Quantitation of soluble aggregates by sedimentation velocity analytical ultracentrifugation using an optical alignment system – Aspects of method validation

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely used for quantitation of soluble aggregates as an orthogonal technique to size-exclusion chromatography (SEC). SV-AUC presents many advantages over the SEC, yet lower precision of aggregate quantitation by SV-AUC often complicates comparison between aggregate values generated by these techniques and subsequent decision making. In an earlier report, we described the development of an optical alignment (OA) system and evaluated the intermediate precision of aggregate quantitation offered by the OA. Here, we determine the limit of detection (LOD) and limit of quantitation (LOQ) which can be achieved with the OA. For a common setup using three cells, the improvement lent by the OA system is almost 2.5-fold compared to the earlier reported limits. In addition, we estimate the contribution of the fitting variability and compare options to further increase the precision of aggregate quantitation by SV-AUC. •Optical alignment improves the LOD/LOQ of aggregate quantitation by almost 2.5-fold.•Fitting variability of Sedfit c(S) is practically constant across the range studied.•More cells per sample yield higher precision than more replicate fits per cell.