ELEVATING EV Characterization with Analytical Ultracentrifugation (AUC)

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Why use analytical ultracentrifugation (AUC) to characterize extracellular vesicles (EVs)?

EVs are an exciting area of research, given their tremendous promise as diagnostic biomarkers for disease, and their potential use in novel therapeutics. Nonetheless, characterizing EVs remains a challenge due to their inherent heterogeneity.

It is crucial to assess attributes such as purity, size distribution, shape, and cargo packaging for these heterogeneous nanoparticles. AUC stands out as an excellent option for characterization, as it enables the measurement of these parameters in a single experiment.
Advantages of UC for AAV purification, Process Quality: Yield and Purity

Unique advantages of AUC analysis of EVs

AUC enables analysis of samples in their native state

  • No substrate or matrix interactions
  • No calibration standards required
It offers an extremely large dynamic range, and can characterize anything from peptides to intact viruses. This extensive range can provide an in-depth understanding of the different EV populations in solution, as well as identification of contaminants and aggregates.

For example, AUC facilitated in the determination that an umbilical EV sample contained 4 prominent species, with the smallest (5 S species) being the most abundant.
Advantages of UC for AAV purification, Process Quality: Yield and Purity

Understanding the shape of EVs

Advantages of UC for AAV purification, Process Quality: Yield and Purity
Using AUC, we can begin to understand the shape of our analytes in solution. This is done using the frictional ratio (f/f0), which is calculated based on the diffusion coefficient measured by AUC. The f/f0 describes the anisotropy (or relative shape) of analytes in solution.

A frictional ratio of 1, as seen for the majority of the particles here, indicates a sphere, while larger f/f0 indicate a more extended or linear structure.

Characterizing biomolecules that make up EVs

If the sample is measured with multiple wavelengths via AUC, insights into the molecular content of the EV sample can start to be developed. Multiwavelength AUC lets us identify and quantify proteins, nucleic acids, lipids, and other biomolecules based on their absorption profiles.
Advantages of UC for AAV purification, Process Quality: Yield and Purity

Summary

The Optima AUC analytical ultracentrifuge enables you to quantitatively identify different populations in EV preparations, and provides crucial insights into their molecular content and shape. It also provides valuable information on sample purity, and can be used for batch-to-batch comparability and stability testing. Such comprehensive characterization can greatly enhance our understanding of EVs and their potential applications in various fields such as medicine, biology and biotechnology.

All figures were generated using UltraScan III software. Third-party analysis software products, including UltraScan, have not been validated by Beckman Coulter for use with an analytical ultracentrifuge. Beckman Coulter does not endorse any third-party analysis software. Beckman Coulter warranties and/or performance guarantees that may apply to our analytical ultracentrifuge instruments do not apply to any third-party software.

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