Measuring low levels of protein aggregation by sedimentation velocity (original) (raw)

Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation (SV-AUC): Sources of variability and their relative importance

Journal of Pharmaceutical Sciences, 2009

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has found application in the biopharmaceutical industry as a method of detecting and quantifying protein aggregates. While the technique offers several advantages (i.e., matrix-free separation and minimal sample handling), its results exhibit a high degree of variability relative to orthogonal size-sensitive separation techniques such as size exclusion chromatography (SEC). The goal of this work is to characterize and quantify the sources of variability that affect SV-AUC results, particularly size distributions for a monoclonal antibody monomer/dimer system. Contributions of individual factors to the overall variability are examined. Results demonstrate that alignment of sample cells to the center of rotation is the most significant contributing factor to overall variability. The relative importance of other factors (e.g., temperature equilibration, time-invariant noise, meniscus misplacement, etc.) are quantified and discussed. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3522–3539, 2009

Assessing sedimentation equilibrium profiles in analytical ultracentrifugation experiments on macromolecules: from simple average molecular weight analysis to molecular weight distribution and interaction analysis

Biophysical reviews, 2016

Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is a particularly powerful tool when used in conjunction with its sister technique, namely sedimentation velocity. Here, we describe key approaches now available and their application to the characterization of antibodies, polysaccharides and glycoconjugates. We indicate how major complications, such as thermodynamic non-ideality, can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor Don Winzor over several decades of research.

A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

European biophysics journal : EBJ, 2018

In establishing the sources of data variability within sedimentation velocity analysis in the analytical ultracentrifuge and their relative importance, recent studies have demonstrated that alignment of the sample cells to the centre of rotation is the most significant contributing factor to overall variability, particularly for the characterisation of low levels of protein aggregation. Accurate mechanical and optical alignment tools have been recently designed. In this study, we (1) confirm the effect of misalignment observed by others on the estimated amounts of bovine serum albumin (BSA) monomer and dimer, and the sedimentation coefficient value for the BSA dimer; and (2) demonstrate the high performance of a mechanical alignment tool and the usefulness of a simple and complementary enhanced manual alignment protocol which should be useful for situations where these tools are not available.

Analytical ultracentrifugation: a powerful 'new' technology in drug discovery

Drug discovery today. Technologies, 2004

Analytical ultracentrifugation (AUC) is a powerful means of characterizing the solution behavior of molecules. Sedimentation velocity analysis, the preferred AUC technique for characterizing complex systems, has higher resolution, broader applicable range and fewer solute/solvent limitations than gel-permeation chromatography. The technique is simple to perform and should become a mainstay for target identification, target validation, lead optimization, formulation in drug development and QA/QC. Recent studies have used AUC to characterize the binding stoichiometry and binding sites of an anti-tumor agent; of a hemoglobin-stabilizing protein, and of a fibril growth inhibitor, and to assess the causes of protein aggregation. The recent addition of fluorescence to the existing absorbance and interference detectors dramatically extends the flexibility of analytical ultracentrifugation.: