Studies on the removal of Escherichia coli cell debris by aqueous two-phase polymer extraction

Abstract

Products of recombinant-DNA technology often need to be purified from complex mixtures. Biochemical engineers thus recognize the need for efficient ‘new’ techniques for product recovery. Such techniques must be easy to scale up and attain high capacities. One promising separation method for purifying both molecular and particulate biological materials is phase partitioning between immiscible aqueous phases, which are formed by aqueous solutions of certain polymers. In this paper we briefly review this technique and present results of our use of polyethyleneglycol (PEG)-potassium phosphate salts in the partitioning of the desired product from the cell debris. The system analysed was that of a genetically engineered product — human growth hormone (hGH). The effects of biomass loading, pH, degree of mixing on mass transfer rates, and the integration of phase mixing, settling and phase separation have been studied. The time taken for hGH, total protein and cell debris, to attain equilibrium distribution between the top and bottom phases was found to be between 5 and 10 s. The top PEG phase from a continuous run contained 68.5% of the total hGH with a purification factor of 8. All cell debris and up to 95% of RNA were confined to the interface and the bottom (potassium phosphate) phase. With multiple step extraction, however, total hGH yields could be increased to 81%.