The SmartFlow® filter WORKS™ E. coli lysate by simultaneous process Optimization Procedure from SFT is intended as a procedure for developing an integrated process for concurrently isolating a target protein from an E. coli lysate and concentrating the protein in a second system. In the first step or the isolation step, the product protein is isolated from the lysate by passing the target molecule through the membrane in the first skid. In the second step or concentration step, the product is concentrated in the second skid. The permeate from this concentration step can then be used as the diafiltration buffer for the isolation step. Significant benefits in time savings, process equipment costs, and buffer costs are realized by performing the processes simultaneously rather than sequentially.
This Isolation of proteins from cell lysate protocol is intended for isolating, concentrating and diafiltering recombinant proteins with a molecular weight of less than 30 kD from lysed cells in two simultaneous processes. This process has been repeatedly implemented with consistent success in E.coli and Pichia fermentations.
The initial step isolates the target molecule from the fermentation broth by using a 150 kD regenerated cellulose membrane to pass the target molecule freely into the permeate and retain the large molecular weight broth components, and the accumulated cell debris. The protocol call for the fermentation broth to be concentrated to 5X prior to starting the diafiltration. The required diafiltration buffer is generated in the second ultrafiltration process.
The target of the second step is to concentrate the target molecule with a 5kD regenerated cellulose ultrafiltration membrane. The permeate from this process is fed back to the recirculation loop of the isolation process to create a closed loop system. The target product is concentrated in the retentate tank of the second loop and recovered when the desired concentration or target yield is achieved.
