See also ethanol fractionation under plasma proteins.  See also precipitation of plasma proteins such as antibodies under “IVIG Preparation” see also Affinity precipitation  See also Polyelectrolyte Precipitation 

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subject of proteins from a protein solution, or as a very specific method to recover a single protein of interest form a purification step ((Current Protocls in Protein Science, “Extraction, Stabilization, and Concentration” Unit 4.5) Typical precipitation agents are ethanol, polyetheylene glycol, lyotropic (i.e., anti-chatropic) salts such as ammonium sulphate and potassium phosphate and carpylic acid (WO 2007/073311).

Precipitation methods can be classified in 2 grouping; those that selectively precipitate antibodies, mainly by altering the properties of the solvent and those that selectively precipitate contaminants by complexation with agents that reduce their solubility. The first group that selectively precipitates antibodies includes precipitation with salts such as ammonium sulfate, with organic polymers such as polyethylene glycol (PEG) and by electrolyte depletion. The second group includes precipitation by organic complexants such as short chain fatty acids and organic bases (Gagnon, “Purificaiton Tools for Monocloanl Antibodies” Validated Biosystems 1996, pp. 1-269). 

Salt Precipitation (“salting out”) See also isolation of IgY by ammonium sulfate precipitation

High salt concentraiton promotes protein aggregation and precipitation. Alhtough the mechanisms is not well understood, the salt is thought to remove the water of solution from the protein, thereby reducing protein solubility. The Hofmeister series represents decreasing anion effectiveness: citrate>phosphate>sulfate>acetate>chloride>nitrate>thiocynate. The salts at the low end of theis series casue structural damage to proteins. The high solubility of ammonium sulfate in water and the position of sulfate in the Hofmeister series make it the most popular choice for slating out proteins. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Precipitation is widely used for product recovery of biomolecules such as immunoglobulins. The most common type of precipitation of proteins is salt induced precipitation. (WO 2007/073311)

Temponi (Hybridoma, (8)(1), 1989, 85-95) discloses that the sequence (NH4)2SO4 – caprylic acid precipitation and caprylic acid precipiation of IgG mAbs from acitic fluid was comparable. 

Caprylic Acid (CH3(CH2)6COOH) See also Precipitation of Plasma proteins with fatty acids

Cell Culture-Caprylic Acid

Arunakumari (WO2010/151632) disclose methods for purifying a protein of interest such as an antibody from a cell culture by precipitation of the contaminants such as host cell DNA with 1% caprylic aicd. Mixtures that were thus depleted of contaminants can then be sued directly in downstream chromatography applications such as IEX. 

Jiao (US 2005/0272917) discloses a method of purifying IgG from a feedstock which can be supernatant from a cell culture that includes adjusting the pH to 4-5.5 then contacting the pH-adjusted feedstock with a mono or polyakanoic acid haivng between 4-12 carbon atoms, preferably caprylic acid. After the addition of alkanoic acid, the supernatant containing the IgG is separated form the precipitate as by centrifugation or filtration. The supernatant is then dialzyed against a buffer haivng a pH of about 4.5-6.0. 

—-Cell Culutre -Caprylic Acid – Allantoin

Gagnon (US 15/121,676, published as US 20160362446) discloses a method of purifying an antibody by contacting a propertin preparation such as a cell culture harvest having an antibody with at least one fatty acid such as enanthic (heptanoic) acid, caprylic acid, pelargonic (nonanoic) acid or capric (decanoic) acid having 7 to 10 carbon atoms to form a mixture, contacting this mixsture with allantoin and then removing solid materials form the IgG containing liquid. 

—-Cell Culture -Caprylic acid – PEG and/or CaCl2:

Jungbauer (US 15/029,682 published as US 2016/0272675; see also WO2015/056237) discloses precpitating a clarified cell culture supernatant using 100 mM sodium citrat, pH 4.5, 1% caparylic acid and removing the precipitate to provide a secondary supernatant to which 21% PEG at pH 7 was added so as to precipitate the antibody. This precipitate can be resuspended and treating using CaCl2 to precipitate further impurities such as DNA which can be removed from the solution coprising antibodies. 

Wien, “Continous precipitation of therapeutic proteins, with an emphasis on monoclonal antibodies” Thesis, December 2013) discloses various sequences for mAb purificaiton from CHO cell culture sueprants using caprylic acid (CA), calcium chloride (CaCL2), polyethyle glycol (PEG) and ethanol (CEP). In one embodiment, a two step precipiation using CA/PEG was performedfwhere CA was used for precipitating HCP and dsDNA and PEG was used for mAb capturing from the supernatant. 

