Analytical ultracentrifugation (AUC) has a rich history that dates back to the early 20th century. The technique
was pioneered by Swedish chemist Theodor Svedberg, who was awarded the Nobel Prize in Chemistry in
1926 for his innovative work on dispersed systems, particularly colloids. His development of the first analytical
ultracentrifuge facilitated the determination of molecular weights and the study of the sedimentation behavior
of macromolecules.1 Since then, AUC has experienced significant advancements in instrumentation and software,
making it an indispensable tool in molecular biology, biochemistry, pharmacology, material science, synthetic
polymer chemistry and various other fields.
The first commercially available AUC was the Model E analytical ultracentrifuge, released in 1947 by SpinCo, who
partnered with and became a new division of Beckman Instruments in 1955. The Model E was used primarily in
molecular biology and biochemistry to determine protein molecule weight.2 However, in the years that followed,
the AUC started to fall out of favor due to the complexity of maintaining the instrument, challenges with data
analysis, and the eventual need for more quantitative results.
Technological innovations in the 1970s and 1980s, such as digital data acquisition and new rotor designs, resulted
in Beckman Coulter releasing, in 1991, the XL-A Analytical Ultracentrifuge equipped with a UV-vis detector, and, in
1996, the Optima XL-I analytical ultracentrifuge equipped with both UV-Vis and Rayleigh interference detectors.
The company later renamed these instruments the ProteomeLab XL-A and XL-I analytical ultracentrifuges. These
innovations enhanced the accuracy and efficiency of AUC experiments and expanded their range of applications.
Then, in 2016, Beckman released the Optima AUC analytical ultracentrifuge, which introduced several improvements
to enhance its capabilities and ease of use. Some of these improvements include the capability of multiwavelength
experiments, which enables precise analysis of complex systems at discrete wavelengths, temperature control
from 4-40°C, remote monitoring capabilities, which enable set up, monitoring, and extraction of data from
virtually any location, improved signal to noise, and increased scan speeds.
Throughout its history, AUC has proven to be a versatile and valuable technique for understanding the fundamental
properties of macromolecules in solution. Over the years, several review articles about using AUC have been
published, highlighting its capabilities.3–7 Here, we will highlight a few examples of recent publications that leveraged
the Optima AUC to explore various aspects of protein research, including membrane proteins, antibodies, protein-nucleic
acid interactions and viral vectors.
Figure 1: AUC over the years: After the development of (a) the original AUC by Svedberg, there have been several commercially available AUCs, including (b) the
Model E, (c) the ProteomeLab XL-I, and (d) the Optima AUC.
* when ambient temperature is < 25°C
Since its inception, AUC has been a foundational technique for the characterization of proteins and protein
therapeutics.8 When developing protein therapeutics such as antibodies, it is critical to minimize the formation
of aggregates, as they can reduce drug efficacy and potentially trigger immune responses.9 It is vital, therefore,
to carefully characterize oligomers and aggregates that might be present in the solution. Size exclusion
chromatography (SEC) is commonly used for aggregate quantification in development and quality control,
but its limitations, such as sample dilution, potential dissociation of aggregates, buffer considerations, and
interactions with the column matrix, necessitate the use of orthogonal techniques such as AUC for more accurate
characterization.9–12 Sedimentation velocity AUC (SV-AUC) is particularly valuable for quantifying biotherapeutic
aggregates and oligomers, as it differentiates species based on hydrodynamic properties without the confounding
effects of stationary phases.
The Optima AUC uses two detection methods: UV-Vis absorbance and Rayleigh interferometry, which can be used
separately or simultaneously to measure radial concentration as species sediment within the instrument. When
using UV-Vis absorbance optics, targeting a concentration between 0.3 – 1 optical density (OD) at the wavelength
measured is typically recommended. The most commonly used centerpiece has a 12 mm pathlength; however,
a centerpiece with a 3 mm pathlength can be used to measure higher concentrations. Additionally, researchers
typically use wavelengths in the 215-230 nm range if low-protein concentrations need to be measured. However,
in this range, the OD change between wavelengths is steep; due to this, a shift in the measured wavelength could
lead to erratic results. This is especially problematic with the older ProteomeLab AUCs, which had a wavelength
accuracy specified at 4 nm.14,15 The Optima AUC contains improved absorbance optics, with the wavelength
accuracy specified at 0.5 nm. This provides confidence in the agreement of the set and actual wavelengths,
resulting in accurate and reliable results. In cases where much higher concentrations are required or the measured
particles do not absorb, the Rayleigh interference optics can be used. The interference detection optics measures
the differential refractive index of the solution compared to the reference solution and has allowed researchers to
measure monoclonal antibodies up to ~45 mg/mL.13
Although SV-AUC has some limitations for protein therapeutic characterization, including lower throughput, it
remains a preferred method for aggregate characterization in monoclonal antibody (mAb) formulations.9 The
Optima AUC also allows for improved throughput by enabling researchers to measure a sample in both channels
of the AUC cell when measuring in intensity mode with the UV-Vis optical system. Bou-Assaf, G., et al. have created
a comprehensive guide to overcome some of the issues that can be encountered, and help with the design,
execution and analysis of aggregates in protein therapeutic preparations with a focus on mAbs.9
Another highly studied area of research is membrane proteins, which are involved in various cell processes and
regulations. Purification of these proteins is challenging, however, with the bottleneck frequently being the stability
of the membrane proteins in detergents.16 Li, D., et al. employed SV-AUC to quantitatively assess the behavior of
their membrane protein, TmrA, under different detergent conditions, offering insights into optimizing membrane
protein purification.16 By combining UV-Vis absorbance and Rayleigh interference detection, they characterized
the protein’s different conformational and aggregation states based on its sedimentation coefficients in various
detergent concentrations, and optimized the step at which high detergent concentrations could be added (Figure 2).
