Microfluidic Diffusion Analysis of the Size Distribution and Microrheological Properties of Antibody Solutions at High Concentrations

Abstract

The size distribution and the rheological properties of dispersions of biological colloids are relevant quality attributes for a variety of industrial applications, including pharmaceutical, food, and cosmetic products. For instance, the biophysical properties of monoclonal antibodies and therapeutic proteins, which represent an important class of drugs in the pharmaceutical market, are important for their safety and efficacy. In this work, we apply a microfluidic diffusion platform to analyze protein sizes and interactions in high-concentration antibody solutions directly in the liquid state with minimal perturbation of the sample. We show that this method provides size distributions in a size range scaling from a few angstroms to hundreds of nanometers. The detection sensitivity of the technique is independent of the particle size, and the method provides number-average distributions, enabling the simultaneous detection of both monomeric species and soluble aggregates. We further show that the same platform can be applied to measure viscosity–scaling effects in crowded environments by probing the Brownian motion of several tracers with different sizes. Such tracers experience a shift from the microviscosity to the macroviscosity of the sample at a critical probe size that is equal to the characteristic dimension of the main components of the dispersions. The technique simultaneously provides quantitative measurement of the microrheological properties and the macroviscosity of the sample, as well as information on the characteristic size of the components of the solution. Overall, these methods represent attractive tools in the context of the analysis of sizes and interactions of proteins in both diluted and high-concentration solutions during development, manufacturing, and formulation.

Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses

“…A first urgent step in this direction is the standardization of characterization techniques for EVs, capable of simultaneously monitoring both physical and biochemical properties with high throughput. In this context, in analogy with analytics for therapeutic proteins, microfluidic technology is emerging as an attractive platform for the characterization of EVs …”

Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence‐Based Microfluidic Diffusion Sizing

“…The experimental concentration profiles were analyzed following the approach previously described. [ 27–29,46,47 ] Briefly, a library of concentration profiles $${B^{{r_i}}}$$ was created for a range of particles of known sizes r i by solving the diffusion advection equation within the channel boundaries. [ 46 ] The experimental concentration profiles P(t) integrated over the channel height were extracted from the acquired images at the 12 positions (Figure 2a,b).…”

“…The relative weights of the linear combination were determined by a basin-hopping algorithm implemented in Python with 2500 random displacements. The coefficients c i were constrained to a sum of two Gaussian distributions [28,29]…”

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event

“… In particular, techniques capable of analysing aggregates in the submicron range are emerging, based for instance on diffusion measurements, light scattering, mass measurement, microfluidics and other techniques [117][118][119][120][121][122][123][124][125][126][127] . This is important since these submicron species appear to be characterized by high immunogenic risks 128,129 , in addition to risks from micron size subvisible particles [130][131][132] .…”