1. Downstream Processes BIE/CEE 5930/6930 Spring 2010

2. Aqueous Two-phase Extraction Made by combining two water-soluble polymers or a polymer and salt in water Above a “critical concentration” phase separation occurs –Two immiscible liquid phases are formed –One phase is enriched in one polymer and other phase is enriched in other polymer or salt

3. Can be used to separate proteins, cells, cell organelles, nucleic acids, polysaccharides. Examples –Polymer/polymer: Poly-ethylene glycol (PEG)/Dextran –Polymer/salt: PEG/(NH 4 ) 2 SO 4 (ammonium sulfate)

4. Aqueous Two-phase Extraction PEG-water phase is typically lighter and floats Water-salt or water- dextran phases are heavier Typical concentrations –PEG/Dextran: 10%:15% –PEG/salt: 15%:15%

5. Factors that Affect Protein Partitioning Protein Molecular Weight Protein charge and surface properties Polymer(s) molecular weight Phase composition of the system Salt effects Affinity ligands attached to the polymers

6. Aqueous Two-phase Extraction Cells and cell debris settle to the bottom phase or stay at the interface Protein can stay in either phase Partition coefficient is a function of protein size and properties

8. Aqueous Two-phase Extraction

9. Below the line is a single phase On the line is two phases The tie-line connect compositions that are in equilibrium

10. Typical concentrations of systems: PEG – Dextran 10% w/w PEG, 15% Dextran PEG – Salt 15% w/w PEG, 15% Salt

11. Effect of Salt Concentration

12. Effect of protein molecular weight

13. Typical process flow diagram