Monitoring polysorbate 80 degradation in protein solutions using Total Holographic Characterization.

The degradation of polysorbate surfactants can limit the shelf life of biologic pharmaceutical products. Polysorbate is susceptible to degradation via either oxidation or hydrolysis pathways which releases free fatty acids (FFA) and other complex polymers. Degradants from Polysorbate 80 (PS80) can form particles and impact drug product quality.

Label-free viability assay using in-line holographic video microscopy.

Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a single-particle characterization technology that determines the size and index of refraction of individual particles using the Lorenz-Mie theory of light scattering. Although assessment of cell viability is a challenge in many applications, including biologics manufacturing, traditional approaches often include unreliable labeling with dyes and/or time consuming methods of manually counting cells.

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 ρ 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).

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.

Holographic characterization and tracking of colloidal dimers in the effective-sphere approximation.

An in-line hologram of a colloidal sphere can be analyzed with the Lorenz-Mie theory of light scattering to measure the sphere's three-dimensional position with nanometer-scale precision while also measuring its diameter and refractive index with part-per-thousand precision. Applying the same technique to aspherical or inhomogeneous particles yields measurements of the position, diameter and refractive index of an effective sphere that represents an average over the particle's geometry and composition. This effective-sphere interpretation has been applied successfully to porous, dimpled and coated spheres, as well as to fractal clusters of nanoparticles, all of whose inhomogeneities appear on length scales smaller than the wavelength of light.

Quantitative Differentiation of Protein Aggregates From Other Subvisible Particles in Viscous Mixtures Through Holographic Characterization.

We demonstrate the use of holographic video microscopy to detect individual subvisible particles dispersed in biopharmaceutical formulations and to differentiate them based on material characteristics measured from their holograms. The result of holographic analysis is a precise and accurate measurement of the concentrations and size distributions of multiple classes of subvisible contaminants dispersed in the same product simultaneously. We demonstrate this analytical technique through measurements on model systems consisting of human IgG aggregates in the presence of common contaminants such as silicone oil emulsion droplets and fatty acids.

The role of the medium in the effective-sphere interpretation of holographic particle characterization data.

The in-line hologram of a micrometer-scale colloidal sphere can be analyzed with the Lorenz-Mie theory of light scattering to obtain precise measurements of the sphere's diameter and refractive index. The same technique also can be used to characterize porous and irregularly shaped colloidal particles provided that the extracted parameters are interpreted with effective-medium theory to represent the properties of an equivalent effective sphere. Here, we demonstrate that the effective-sphere model consistently accounts for changes in the refractive index of the medium as it fills the pores of porous particles and therefore yields quantitative information about such particles' structure and composition.

Holographic Characterization of Protein Aggregates in the Presence of Silicone Oil and Surfactants.

Characterizing protein aggregates in the presence of silicone oil is a long standing challenge for the pharmaceutical industry. Silicone oil is often used as a lubricant in devices that deliver and store therapeutic protein products and has been linked to protein aggregation, which can compromise a drug's effectiveness or cause autoimmune responses in patients. Most traditional technologies cannot quantitatively distinguish protein aggregates and silicone oil in their native formulations for sizes less than 5 μm.

Lifting degeneracy in holographic characterization of colloidal particles using multi-color imaging.

Micrometer sized particles can be accurately characterized using holographic video microscopy and Lorenz-Mie fitting. In this work, we explore some of the limitations in holographic microscopy and introduce methods for increasing the accuracy of this technique with the use of multiple wavelengths of laser illumination. Large high index particle holograms have near degenerate solutions that can confuse standard fitting algorithms.

Holographic characterization of contaminants in water: Differentiation of suspended particles in heterogeneous dispersions.

Determining the size distribution and composition of particles suspended in water can be challenging in heterogeneous multicomponent samples. Light scattering techniques can measure the distribution of particle sizes, but provide no basis for distinguishing different types of particles. Direct imaging techniques can categorize particles by shape, but offer few insights into their composition.

