(155a) The Development Of A Macro-Scale Process To Release Periplasmic Proteins Using Osmotic Shock

The conventional laboratory approach of small scale batch osmotic shock to release proteins from microbial cells consists of mixing and equilibration of cell paste with high osmolarity buffer, followed by dilution in a low molarity buffer. This method is time-consuming, inefficient, difficult to control and scale up. This presentation will describe development of a novel approach which contacts in a continuous flow, high osmolarity fermentation broth with an appropriate low osmolarity shock buffer through a static mixer coupled to a continuous centrifuge for simultaneous solid-liquid separation. This method has a high throughput, the mixing time is easier to control, and scale-up is straightforward and quantitative. We successfully demonstrated the applicability of this operation using human growth hormone expressed in P. fluroescens as a model system. Under best conditions, high step yields (~90% and above) and purity comparable to the batch protocol were achieved. This development is a successful example of an integrated approach to biological product recovery and solid-liquid separation.