(326d) Concentration-Controlled Single-Step Protein Purification Using a Tunable Self-Cleaving Affinity-Tag
Downstream processing is an essential step in the manufacture of pharmaceuticals such as antibodies and other protein therapeutics. A reduction in production costs can be achieved by the use of a simplified one-step purification method, based on self-cleaving inteins. Development and demonstration of this method is the focal point of this study. Our purification technique involves intein-mediated cleaving of a chitin affinity tag, allowing simple product recovery from a conventional affinity column by a simple shift in temperature and pH. Further, by carefully controlling the temperature and pH within the column during cleaving, it is possible to regulate the concentration profile of the protein exiting the column over time. This ability is unique to pH-induced intein cleaving, and has the potential to allow greater control over product aggregation and buffer usage during downstream processing. The concentration of the protein exiting the column, as well as rate of the cleavage reaction throughout the column, can be predicted by an empirically designed mass transport and reaction model. To demonstrate the model, we have expressed the Green Fluorescent Protein (GFP) in E. coli, and have conducted several experiments where we have generated various time vs. concentration profiles at the column effluent by manipulating the pH of the buffer entering the column over time.