Analytical Ultracentrifugation Overview

Analytical ultracentrifugation (AUC) for macromolecule characterization in solution

Analytical ultracentrifugation (AUC) is the most versatile, rigorous and accurate technology available for determining the molecular weight, hydrodynamic and thermodynamic properties of a protein or other macromolecule. 

Although its history dates back nearly a century, AUC is:

  • A stand-alone, first-principle technique performed in a matrix-free environment that does not require standards.
  • Unsurpassed by other techniques at providing the same range of information with a comparable level of precision and accuracy. 
  • The gold-standard method for in-solution protein molecular weight determination2

AUC 101: providing insights based on velocity and equilibrium

In contrast to other methods used to analyze macromolecules, analytical ultracentrifugation (AUC) enables characterization of samples in their native state under biologically relevant solution conditions. AUC can be used to investigate nearly any type of molecule or particle over a wide range of concentrations and in a diverse variety of solvents. For many research questions, there is no satisfactory analytical substitute for AUC.1

AUC gives researchers two complementary views of solution behavior. Sedimentation velocity provides first-principle, hydrodynamic information about the size and shape of molecules. Sedimentation equilibrium provides the same type of information about the solution molar masses, stoichiometries, association constants and solution nonideality.

More questions? More answers.

In one experiment, AUC technology can provide more answers to critical questions than any other comparable technique:

  • Shape:How spherical is my protein?
  • Diameter: What’s the size of my particle?
  • Mass: What’s the molecular weight of my protein or complex in solution?
  • Stoichiometry: How many subunits comprise my protein?
  • Purity: Are there other particles in my sample?
  • Formulation: How does my protein behave in this buffer?
  • Heterogeneity: Is my protein bound to other molecules, and what’s the configuration of the complex?
  • Aggregation: Is my protein still in a usable form? Should I expect an immune response with my drug formulation?
  • Association: Does my protein associate and/or dissociate with other proteins?
  • Conformation:  Does the conformation of my protein change upon binding to a ligand?

Learn even more about AUC in our Introduction to Analytical Ultracentrifugation handbook.

References

1 Cole JL, Lary JW, Moody T, et al. Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium. Methods Cell Biol 2008;84:143–79.

2 Berkowitz S, Philo J. Characterizing Biopharmaceuticals using Analytical Ultracentrifugation. In: Houde D, Berkowitz S, editors. Biophysical Characterization of Proteins in Developing Biopharmaceuticals. Amsterdam: Elsevier; 2015. p. 211-260.

 

 

Content and Resources

Advances in Sedimentation Analysis by Dr. Borries Demeler This presentation covers advances in sedimentation velocity (SV) analysis including methods for accurately characterizing heterogeneous systems at high resolution; approaches to extract rich information from SV experiments and techniques to measure the strength of macromolecular interactions with discrete binding models to whole boundary SV experiments in analytical ultracentrifugation.
The Use of the Analytical Ultracentrifuge to Characterize Very Large Complex Biopharmaceuticals This webinar covers the key attributes of AUC as a highly effective technique for characterizing viral vectors for viral gene therapy delivery systems
Analytical Ultracentrifugation in the Biopharmaceutical Industry Dr. Alexander Bepperling focuses on the data analysis tools to achieve high precision when analyzing biopharmaceutical products due to an interest in minor species of about 1-2% usually comprising of the total protein content. He focuses on the data analysis done in SEDFIT. Learn how to set up continuous c(S) distribution analysis as implemented with SEDFIT software for enhanced reproducibility during a study of many batches of one protein. Also covers applications of sedimentation equilibrium (SE) for screening weak interactions and determining detergent binding to proteins.
Addressing the Challenges of Purification & Quality Control in Gene Therapy Our panelists will focus on several pertinent topics in rAAV process development including current challenges, technologies for overcoming quality control hurdles, best practices for downstream processing, as well as discuss the current trends and future outlook of this rapidly growing therapeutic frontier.
Looking Under the Hood: Getting Started with the Optima AUC In the webinar we discuss the fundamentals of analytical ultracentrifugation and look at a few sample applications.

What is an analytical ultracentrifuge?

Optima AUCAn analytical ultracentrifuge is similar to a high-speed preparative centrifuge, except that the particles in the sample being spun are monitored in real time by an integrated optical detection system. These optical systems (UV-Vis absorbance and Rayleigh interference) enable precise observation of the solution behavior of molecules as they undergo sedimentation. Using state-of-the-art software, data can be used for a comprehensive analysis of molecules in the sample being studied.

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