Analytical ultracentrifugation (SV-AUC / SE-AUC)
AUC: A first principle method with many benefits
Analytical ultracentrifugation – or more precisely the sedimentation velocity (SV-AUC) and sedimentation equilibrium (SE-AUC) method – is based on a simple principle: when (macro)molecules in solution are subject to a centrifugal force, they begin to settle at a certain velocity. With this direct correlation, scientists can determine many critical parameters of a drug substance or drug product for a variety of biopharmaceutical applications.
Coriolis is a globally operating service provider and one of the world leaders for SV-AUC and SE-AUC services. We operate the latest AUC technology equipped with a best-in-class selection of detection methods. Our dedicated AUC team is highly experienced in a number of (bio)pharmaceutical applications and has developed a set of generic methods, ready for application with many standard samples. Above that, our scientists love to push the limits of the AUC technology during challenging projects.
Our AUC services cover a large variety of applications for proteins, peptides, viruses, nanoparticles and more. We work with samples up to biosafety level S2 and can implement enhanced documentation (e.g., method qualification under GxP), tailored to your needs. Please find more details about our AUC services below.
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Applications of AUC
Analytical ultracentrifugation is a highly versatile analytical method and brings valuable benefits during the research and development of (bio)pharmaceutical drug products. Coriolis is one of the world leaders for SV-AUC and SE-AUC services and offers them for a variety of (bio)pharmaceutical applications:
Analytical ultracentrifugation is highly suited for the accurate determination of important parameters of a drug substance, including molecular weight Mw, diffusion coefficient and sedimentation coefficient. As a first principle method, AUC does not require reference standards for such determination. For the demanding data processing by UltraScan, Coriolis uses its own dedicated in-house computer cluster, so none of your data goes to third-party servers.
SV-AUC determines the content of low- and high molecular-weight species without any change in solution condition (if formulation components do not interfere with the detection). Thus, SV-AUC is the ultimate standard for determining the content of fragments, oligomers, aggregate and even nanoparticles in (bio)pharmaceutical solutions.
HP-SEC and AF4 are commonly used methods for determining aggregate content in (bio)pharmaceutical samples. While both techniques have numerous advantages, they also have a key fundamental limitation: a change in solution condition (i.e., mobile phase) is often required. This may alter the ‘true’ aggregate population in a given sample prior to detection.
SV-AUC can detect aggregates without any change in solution condition (if formulation components do not interfere with the detection). Thus, SV-AUC is often performed as an orthogonal method during the development of HP-SEC and AF4 to ensure the validity of their results.
While many analytical techniques are unable to directly measure highly concentrated samples (such as > 100 mg/ml antibody solutions), Coriolis can apply AUC for highly concentrated samples, e.g., during formulation screening, without the need for sample dilution. By applying our interference optics, highly concentrated sample can often be analyses by AUC as-is and enable an unaltered view on the sample’s properties.
The size range of AAVs (around 25 nm) is ideal for the analytical characterization by SV-AUC, particularly the quantification of empty vs. full capsids. Coriolis offers SV- and SE-AUC for AAVs, biological agents and GMOs up to biosafety level S2.
Analytical characterization of viruses, such as retroviruses, lentiviruses and adenoviruses by SV-AUC for sizes up to 200 nm. We offer stability testing of viruses by SV-AUC as a stand-alone service or as part of a virus formulation development project. Our AUC experts can also combine the power of SV-AUC with orthogonal method, such as AF4, for enhanced insights.
AUC is a first principle method and does not rely on (internal) standards. Coriolis uses this benefit and offers AUC for the reliable determining of interaction parameters (KD, Stoichiometry).
Coriolis applies the latest in AUC technology and two different sample-rotor types. This allows our scientists to apply centrifugal forces, which can sediment peptides down to 1 kDa and, thus, enable their analytical characterization.
SE-AUC is a very powerful technique delivering the most accurate information on the molecular weight of the main species in solution.
We are proud to offer fluorescence detection as an option for AUC analysis. This allows for an exclusive analysis of labeled molecules in a sample mixture, excluding interferences from other (unlabeled) species.
Coriolis employs AUC instruments with fluorescence detection. This enables in-depth analysis of protein-protein interactions with fluorescent tracers. With this, a multitude of application scenarios and assays tailored to specific research questions becomes available.
Low concentration samples are often challenging for analytical techniques such as HP-SEC and AF4, because samples are further diluted during the analytical run and the analyte can interact with column or membrane surfaces. AUC measures samples undiluted, does not have a stationary phase and utilizes high-sensitivity UV or fluorescence detection. Thus, analytical characterization of low-concentration samples (e.g., diluted virus preparations) is possible by AUC.
