A method to determine confidence limits for the area, sedimentation coefficient, and molar mass of individual peaks...

The c(s) sedimentation distribution method implemented in the program SEDFIT (Biophys J 78:1606-1619, 2000) is widely used for analyzing sedimentation velocity data, and is particularly useful for detecting low levels of aggregates or other minor components in protein pharmaceuticals. Unfortunately, this method does not provide confidence limits for the area or sedimentation coefficient of each resolved peak, which makes it difficult to assess whether differences from one sample to another are statistically significant. This paper describes a new method to obtain such confidence limits using the program SVEDBERG (Biophys J 72:435-444, 1997) by automatically translating a saved c(s) distribution into a discrete species model where the molar masses of all species are constrained to keep the f/f₀ ratio constant for all species. This approach also then allows relaxing the constant f/f₀ ratio constraint on one or more minor species to determine their true molar masses (independent of assumptions about hydrodynamic shape), and also determining the confidence limits on that molar mass. It is demonstrated that this approach will work for samples containing up to five minor components (six total species), and even when multiple minor species are present at levels of only a few tenths of 1%.