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AUC sedimentation velocity (SV) has a longstanding reputation as being the gold standard for the matrix free quantification of aggregates in biopharmaceuticals. The continuous c(S) distribution analysis as implemented in the software SEDFIT is the most commonly used data evaluation method in the biopharma environment due to regulatory requirements and the reliable reporting of very minor species by the algorithm. I will show how to set up such an analysis in order to achieve enhanced reproducibility during a study of many batches of one protein. Attention will be also paid to the recently developed calibration procedures regarding the temperature, scan time and the radial calibration of the centrifuge.
In the second part examples are shown apart from the generic method of quantitating aggregates in dilute solutions under standardized conditions. These examples will include the determination of the extinction coefficient and concentration of protein formulations, a task far from being trivial in the biopharmaceutical field and the determination of the second virial coefficient, which is known as a predictor for aggregation propensity. The presentation will conclude with some applications of sedimentation equilibrium (SE) for the screening of weak interactions and the determination of detergent binding to proteins.
