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Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC)

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Sedimentation velocity analytical ultracentrifugation (SV-AUC) is the gold standard in aggregation analytics. The importance of SV-AUC results is growing, especially in biosimilarity studies, and also within the development of gene therapy products.   ZentriForce Pharma offers SV-AUC services for a wide sample spectrum. For more information please follow the link.

Even though SV-AUC is with its more than 100 years of history a by far older technique than most others currently used within biopharmaceutical development. It is one of the most complex in regards of data evaluation. We have setup our own supercomputer cluster to be able to offer you several different data evaluation approaches, without the need of your data leaving our premises. We currently offer Sedfit and Ultrascan data evaluations. However, if we see the need to use a different software for your very molecule we have the knowledge base to do so. Please follow the link for more information. 

While sedimentation velocity is the standard AUC experiment, sedimentation equilibrium analytical ultracentrifugation (SE-AUC) nonetheless offers valuable information on an analyte.  SE-AUC is a first principle method for the determination of the molecular weight. Therefore, it is the method of choice to determine the molecular weight of your API in solution. Follow this link to get more information about SE-AUC theory.

Light scattering is a well defined effect. The physical properties of light scattering are used for several applications, such as dynamic and static light scattering. Please follow the link to a detailed description of light scattering theory. 

With the increasing interest in gene therapy products also the interested in AF4 is growing. However, AF4 methods are comparably complex to develop. While most people use AF4 only as separation technique. We are experts in AF4 theory and use the theory to get more information out of the technique (e.g. particle size distributions). If you want to get more information about AF4 theory please follow this link.


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Sedimentation Velocity Analytical Ultracentrifugaton (SV-AUC)

Sedimentation velocity analytical ultracentrifugation (SV-AUC) method is the established standard experiment when it comes to AUC. It is carried out at high rotational speed and the easiest way to obtain sample composition and species characteristics (e.g. molecular weight and frictional ratio). SV-AUC measurements are comparably fast experiments. SV-AUC measurements may run between one hour and one day, depending on the solute’s size and the angular velocity. The maximum possible rotational speed is 60 krpm, which corresponds to 260,000 g. Depending on the chosen rotational speed, the size range of SV-AUC can be adapted to the requirements of the analyzed products. With the correct rotational speed settings, SV-AUC measurements can be applied to samples ranging from small peptides (1 kDa) to complete viruses (up to 200 nm).

Theoretical Description of SV-AUC Experiments

Within a sedimentation velocity analytical ultracentrifugation experiment, the time dependent concentration distribution is measured. The sedimentation of solutes toward the cell bottom is monitored. The speed of sedimentation correlates with size, mass and shape of the monitored species.  Modern softwares such as Ultrascan and Sefit use a finite element method approach to compute information such as molecular weight, partial concentration and the frictional ratio for all obtained species. For information about the differences between Sedfit and Ultrascan, please proceed to our AUC software page.     

In an SV-AUC experiment the rotational speed is high enough to reduce the back diffusion of the solute to a low level. Peak broadening in SV-AUC is mostly caused by back diffusion along the concentration gradient caused by sedimentation. Even though modern algorithms such as Sedfit c(s) and Ultrascan PCSA apply a diffusion correction, a low level of back diffusion is essential for a high sensitivity.

Sedimentation Velocity Analytical Ultracentrifugation Optima AUC
Setup of an Optima Analytical Ultracentrifuge.

Comparison to other Aggregation Detection Techniques

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is regarded as the gold standard in aggregation analytics. SV-AUC is a first principle method.

In contrast to size exclusion chromatography (SEC), it does not rely on a stationary phase. Interactions with the stationary phase might have an influence on the measured sample composition. It may, for example, specifically absorb species, split oligomers, or form such. Additionally, in SV-AUC, sample is not diluted with a mobile phase during the measurement. Therefore, SV-AUC can be used for the determination of the “real” aggregate/fragment analysis. For these reasons, regulatory bodies like FDA and EMA show steadily increasing interest in SV-AUC results. It is recommended to verify your SEC / AF4 results by SV-AUC measurements.

