Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation: Accuracy, precision, and limits for a monoclonal antibody

BIOT 206

Michael Stoner, mstoner@amgen.com1, Kelly Macdonald1, John Gabrielson2, and Brent Kendrick1. (1) Analytical Sciences, Amgen Inc, 4000 Nelson Rd., Longmont, CO 80503, (2) Department of Chemical and Biological Engineering, University of Colorado, Boulder, Boulder, CO 80309
The use of sedimentation velocity analytical ultracentrifugation (SV-AUC) to characterize protein self association and size distribution has become widespread within the biotechnology industry. Unlike alternative techniques such as size exclusion chromatography (SEC), and polyacrylamide gel electrophoresis (PAGE), SV-AUC involves minimal matrix interactions and can be carried out in a wide variety of buffers. SV-AUC also provides much higher size distribution resolving power than light scattering techniques. However, diverse factors (molecule-, formulation-, instrument-, technique-, and software-related) make it unusually difficult to establish meaningful estimates of accuracy, precision, and detection/quantitation thresholds for the SV-AUC technique. This study describes a multi-pronged approach to this problem for a monoclonal antibody therapeutic, where data simulations and an experimental campaign were combined to uncover sources of variability and determine method capabilities.