result is that appropriate thresholds are automatically set for solutions with a range of particle properties, and that improved boundaries will allow for more accurate sizing results and potentially improved particle classification studies.

Particle shape effects on subvisible particle sizing measurements.

Particle analysis tools for the subvisible (<100 µm) size range, such as light obscuration, flow imaging (FI), and electrical sensing zone (ESZ), often produce results that do not agree with one another, despite their general agreement when characterizing polystyrene latex spheres of different sizes. To include the effect of shape in comparison studies, we have used the methods of photolithography to create rods and disks. Although the rods are highly monodisperse, the instruments produce broadened peaks and report mean size parameters that are different for different instruments. We have fabricated a microfluidic device that simultaneously performs ESZ and FI measurements on each particle to elucidate the causes of discrepancies and broadening. Alignment of the rods with flow causes an oversizing by FI and undersizing by ESZ. FI also oversizes rods because of the incorrect edge definition that results from diffraction and imperfect focus. We present an improved correction algorithm for this effect that reduces discrepancies for rod-shaped particles. Tumbling of particles is observed in the microfluidic ESZ/FI and results in particle oversizing and breadth of size distribution for the monodisperse rods.

Nanoparticles for cancer treatment: role of heat transfer.

An overview is presented of an approach for treating cancer that uses nanoparticles to deliver heat to diseased areas after absorbing energy from a laser of the appropriate wavelength. The implications are discussed of the relationship of parameters necessary to raise the temperature to therapeutically beneficial levels. Tight focusing is required for a continuous-wave laser to sufficiently heat individual nanoparticles because of heat loss to the surrounding fluid during the period of exposure. The natural thermal confinement of pulse lasers minimizes this effect because of the finite thermal diffusion time, which restricts the absorbed energy to a region around the particle, that offers the potential for achieving high temperatures that can promote phase change on the surface of a nanoparticle or even melting of the particle. A discussion of a way to potentially measure temperature on the scale of an individual nanoparticle is included based on using a single-walled nanotube (SWNT) of carbon as a thermistor. The challenges of this undertaking are that SWNTs do not always follow Ohm's law, they may exhibit metallic or semiconductor behavior with an often unpredictable result in manufacturing, and no two SWNTs behave identically, which necessitates calibration for each SWNT. Some results are presented that show the electrical characteristics of SWNTs and their potential for exploitation in this application.

Bubble nucleation and growth anomaly for a hydrophilic microheater attributed to metastable nanobubbles.

Nanobubbles on a hydrophilic surface immersed in water and ethanol are inferred from the response of the surface to two consecutive heat pulses with a variable separation time. Bubble nucleation occurs at specific positions on the surface during the first heat pulse but at lower nucleation temperatures and random locations on the second. Nanobubbles are hypothesized to form on collapse of the bubble from the first pulse.

Nanosecond imaging of microboiling behavior on pulsed-heated au films modified with hydrophilic and hydrophobic self-assembled monolayers.

Fast transient microboiling has been characterized on modified gold microheaters using a novel laser strobe microscopy technique. Microheater surfaces of different hydrophobicity were prepared using self-assembled monolayers of hexadecane thiol (hydrophobic) and 16-mercaptohexadecanol (hydrophilic) as well as the naturally hydrophilic bare gold surface. The microheater was immersed in a pool of water, and a 5-micros voltage pulse to the heater was applied, causing superheating of the water and nucleation of a vapor bubble on the heater surface. Light from a pulsed Nd:Yag laser was configured to illuminate and image the sample through a microscope assembly. The timing of the short duration (7.5 ns) laser flash was varied with respect to the voltage pulse applied to the heater to create a series of images illuminated by the flash of the laser. These images were correlated with the transient resistance change of the heater both during and after the voltage pulse. It was found that hydrophobic surfaces produced a bubble that nucleated at an earlier time, grew more slowly to a smaller maximum size, and collapsed more rapidly than bubbles formed on hydrophilic surfaces.