On the use of size exclusion chromatography for the resolution of mixed amyloid aggregate distributions: I. Equilibrium partition models

In this study, we investigated the theoretical potential of size exclusion chromatography (SEC) for resolving mixtures of protein aggregates (of various sizes and shapes) produced in the generation of amyloid fibrils. We present our findings in the form of an equilibrium partition model. We first review the general characteristics of SEC and discuss the physicochemical features affecting solute transport and partition. We then develop new methods for estimating the transport and partition coefficients of protein aggregates on the basis of their molecular dimensions and the SEC column properties. We detail how these calculated properties can be used to estimate the likely resolving power of an SEC column. Model predictions were found to be in general agreement with experimental data gained from the measurement of the elution profile of sheared amyloid fibrils prepared from bovine insulin and passed through a Superose 6 precision SEC column. Our formalism should provide a basic appreciation of the competing factors at work and allow an informed choice to be made for optimal selection of SEC column medium to separate a desired size range of aggregate.