Analytical ultracentrifugation (SV-AUC / SE-AUC)

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Analytical ultracentrifugation (AUC), or more precisely, the sedimentation velocity (SV-AUC) and sedimentation equilibrium (SE-AUC) methods, allows the determination of aggregate content and molecular weight.

AUC is a first principle method and therefore ideal for the verification of results from HP-SEC, AF4 and HF5.

As a leading service provider for SV-AUC and SE-AUC, Coriolis operates 4 AUC instruments (Optima AUC, XL-I, XL-A) and offers data processing by either SEDFIT or UltraScan using a dedicated in-house high-computing cluster. Since, Coriolis operates AUC instruments under Biosafety Level 1, it can be employed for AAV purity testing, i.e. assessment of filled and empty capsids in AAV preparations.

Principle of SV-AUC

When macromolecules in solution are subjected to a centrifugal force, they will begin to settle at a certain velocity.

The sedimentation velocity depends on the instrument settings (angular velocity), the molecule (mass, density and shape) and the carrier solution (density, viscosity).

Figure 1 provides a cross section of a 2 sector SV-AUC center piece, filled with a blank matching the carrier macromolecule solution (left) and filled with the macromolecule/protein solution (right). When a centrifugal force is applied, molecules are depleted from the solution at the top, and a boundary is formed between the solution containing the macromolecules and solution depleted of macromolecules.

Figure 2 illustrates how a sample subjected to a centrifugal force evolves over time.

The cross sections of the center pieces at different time points show the depleted area expanding and the boundary layer moving towards the bottom. This time dependent concentration profile is detected by absorbance and interference during an SV-AUC run. The distance that the boundary layer travels between time points depends on the size, shape and mass of the molecules being sedimented. The shape of the boundary layer provides information about the distribution of differently sized molecules in solution (i.e. amount aggregates).

Comparison with other aggregate detection methods

The most commonly used methods for determining the aggregate content in biopharmaceutical protein solutions under non-denaturing conditions are size-exclusion chromatography (HP-SEC) and field-flow fractionation (FFF, including AF4 or HF5).

Although both aggregate detection methods have numerous advantages (easy to use, high sensitivity, good reproducibility, relatively lost cost, high throughput, etc.), both have fundamental limitations.

The key limitation with both HP-SEC and FFF is that a change in solution condition (i.e., mobile phase) is often required which may alter the ‘true’ aggregate population in a given sample prior to detection.

SV-AUC can detect aggregates without any change in solution condition as long as formulation components do not interfere with the detection by absorbance or interference. For this reason, SV-AUC is the ultimate standard for determining soluble aggregate content in biopharmaceutical solutions. Often, as part of method development for HP-SEC and FFF, SV-AUC is performed in parallel to ensure that these routine methods for aggregate detection are not altering the true aggregate population prior to detection.

Principle of SE-AUC

SE-AUC uses the same physical principles and detection mechanisms as SV-AUC but is performed at a lower rotation speed to allow significant back diffusion from the bottom of the centrifuge cell. This back diffusion leads, after equilibrium of all particle movements is reached, to a stable concentration profile, which is recorded at runtime. The profile contains most accurate information on the molecular weight of the main species in solution, as shape-related parameters that influence the particle behavior during SV-AUC are not relevant in SE-AUC. Coriolis Pharma has used its instruments to investigate molecules as large as MDa and as small as 1 kDa monomeric peptides by this approach. SE-AUC is currently the gold standard method for determining molecular weights of macromolecules in their native buffer solution.

SE-AUC is best performed to characterize homogeneous particle solutions, but also heterogeneous solutions can be analyzed. The analysis methods also allow characterizing binding equilibria, like monomer-dimer or monomer-oligomer transitions or interactions between different proteins. In combination with SV-AUC, information on the heterogeneity and more accurate information on the molecular weight can be combined to give a comprehensive description of the particles in solution.

Figure 1 provides a cross section of a 2 sector SV-AUC center piece

Coriolis Pharma AUC Services

Coriolis Pharma combines high-level expertise in biopharmaceutical formulation and stability with a broad spectrum of analytical tools, including AUC, to assess aggregation and particle formation. This unique combination allows a most comprehensive interpretation of the generated data in the context of drug development.

We feature the Beckman Optima AUC, as well as ProteomeLab XL-I and XL-A analytical centrifuges for SV-AUC and SE-AUC analysis. Instruments are equipped with An-50Ti 8-hole rotor capable of analyzing up to 7 samples per run or with An-60Ti 4-hole rotor capable of analyzing up to 3 samples per run. The UV/Vis absorbance optics allow for detection across a wide range of wavelengths.

Figure 2 illustrates how a sample subjected to a centrifugal force evolves over time

Rayleigh Interference optics provides the capability to measure the change in refractive index resulting from changes in sample concentration. This delivers increased accuracy and the ability to examine a greater concentration range with a wider selection of samples.

SV-AUC experimental set-up and data analysis are not always straight forward and depend heavily on sample type. Our team of scientists can customize methodology appropriate to your sample.

Data analysis is performed by modeling the experimentally obtained boundary data using SEDFIT or UltraScan software. The typical results obtained are a continuous c(s) distribution, which is a distribution of the relative amounts of the differently sedimenting species in a given sample.

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