PEG/Salt Systems

PEG-salt two phase system: has been known for some time. Kohler formed 7.5% w/w PEG 1500 and 14% potassium phasphate two phase systems directly in a bioreactor and used them to purify recombinant protein in E coli. 

Andrews (Partitioning and purification of monoclonal antibodies in aqueous two phase systems, Bioseparation 6, 1996, 306-313) disclose systems for antibody partitioning such as 75 w/w PEG 1450, 14% NaPhosphate and 12% NaCl. Such systems gave antibody partiion k (ratio of protein concentration in upper phase over lower phase) values of 100. Andrews noted a drawback is low protein solutbility due to high salt concentrations. 

A typical PEG/salt system contains 20-30% PEG plus salt in the top phase, 10-15% phosphate or sulphate salt in the bottom phase (Johansson, “Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification” Biotechnology and bioengineering, 1999, 66(4), 247-257). 

Tran (US2011/0257378) disclose an aqueous two pahse extraction (ATPE) preciptiation process used to recover therapeutic proteins such as antibodies from a crude mult-component mixture which innvolves the formation of a forward extraction PEG-Phosphate ATPE system in which the target is preferentially partitioned to the polymer rich phase. A second ATPE back extraciton system is then formed by introducing the polymer rich phase to a new phosphate salt rich phase, causing the product to precipitate at the interface between the two phases. This precipitate is then recovered and resolubilised.

PE62-phosphate salt: aqueous two phase system has been used to purify beta-xylosidase (Pan, J. Chromatography B, 754 179-184 (2001). 

PEG/Polymer Systems

PEG-Poly(acid)-Salt: 

–PEG – Poly(acrylic acid) (PAA): Daravaanen (J. Chem. Eng. Data 2006, 51, 1246-1249) disclsoes effect of temeprature on the densities and viscosities of aqueous solution of poly(acrylic acid) (PAA) of different mass fraction and liquid-liquid equilibrium for the aqueous two phase PEG-6000 + PAA + water system at equilibrium. 

–PEG – Poly(acrylic acid) (PAA) or EOPO – Salt: Hjorth (WO2008/156409 and US2010/0174052) dicloses partitioning in a multiphase system with a  1) first polymer which is synthetic poly(acid) such as poly(acrylic acid) (PAA) or polyacrylate, 2) a second synthetic polymer which is poly(ether) such as poly(ethylene)glycol (PEG)  or ethylene oxide properylene oxide (EOPO) and 3. at least one salt. In one embodiment a multiphase system is used to isolate an antibody in an aqueous polymer two phase system comprising about 4-8% PEG and 4-8% poly(acid) such as NaPAA. When EOPO is used, the system may form a three phase system since EOPO separates into two phases itself upon heating.