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. PS80 degradation products appear at low concentrations, and their refractive indexes are similar to that of the buffer, making them very challenging to detect. Furthermore, aggregates of FFA are similar in size and refractive index to protein aggregates adding complexity to characterizing these particles in protein solutions. Total Holographic Characterization (THC) is used in this work to characterize FFA particles of oleic acid and linoleic acid, the two most common degradation products of PS80. We demonstrate that the characteristic THC profile of the FFA oleic acid emulsion droplets can be used to monitor the degradation of PS80. THC can detect oleic acid at a concentration down to less than 100 ng/mL. Using the characteristic THC signal of oleic acid as a marker, the degradation of PS80 in protein solutions can be monitored quantitatively even in the presence of other contaminants of the same size, including silicone oil emulsion droplets and protein aggregates.

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.

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. Holographic video microscopy also clearly identifies metal particles and air bubbles. Being able to differentiate and characterize the individual components of such heterogeneous dispersions provides a basis for tracking other factors that influence the stability of protein formulations including handling and degradation of surfactant and other excipients.

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. We use holographic video microscopy to study protein aggregation to demonstrate its capability to quantitatively distinguish protein aggregates and silicone oil in the presence of varying amounts of the surfactants SDS and polysorbate 80 in the size range of 0.5-10 µm without the need for dilution or special sample preparation. We show that SDS denatures proteins and stabilizes silicone oil. We also show that polysorbate 80 may limit protein aggregate formation if it is added to an IgG solution before introducing silicone oil.

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. Using a model based on diffraction from a phase disk, we explain the source of these degeneracies. We introduce multiple color holography as an effective approach to distinguish between degenerate solutions and provide improved accuracy for the holographic analysis of sub-visible colloidal particles.

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 meets this need by directly measuring the size, refractive index, and three-dimensional position of individual particles in a suspension. The ability to measure an individual colloidal particle's refractive index is a unique capability of holographic characterization. Holographic characterization is fast enough, moreover, to build up population distribution data in real time, and to track time variations in the concentrations of different dispersed populations of particles. We demonstrate these capabilities using a model system consisting of polystyrene microbeads co-dispersed with bacteria in an oil-in-water emulsion. We also demonstrate how the holographic fingerprint of different contaminants can contribute to identifying their source.

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.

Holographic Characterization of Protein Aggregates.

We demonstrate how holographic video microscopy can be used to detect, count, and characterize individual micrometer-scale protein aggregates as they flow down a microfluidic channel in their native buffer. Holographic characterization directly measures the radius and refractive index of subvisible protein aggregates and offers insights into their morphologies. The measurement proceeds fast enough to build up population averages for time-resolved studies and lends itself to tracking trends in protein aggregation arising from changing environmental factors. Information on individual particle's refractive indexes can be used to differentiate protein aggregates from such contaminants as silicone droplets. These capabilities are demonstrated through measurements on samples of bovine pancreas insulin aggregated through centrifugation and of bovine serum albumin aggregated by complexation with a polyelectrolyte. Differentiation is demonstrated with samples that have been spiked with separately characterized silicone spheres. Holographic characterization measurements are compared with results obtained with microflow imaging and dynamic light scattering.

Universal, strong and long-ranged trapping by optical conveyors.

Optical conveyors are active tractor beams that selectively transport illuminated objects either upstream or downstream along their axes. Formed by the coherent superposition of coaxial Bessel beams, an optical conveyor features an axial array of equally spaced intensity maxima that act as optical traps for small objects. We demonstrate through measurements on colloidal spheres and numerical calculations based on the generalized Lorenz-Mie theory that optical conveyors' interferometric structure endows them with trapping characteristics far superior to those of conventional optical tweezers. Optical conveyors form substantially stiffer traps and can transport a wider variety of materials over a much longer axial range.

Optical conveyors: a class of active tractor beams.

We experimentally demonstrate a class of tractor beams created by coherently superposing coaxial Bessel beams. These optical conveyors have periodic intensity variations along their axes that act as highly effective optical traps for micrometer-scale objects. Trapped objects can be moved selectively upstream or downstream along the conveyor by appropriately changing the Bessel beams' relative phase. The same methods used to project a single optical conveyor can project arrays of independent optical conveyors, allowing bidirectional transport in three dimensions.

Measuring the size of individual particles from three-dimensional imaging experiments.

Often experimentalists study particulate samples that are nominally monodisperse. In reality, many samples have a polydispersity of 4-10%. At the level of an individual particle, the consequences of this polydispersity are unknown as it is difficult to measure an individual particle size from images of a dense sample. Here we propose a method to estimate individual particle radii from three-dimensional data of the particle positions. We first validate our method with simulations. We then apply our method to experimental data of colloidal suspensions observed with confocal microscopy. We demonstrate that we can recover the full particle size distribution in situ. Finally, we use our method to study the relationship between homogeneous colloidal crystal nucleation and particle sizes. We show that nucleation occurs in regions that are more monodisperse than average.

Optical forces and torques in nonuniform beams of light.

The spin angular momentum in an elliptically polarized beam of light plays several noteworthy roles in optical traps. It contributes to the linear momentum density in a nonuniform beam, and thus to the radiation pressure exerted on illuminated objects. It can be converted into orbital angular momentum, and thus can exert torques even on optically isotropic objects. Its curl, moreover, contributes to both forces and torques without spin-to-orbit conversion. We demonstrate these effects experimentally by tracking colloidal spheres diffusing in elliptically polarized optical tweezers. Clusters of spheres circulate deterministically about the beam's axis. A single sphere, by contrast, undergoes stochastic Brownian vortex circulation that maps out the optical force field.