Sedimentation Velocity Analytical Ultracentrifugation Measurement Cells
Design of AUC Cells used for SV-AUC Measurements

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is the gold standard in aggregation analytics. The importance of SV-AUC results is growing, especially in biosimilarity studies, and also within the development of gene therapy products.   ZentriForce Pharma offers SV-AUC services for a wide sample spectrum. For more information please follow the link.

Even though SV-AUC is with its more than 100 years of history a by far older technique than most others currently used within biopharmaceutical development. It is one of the most complex in regards of data evaluation. We have setup our own supercomputer cluster to be able to offer you several different data evaluation approaches, without the need of your data leaving our premises. We currently offer Sedfit and Ultrascan data evaluations. However, if we see the need to use a different software for your very molecule we have the knowledge base to do so. Please follow the link for more information. 

While sedimentation velocity is the standard AUC experiment, sedimentation equilibrium analytical ultracentrifugation (SE-AUC) nonetheless offers valuable information on an analyte.  SE-AUC is a first principle method for the determination of the molecular weight. Therefore, it is the method of choice to determine the molecular weight of your API in solution. Follow this link to get more information about SE-AUC theory.

Light scattering is a well defined effect. The physical properties of light scattering are used for several applications, such as dynamic and static light scattering. Please follow the link to a detailed description of light scattering theory. 

With the increasing interest in gene therapy products also the interested in AF4 is growing. However, AF4 methods are comparably complex to develop. While most people use AF4 only as separation technique. We are experts in AF4 theory and use the theory to get more information out of the technique (e.g. particle size distributions). If you want to get more information about AF4 theory please follow this link.

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For gene therapy product development, traditional quality attributes as well as new quality attributes must be addressed. Our scientists have worked with a wide range of different gene therapy products, ranging from small DNA / RNA complexes over lyposome-based carrier systems and adeno-associated viruses (AAV) to viruses that are several hundred nanometers in size.
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Within biosimilarity studies attention is on the collection of evidence that proves that no differences (larger than method variability) in structure and purity exist between a proposed biosimilar and the reference product. Evidence is collected through extensive analytical testing.
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Despite being far less complex than full proteins, analytical handling of peptides is challenging. Species as small as peptide-based APIs require analytical instrumentation to be run at its physical limits. Expertise and extensive experience in working with such materials is imperative for successful method development. Our scientists have worked with a large number of peptide-based APIs, with sizes down to 1 kDa.
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ZentriForce Quality System
ZentriForce Pharma has established a laboratory quality system that closely follows the extensive and detailed guidelines provided by the WHO and the principle of “writing what you do and doing what you write”. ZentriForce implements procedural, equipment and analytical SOPs. To date, close to 30 SOPs are in place.
Click here for more information
Gene Therapy
For gene therapy product development, traditional quality attributes (e.g. purity) as well as new quality attributes (e.g. the ratio of particles that carry nucleic acids to empty particles) must be addressed. The diversity of approaches for gene therapy API analysis adds a further challenge to product characterization. Our scientists have worked with a wide range of different gene therapy products, ranging from small DNA / RNA complexes over lyposome-based carrier systems and adeno-associated viruses (AAV) to viruses that are several hundred nanometers in size.
Click here for more information
Biosimilarity
Within biosimilarity studies attention is on the collection of evidence that proves that no differences (larger than method variability) in structure and purity exist between a proposed biosimilar and the reference product. Evidence is collected through extensive analytical testing. The different focus between development and biosimilarity testing require an adjusted mindset and a customized study design for the respective task. ZentriForce Pharma can be a valuable partner on your way to release a biosimilar
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Peptides
Despite being far less complex than full proteins, analytical handling of peptides is challenging. Species as small as peptide-based APIs require analytical instrumentation to be run at its physical limits. Expertise and extensive experience in working with such materials is imperative for successful method development. Our scientists have worked with a large number of peptide-based APIs, with sizes down to 1 kDa. They will be valuable assets where expertise and consultation in analytical method development are concerned.
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