Optimized Single-Step Affinity Purification with a Self-Cleaving Intein Applied to Human Acidic Fibroblast Growth Factor

To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a controllable self-splicing protein element in the form of a mini-intein was used to optimize the recovery of proteins for both batch and flow purification strategies. The ability to recover purified proteins was demonstrated using a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To characterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant dispersion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). Insight into the nature of the cleavage reaction and its regulation was obtained via temperature- and pH-dependent kinetic data.