Downstream Processes BIE/CEE 5930/6930 Spring 2010.

Downstream Processes

BIE/CEE 5930/6930Spring 2010

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

• Can be used to separate proteins, cells, cell organelles, nucleic acids, polysaccharides.

• Examples– Polymer/polymer: Poly-ethylene glycol (PEG)/Dextran– Polymer/salt: PEG/(NH4)2SO4 (ammonium sulfate)

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%

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

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

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Aqueous Two-phase Extraction

• Below the line is a single phase

• On the line is two phases

• The tie-line connect compositions that are in equilibrium

Typical concentrations of systems:

• PEG – Dextran 10% w/w PEG, 15% Dextran

• PEG – Salt 15% w/w PEG, 15% Salt

Effect of Salt Concentration

Effect of protein molecular weight

Typical process flow diagram