Figure 2: The membrane protein
TmrA’s sedimentation coefficient
when purified or supplemented with
DDM at various concentrations. A and B show the pairwise comparison
results for the UV-vis detector for TrmA
at two different concentrations.
C and D show the pairwise comparison
results from the interference detector. For more information, please see Li et al.
2021 doi: 10.3390/membranes11100780,
https://creativecommons.org/licenses/
by/4.0/ images were not altered.
Protein nucleic acid interactions are another area of research that benefits from the characterization powers of
AUC, especially since the addition of multiwavelength (MW) capabilities in the Optima AUC. Multiwavelength AUC
has been pivotal in understanding the stoichiometry and thermodynamics of protein-nucleic acid interactions.
It allows for the decomposition of analytes in solution based on their hydrodynamics and spectral properties
to determine their molar quantities and stoichiometry, thus offering a more comprehensive characterization of
their interactions.17 In a notable study, Horne, C., R., et al., used MW AUC to aid a functional understanding of how
transcriptional regulators mediate the regulation of genes in bacteria as a response to environmental changes.18
To do so, it was essential to validate the stoichiometry involved in the protein–DNA binding to gain insight into
the thermodynamics of these interactions. By hydrodynamically separating the interacting and non-interacting
analytes due to the centrifugal force, and optically separating the spectral signals from the protein and the DNA, the
researchers determined the molar concentrations of the analytes at each sedimentation coefficient. This revealed
that three dimers of their protein, NanR, sequentially bind to their DNA repeat operator with low nanomolar
affinity (Figure 3). This experiment and several others,17,19–21 underscore the added experimental knowledge that
can be obtained through multiwavelength AUC, highlighting its invaluable role in advancing our understanding of
complex biological interactions.
Figure 3: Optically deconvoluted multiwavelength AUC interaction study of NanR and DNA. (a) The sedimentation coefficient distributions of the controls NanR (blue) and the DNA (black) were measured at 280 and 260 nm,
respectively. (b-e) Show the sedimentation coefficient distributions from the titration of increasing concentrations of NanR into the DNA,
resulting in a shift in sedimentation coefficient consistent with complex formation. The shaded areas highlight heterocomplexes, and each
heterocomplex‘s molar ratio is shown. For more information, please see Horne et al. 2021. Doi: 10.1038/s41467-021-22253-6,
https://creativecommons.org/licenses/by/4.0/ images were not altered.
Finally, AUC has seen significant adoption in the characterization of viral vectors used for drug delivery,
particularly adeno-associated viruses (AAVs).22 The safety and efficacy of AAV treatments can be compromised
by quality issues such as empty or partially filled capsids or other contaminants such as endotoxins, residual
host cell DNA, and defective particles, which can affect large-scale production and patient safety.23 Due to
AUC’s ability to fractionate particles in solution based on their size, density, and shape during the detection,
it can characterize several attributes of AAVs, including their sedimentation coefficients, the percentage of
loaded, empty and partially filled capsids, and the presence of other contaminants (Figure 4).24,25
Figure 4: Experimental data highlighting AUC capabilities to differentiate AAV loading states. (a) Graphical explanation of the combination of different capsid (ncapsid) and full-length ssDNA (nssDNA) on the left and the theoretically
correlated sedimentation coefficient on the right. Correlation plot between the number of nucleotides per capsid and s-value for each
association state of particles (right). (b) Highlights the author‘s ability to calculate the number of nucleotides in an AAV capsid using multiple
regression analysis with each component‘s molar extinction coefficient spectra. For more information, please refer to the publication.
(c) Sedimentation velocity results for an AAV1 sample, highlighting empty, partially filled, full, and aggregated capsids. The aggregated
capsid illustration is a representation of possible components. For more information, please see Maruno et al. 2021.doi: 10.1016/j.
xphs.2021.06.031, https://creativecommons.org/licenses/by/4.0/ images were not altered.
Discussion
Analytical ultracentrifugation remains a versatile and valuable technique for understanding the fundamental
properties of macromolecules in solution. The continuous advancements in AUC technology demonstrate its
indispensable role in various fields of research. The most recent AUC instrument, the Optima AUC, introduced
improvements such as enhanced wavelength accuracy, allowing more confidence in your measurements. This is
especially useful when measuring low-concentration proteins in the 215-230 nm range, where the OD change is
steep between wavelengths. Faster scanning capabilities enable the collection of dense raw datasets, which can
help improve RSMDs and be useful when collecting information on fast sedimenting analytes. Multiwavelength
capabilities, which allow for protein and DNA overlapping signals to be deconvoluted into separate profiles
based on their spectral differences, provide direct access to the molar stoichiometry of interacting complexes
and improve the characterization of viral vector loading states. Increased throughput can also be achieved
when measuring with the UV-Vis detector system, as both channels can be used for samples when measuring
in intensity.* Here, we highlighted select publications that demonstrate the strengths of the Optima AUC. From
optimizing protein purification to characterizing complex interactions and ensuring the quality of gene therapy
and drug delivery vectors, AUC continues to provide critical insights that drive scientific progress. Through the
review of recent studies, this paper highlights the enduring strengths and evolving capabilities of AUC in modern
scientific research.
* The higher concentrated sample should still be loaded into the sample channel (Channel A).
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This application note on macromolecule characterization with the AUC is for demonstration only, and is not
validated by Beckman Coulter. Beckman Coulter makes no warranties express or implied with respect to this
protocol, including warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing.
Your use of the method is solely at your own risk, without recourse to Beckman Coulter.
For Research Use Only. Not for use in diagnostic procedures.