Continuous refolding of a biotech therapeutic in a novel Coiled Flow Inverter Reactor

Abstract

A novel coiled flow inverter (CFI) based plug flow reactor has been developed for continuous refolding of granulocyte colony stimulating factor (GCSF), a biotech therapeutic product. Solubilized inclusion bodies containing the denatured and reduced forms of GCSF were continuously diluted with the refolding buffer using an inline mixing unit. This was followed by protein refolding into a CFI based tubular reactor in which a helical coil was bent at equidistant right angles to cause flow inversion at each bend. This configuration effectively provided substantial cross sectional mixing while maintaining a favourable distribution of residence time. Design of experiments (DOE) based studies was performed to optimize the refolding protocol with respect to redox conditions, pH and dilution ratio. The performance of the continuous refolding protocol has been compared with an optimized batch refolding protocol. It has been demonstrated that enhanced mixing in CFI allows for operation at higher protein concentrations (0.38mg/ml as compared to 0.19mg/ml in batch) and results in comparable purity (84% vs. 83% in batch), thereby resulting in a nearly 15 times increase in productivity. This will result in a significant reduction of costs related to downstream purification as well as no need for the large tank that is otherwise required for dilution based batch refolding. The proposed configuration is likely to perform favourably in other biotech unit operations that require mixing and/or sharp residence time distribution such as precipitation.