Holographic characterization of colloidal fractal aggregates.

In-line holographic microscopy images of micrometer-scale fractal aggregates can be interpreted with an effective-sphere model to obtain each aggregate's size and the population-averaged fractal dimension. We demonstrate this technique experimentally using model fractal clusters of polystyrene nanoparticles and fractal protein aggregates composed of bovine serum albumin and bovine pancreas insulin.

Rapid, high-throughput tracking of bacterial motility in 3D via phase-contrast holographic video microscopy.

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria.

Holographic deconvolution microscopy for high-resolution particle tracking.

Rayleigh-Sommerfeld back-propagation can be used to reconstruct the three-dimensional light field responsible for the recorded intensity in an in-line hologram. Deconvolving the volumetric reconstruction with an optimal kernel derived from the Rayleigh-Sommerfeld propagator itself emphasizes the objects responsible for the scattering pattern while suppressing both the propagating light and also such artifacts as the twin image. Bright features in the deconvolved volume may be identified with such objects as colloidal spheres and nanorods.

Holographic particle-streak velocimetry.

We present a way to measure the positions and instantaneous velocities of micrometer-scale colloidal spheres using a single holographic snapshot obtained through in-line holographic video microscopy. This method builds on previous quantitative analyses of colloidal holograms by accounting for blurring that occurs as a sphere moves during the camera's exposure time. The angular variance of a blurred hologram's radial intensity profile yields both the magnitude and direction of a sphere's in-plane velocity.

Strategies for three-dimensional particle tracking with holographic video microscopy.

The video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution. In this work, we compare the performance of two complementary analysis techniques, one based on fitting to the exact Lorenz-Mie theory and the other based on phenomenological interpretation of the scattered light field reconstructed with Rayleigh-Sommerfeld back-propagation. Although Lorenz-Mie tracking provides more information and is inherently more precise, Rayleigh-Sommerfeld reconstruction is faster and more general.

Rotational and translational diffusion of copper oxide nanorods measured with holographic video microscopy.

We use holographic video microscopy to track the three-imensional translational and rotational diffusion of copper oxide nanorods suspended in water. Rayleigh-Sommerfeld back-propagation of a single holographic snapshot yields a volumetric reconstruction of the nanorod's optical scattering pattern, from which we estimate both its dimensions and also its instantaneous position and orientation. Analyzing a video sequence yields measurements of the freely diffusing nanorod's dynamics, from which we estimate the technique's resolution.

Flow visualization and flow cytometry with holographic video microscopy.

The video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution, and simultaneously to measure their sizes and refractive indexes. Through a combination of hardware acceleration and software optimization, this analysis can be carried out in near real time with off-the-shelf instrumentation. An efficient particle identification algorithm automates initial position estimation with sufficient accuracy to enable unattended holographic tracking and characterization.

Switchable self-protected attractions in DNA-functionalized colloids.

Surface functionalization with DNA is a powerful tool for guiding the self-assembly of nanometre- and micrometre-sized particles. Complementary 'sticky ends' form specific inter-particle links and reproducibly bind at low temperature and unbind at high temperature. Surprisingly, the ability of single-stranded DNA to form folded secondary structures has not been explored for controlling (nano) colloidal assembly processes, despite its frequent use in DNA nanotechnology.

Multicolored carbon nanotubes: decorating patterned carbon nanotube microstructures with quantum dots.

In this work, techniques to create patterned array of multiwalled nanotube (MWNT) microstructures decorated with quantum dots (QDs) were presented. Using aligned array of intertwined MWNTs as the supporting template, a droplet of solution comprising QDs was deposited onto the MWNTs. When the solution evaporated away, QDs were left behind on the MWNT template.

Size selective assembly of colloidal particles on a template by directed self-assembly technique.

We report a simple and effective approach to organize micron- and submicron-sized particles in a size selective manner. This approach utilizes the template assisted directed self-assembly technique. A topographically patterned photoresist surface is fabricated and used to create an ordered array of colloidal particles from their aqueous suspensions.