The benefits of AUC by Coriolis
A handful of service providers offer Analytical Ultracentrifugation. But only Coriolis offers you the quality services of a world leader. Learn why Coriolis is your best choice when it comes to AUC:
With the latest AUC instruments, various rotor options and the broadest range of detectors, our AUC facilities meet even the highest standards.
With our in-house computer cluster, we can perform even the most demanding data processing (SedFit & UltraScan) without relying on external / third-party services. Your data will be handled, processed and stored securely and confidentially with us.
Our dedicated AUC team handles many challenging projects every year and has experience with a large variety of (bio)pharmaceutical drug products. Supported by our distinguished scientific advisory board, our scientists are eager to push the limits of the AUC technique.
AUC is often considered a low-throughput technique. But with our instrumentation and expertise, we can process up to 14 samples per run. This is not much different to the throughput of many HPLC sequences.
Coriolis offers a set of generic methods, ready for application with many standard samples. Above that, our AUC scientists can develop tailor-made methods for more challenging samples and perform a method qualification, if desired.
With 5 AUC systems and a dedicated AUC team, Coriolis offers many resources for AUC projects. This means that we can accommodate most projects with quick response times.
AUC is a valuable technique for the analysis of virus formulations, GMOs and other biological agents. Thus, Coriolis offers AUC services now also for samples up to biosafety level S2.
Our AUC equipment
Coriolis is one of the world leaders for SV-AUC and SE-AUC services and we operate the latest AUC technology equipped with a best-in-class selection of detection methods. Coriolis processes and stores all data in-house by using our own AUC computer cluster. Neither your samples nor your data will be sent to third parties.
Our AUC facilities include:
5 AUC instruments of various types
- 2x Beckman Optima AUC
- 2x Beckman XL-I
- Beckman XL-A
3 Detection methods
- Rayleigh interference (RI)
2 Data processing methods on dedicated in-house computer cluster!
Laboratory facilities up to BioSafety Level S2
Principles of AUC
Analytical ultracentrifugation is a powerful technique for the analysis of (bio)pharmaceuticals. Below, we will introduce you into the two principle operation modes of AUC: the more common sedimentation velocity (SV-AUC) method and the sedimentation equilibrium (SE-AUC) method for accurate molecular weight information.
Sedimentation velocity method (SV-AUC)
When macromolecules in solution are subjected to a centrifugal force, they will begin to settle at a certain velocity. This sedimentation velocity depends on the instrument settings (mainly the speed of rotation), the solution / formulation (density, viscosity) and – of course –the macromolecule itself (mass, density and shape).
cross section of an SV-AUC center piece with two sectors is shown. One sector is usually filled with a blank or placebo, matching the macromolecule solution / formulation. The other sector is filled with the molecule sample. When a centrifugal force is applied, macromolecules move towards the bottom of the cell and are thus depleted from the solution at the top. This way, a boundary is formed between the solution containing the macromolecules and solution depleted of molecules.
During an SV-AUC run, the time-dependent concentration profile of the macromolecule along the entire length of the centerpiece is measured using one or more of the following detection methods: UV absorbance, Rayleigh interference or fluorescence detection. In Figure 2, you can see how this signal changes over time, reflecting the depletion of the macromolecule during the run.
The so obtained time-dependent concentration profile is then processed by computer algorithms that fit these data to appropriate mathematical models. Advanced algorithms – such as those performed by the UltraScan software – require a high-performance computer cluster. Coriolis analyzes all data on its own computer cluster to ensure the highest level of confidentiality over the data and control over the processing methods.
During analysis, the travel speed of the boundary provides information about the size, shape and mass of the macromolecules. The shape of the boundary layer provides quantitative information about the distribution of differently sized macromolecules in solution (e.g., amount of dimers / aggregates).
Coriolis operates specially designed rotors and modern AUC systems, which can create centrifugal forces of up to 290,000 g. This allows for the sedimentation and, thus, characterization of molecules with a mass as low as 1 kDa (e.g., peptides).
Sedimentation equilibrium method (SE-AUC)
SE-AUC uses the same physical principles and detection mechanisms as SV-AUC, but with a crucial difference: it is performed at a lower rotation speed where back-diffusion – from the bottom of the cell (high concentration) towards the top (low concentration) – becomes significant.
Over time, this back-diffusion reaches an equilibrium with the sedimentation movement and forms a stable concentration profile. For an extended period, the formation and stable position of this profile is recorded. This delivers most accurate information on the molecular weight of the main species in solution, because the molecule-shape is not influencing the results of SE-AUC (in contrast to SV-AUC).
Coriolis has used its instruments to successfully investigate molecules as large as MDa polymers and as small as 1-kDa peptides by this approach. SE-AUC is currently the gold standard method for determining molecular weights of macromolecules in their native buffer solution.