See also Plasma Protein Precipitation by Various Agents 

Nonionic, water-soluble polymers induce protein precipitaiton by excluding water from the solvation structure of a protein. PEG is the most widely studied and widely used polymer, but dextrans are also used for this purpose. Thylic acid, caroxymethl cellulose and polyethyleneimines precipitate proteins at much lower concetnration (usually e effect of polymers as precipitating agents is similar to partitioning in aqueous two-phase polymer systems. High PEG concentraitons are required to preciptiate low-molecular weight proteins, wehreas low concentraitons are required for high molecular weight proteins. Polyelectorlytes such as polyacrylic acid, caryleneimines preciptiate proteins at a much lower concentraiton than nonionic polymers. They act more like flocculants and adsrob to the prtoein. Thus, polyelectrolytes, unolke PEG, coprecipitate with the protein and can cause irreversible denaturation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)boxymethl cellulose and polyeth

In the mid 1960s Polson (Biochem. Biophys. Acta 82, 463-475 (1964) analyzed a variety of high molecular weight polymers for purifying proteins include polyethylne glycol (PEG), dextran and polyvinylpyrrolidone (PVP).

Antibodies can also be co-precipiated with negatively charged polymers. Selectivity parallels CEC Co-precipitation of non-IgG contaminants with positively charged polymers parallels the selectivity of AEC (Gagnon “Technology trends in antibody purificaiton, J of Chromatography A, 1221, 57-70 2012). See precipitation of antibodies with polyelectrolytes 

Precipitation with Non-ionic Organic polymers: 

Non-ionic organic polymers refers to a naturally occurring or synthetic hydrocarbon composed of linked repeating organic subunits that lack charged groups. It may be linear, cominatly linear with some branching, or dominantly branched. Examples include PEG, polypropylene glycol, polyvinylpyrrolidone (PVP). (Gagnon, US 14/766,131). 

1. Polyethylene glycol (PEG): 

Purification of proteins such as antibodies by precipitation with polyethylene glycol (PEG) has been described. It is typically performed as an aqueous phase technique, where PEG is dissolved in an aqueous protein preparation and causes the protein to precipitate from that solution. The size and concentration of PEG are known process variable, as is pH (Gagnon, US 14/766,131 published as US 2015/0376231). 

PEG is a nonionic polymer of ethylene glycol. It has been used for industrial prepariton of polyclonal antibodies for decades. In parallel with its polyclonal applicaitons, it is applied more frequently to m IgMs than to IgGs. (Gagnon, “Purificaiton tools for monoclonal antibodies” Validated Biosystems, Inc., 1996, pp. 1-269). 

Knevelman “High-Throughput screening techniques for rapid PEG-based precipitaiton of IgG4 mAb from clarified cell culture supernatant” pp. 697-705) discloses operating conditions of PEG precipitation of IgG4 as a function of mAb concentraiton, precipitatnt concentration and pH. 

Lain (“EPG Precipitaiton: A Powerful Tool for onoclonal antibody purificaiton” BioPharm, 2010, pp. 108) discloses optimization of PEG concentraiton and pH for precipitation of mAB. PEG was added as a 40% (w/w) stock soltuion to the desired final conetration and pH of the clarified media was adjusted to desired level with 2-M Tris in a 15 mL conical tube. The tube was then centrifuged and supernatant decanted. The pellet was redissolved in PBS. The final process resulted in a product yield of 90% and HCP reudction fo about 1 LRV. 

PEG in Combination with Protein-Precipitating salts (“kosmotropic salts”):

Common examples of “protein-precipitating salts” are ammonium sulfate, sodiu or potassium citrate, sodium or potassium pohsphate.  Such salts are commonly referred to as kosmotropic salts. Gagnon (US 14/766,131, published as US 2015/0376231) discloses precipitatin of an antibody with PEG in the presence of a non-protein precipitating salt

–PEG–Ammonium Sulfate:

Brooks (J. Immunoglogical Methods 155(I) 1992, pp. 129-132) discloses a emthod for the purificaiton of mouse monoclonal antibodies from hybridoma culture supernatants using polyethylene glycol 6000 (PEG 6000) and finally reprecipitating using an ammonium suiphate procedure. 

–PEG-Sodium Phosphate (antibody precipitated)

Gervais (WO 2009/016449) disclose purificaiton of antibodies using the steps of precipitation of the antibody using a PEG solution or sodium phosphate.

PEG in Combination with Non-Protein Precipitating salts (“Chatropic salts”):

“Non-protein-precipitating salts” refers to a salt that lacks the ability to mediate precipitation of a desired protein. Common examples include sodium or potassium chloride, sodium or potassium acetate, sodium or potassium thiocyantateor gaunidinium chloride. Gagnon (US 14/766,131, published as US 2015/0376231)

Gagnon (US 14/766,131, published as US 2015/0376231) discloses precipitatin of an antibody with PEG in the presence of non-protein precipitating salts such as sodium chloride, potassium chloride, sodium acetate, potassium acetate, sodium thiocyanate, potassium thiocyanate and guanidine chloride. 

–PEG + NACL:

Gagnon (US 14/766,131, published as US 2015/0376231) discloses precipitatin of an antibody with PEG in the presence of a non-protein precipitating salt such as NACL at a concentration of from about 0.2 – 2.0 M. 

PEG in Combination with Various Types of Chromatographies:

–PEG-AEX:

Tscheliessnig (J. Chromatography A, 1216 (2009) 7851-7864) discloses a two step purification strategy comprising PEG precipitation and AEX for a panel of IgM. 

–PEG-MF/TFF – CEX (bind and elute mode) –AEX –VF — VI –UF/DF:

Kuczewski (Biotechnolgy Journal 2011, 6, pp. 56-65) disclose expression of mAb in a cell line in an extreme density bioreactor process, clarification with enhanced cell settling, PEG precipitation, capturing the precipitate and washing using microfiltration, redissolving in an appropriate buffer at high concentration, CEX with a weak membrane in bind and elute mode, AEX membrane, viral inactivation. 

Coditions/Parameters for PEG Precipitation:

–PH:

Ramanam (US 2008/0214795) disclsoes isolating antibodies by precipitation using various precipitatns such as PEG. The pH of the solution comrising an antibody is adjusted to 0.5 pH unit of the pI of the antibody and PEG is added. 

PEG derviatives which can bind metal ions

Arnold discloses PEG derivatives that can bind metal ions such as Cu2+, Zn2+, Ni2+, Fe3+, Fe2+, Co2+ and Ca2+. The metallated derivatives can, in turn, be used in the extraction or precipitaiton of proteins that interact with these metals. For example, since copper and to some extent nicekl are known to interact with histidines, these metals are good hocies for separation of proteins that contain accessible histidines. If a metal chelate is used that carries a net positive harge, this chelate would be exxpected to interact more faorably with a protein whose surface metal binding sites are adjacent to regions of net negative charge.