Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC)
As a specialist in biopharmaceutical aggregation and particle characterization Coriolis Pharma offers sedimentation velocity analytical ultracentrifugation (SV-AUC) contract laboratory services. Sedimentation velocity analysis allows the determination of aggregate content and molecular weight as an orthogonal method to size exclusion chromatography and field flow fractionation. AUC is a first principle method and therefore ideal for the verification of SEC/FFF results.
Coriolis operates 3 AUC instruments (Optima AUC, XL-I, XL-A) and offers data procesing by either SEDFIT or UltraScan using a dedicated in-house high-computing cluster.
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 centrifugalforce 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 optics during a 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 of these 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 is often required which may alter the ‘true’ aggregate population in a given sample prior to detection.
SV-AUC is capable of detecting aggregates without any change in solution condition as long as formulation components do not interfere with the detection with absorbance optics. 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 in order to ensure that these routine methods for aggregate detection are not altering the true aggregate population prior to detection.
Coriolis Pharma SV-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 analysis. Instruments are equipped with An-50Ti 8-hole rotor capable of analyzing up to 7 samples per run or 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. 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 final results obtained are a continuous c(s) distribution (Figure 3), which is a distribution of the relative amounts of the differently sedimenting species in a given sample.