Analytical ultracentrifugation (AUC) is an extremely versatile and powerful technique for characterizing the solution-state behavior of macromolecules. Coupled with appropriate software and data analysis methods, experiments performed via Analytical Ultracentrifugation are capable of rigorously determining true solution sample size (Svedberg and molecular weight), shape (axial ratio), purity and identifying the presence of aggregates and different oligomerization states.
Furthermore, sedimentation equilibrium and velocity experiments can identify non-ideal interactions with detergent and formulation excipients, characterize assembly and disassembly mechanisms of biomolecular complexes, determine subunit stoichiometries, detect and characterize macromolecular conformational changes, and estimate equilibrium constants and thermodynamic parameters for self- and hetero-associating systems.
Percentage monomer (% monomer), percentage dimer (% dimer) and percentage n-mer can also be estimated.
Analytical Ultracentrifugation relies on the proper design of experiments, and careful interpretation of data derived from relevant fitting models. Blue Stream utilizes software such as SEDNTERP, SEDFIT and SEDPHAT which provides:
- Analysis of data from: sedimentation velocity, dynamic light scattering, and sedimentation equilibrium. Sedimentation velocity data can be acquired using absorbance or interference optics, in conventional loading configuration or synthetic boundary, analytical zone centrifugation, and different cell geometries
- Non-interacting discrete species, self-association models, non-ideal sedimentations and other geometries
- Size-and-shape distributions c(s) distribution and C(M) distribution. For example, Continuous size-distributions c(s) with prior knowledge of sample size for sedimentation velocity analysis with maximum entropy regularization
- Bayesian incorporation of prior for enhanced resolution of distributions
- Lamm equation models for different solution oligomerization states: discrete non-interacting species, and self-associating systems (1-2, 1-3, 1-2-4, 1-n-mer)
- Apparent sedimentation coefficient distribution ls-g*(s) and van Holde-Weischet analysis G(s) (both for absorbance and interference data)
- All sedimentation velocity models for direct boundary modeling with algebraic noise elimination
- Corrections for water compressibility (and compressible organic solvents)
- Continuous size-distribution models for dynamic light scattering and sedimentation equilibrium
- Numerous statistical functions, loading options, and general purpose tools
- Extension to global and statistical analysis in SEDFIT and SEDPHAT
Blue Stream offers Analytical Ultracentrifugation services as one component of a complete portfolio of Therapeutic protein development services, including both structural and functional characterization and method development services. Please contact Blue Stream for more information regarding AUC.