(263b) Adsorptive Protein Bio-Separation and Refolding in Circulating Fluidized Bed

Biotechnology offers an increasing potential for the production of a wide range of commodities, ?green chemicals? and speciality products for human need. An important part of biotechnology is to develop simplified schemes for fermentation and downstream processing of recombinant proteins. Refolding or protein renaturation is the most critical and intricate step in the efficient recovery of active proteins from inclusion bodies. The yields from refolding by traditional methods are usually very low, typically ~5-20%. After separation from cell debris, inclusion body proteins along with contaminants are usually solubilised using high concentrations (2-8 M) of suitable chaotropic salts. The quickest and most convenient way to refold a protein is by diluting the denatured protein with the renaturation buffer containing suitable reducing agents. However, this is not the most efficient or economical approach when considering the large scale purification or refolding. Binding of proteins to the membranes significantly affects the renaturation yields when using dialysis or diafiltration via ultrafiltration membranes. Use of packed bed chromatography columns for refolding of proteins has drawbacks such as ineffective solvent utilization, insufficient media utilization and high level of dilution. These limitations can be addressed by the use of circulating fluidized bed chromatography. A twin-riser circulating fluidized bed refolding system, operating on pressure balance is explored in our lab as a promising alternative to the conventional route of dilution and single column refolding/purification system. Previous work has shown an overall productivity of 21 mg/h.g. On-going experiments are aimed at improving the productivity and refolding yield of the process by further optimizing the refolding conditions and assessing its robustness using two different proteins: lysozyme and citrate synthase.The present work explores the use of circulating fluidized bed ion-exchanger as one-step adsorptive refolding/purification system and elaborates its applicability in purification and refolding of lysozyme and citrate synthase.