 –Cell Culture – Electropositive multivalent ions  (e.g., Tren) + Fatty Acids (electronegative)

Gagnon (US 14/769,098, published as US 2016/0009762 and US 9,994611) also discloses a method of purifying a protein such as an antibody by conditioning an impure protein preparation by contacting it with (i) at least one electropositive organic additive such as methylene blue, ethacridine, chlorhesidine, benzalkonium chloride, cetyl trimethyl ammonium bromide, (Tris(2-aminoethyl) amine (TREN) AND (ii) at least one electronegative organic additive such as  octanoic acid and subsequently removing solides thereby removing at least 95% of chromatin and then applying the conditioned preparation to an adsorptive chromatography medium.   In one embodiment the method includes adding allantoin to carify chromatin from a cell culture harvest, then methylene blue or ethacridine or cetyl trimethyl ammonium bromide or chlorhexidine is added. Then particles bearing an electropositive metal affinity ligand tris (2-aminoethyl)amine (TREN) is added. In a separate embodiment allantoin is added to a cell culture and then heptanoic acid  or octanoic acid or pelargonic (nonanoic) or capric acid is added. The mixture is then stirred for 2 hours and the electropositive metal affinity ligand tris(2-aminoethyl)amine (TREN) are added. In some embodiments the method reduces the contaminants that interfere with a subsequet purificaiton Protein A purificaiton step. 

–Cell Culture — Electropositive multivalent ion (e.g., ethacridine) OR Fatty (electronegative such as caprylic acid) — PEG

Gagnon (US 14/766,131, published as US 2015/0376231 and US 9,988419) discloses a method for purifying a desired protein such as an antibody by conditioning the preparation with ethacridine or an electronegative organic additive such as octanoic acid, thereby removing at elast 90% of chromatin and then precpitating the antibody with PEG in the presence of non-protien-recpitating salts to provide a precipitate with the antibody.  

–Cell Culutre -Fatty acid (e.g., caprylica cid) –substrate with metal binding functional group + allantoin

Gagnon (14/894,993 published as US 2016/0115194 and US 10,174,076) also discloses pufication of a protein such as an antibody with caprylic acid followed by porous particle treatment and further purificaiton by precipitation with polyethylene glycol. In one embodiment a cell culture harvest is contacted with caprylic acid and with a functionalized substrate such as a metal binding funcitonal group such as tris(2-aminoehyl)amine). “TREN’ refers to Tris(2-aminoethyl) amino and is an electropostive compound that is known for strong affinity for metal ions. It may be chemically affixed to various soluble and insoluble materials. In one embodiment allantoin is also present. 

Protein A – Caprylic Acid

Brodsky (Biotechnolgy and Bioengineering, 109(1), 2012) discloses subjecting samples of protein A purified mAbs having been eluted at pH 3.6 which were then dialyzed into 50 mM acetate or 5-0 mM citrate at different pHs between 4.5-5.5 and then precipitation of contaminants by adding 10 mL of caprylic acid to 10 mL of dialyzed mAb solution at the same pH, and removing the precipitate.

–Protein A — Caprylic acid -IEX:

Zheng (US 15/523632), published as US2107/0313742) disccloses a process of purifying a prtoien of interest such as an antibody by subjecting a mixture containing the antibody to Protein A affinity chromatography, recvocering the protein in an eluate, adding caprylic acid to precipitate contaiminants, removing the contaminant precipitate and subjecting the solution to a second chromatography step such as IEX. 

Di and Trivalent Metal Cation Precipitants (see also removing aggregates)

Metal ions such as Mn2+, Fe2+, Ca2+, Mg2+ and Ag+ bind to different protein funcitonal groups and can cause them to preciptiate. They act at much lower concentrations than the ions of Hofmeister series (see aobve) and are easily removed by ion-exchange adsorption or chelating agents. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Di and trivalent metal cations (e.g., Zn2+, Mn2+, Ca2+ and Al3+) provide two means for precipitating proteins; they may act directly or often are used as auxiliary agents for other precipitation methods such as the crowding method using neutral polymers ((Lovrien, Unit 4.5 from Current Protocols in Protein Science). Flocculation reagents such as calcium chloride and potassium phosphate have been studies to vaoid potential issues related to polymer or residual monomer toxicity and the need for their removal through subsequent purification steps. The amount required can be very low (e.g., 20-60 mM calcium chloride with an equimolar amount of phosphate added to form clacium phosphate. The mechanism is thought to be related to the co-precipitation of clacium phosphate with cells, cell debris and impurities. (Liu, mAbs, 2:5, 480-499, 2010)

Thio-heterocyclic cations: refers to an organic cation having 3 rings in a coplanar arrangement bearing at least one thiol group and at least one amino goup, bearing at least one positive charge where the positive charge may reside with the thiol or with an amino group. 

–Methylene blue (IUPAC name: [7-(dimethylamino)phenothiaziin-3-ylidene]-diemthylazanium] (AKA “basic blue 9): 

Gagnon (US 14/766, 123, published as US 2015/0376230) and WO 2014/123486) discloses a method of reducing aggregate content in a protein preparartion haing a target protein such as an antibody by contacting the preparation with a thio-heterocyclic cation such as methylene blue to form a mixture and then contacting the mixture with at least one functionalized solid such a negatively charged solid used for CEX to remove excess thio-heterocyclic cations. In certain embodiments, the sample is additionally contacted with an antiviral agent such as benzalkonium chloride, chlorhexidine or ethacridine and/or a soluble organic modulator such as a noionic organic polymer along with an electropositive organic additie. 

Multivalent metal ions with monovalent/multivalent anions

Calcium/Potassium Phosphate

Coffman (US7,855280; See also WO 2007/035283) discloses using calcium and phosphate as precipitants of mAbs produced in CHO cells. 2M potassium phosphate is added to the culture medium and 5M calcium chloride was added to a concentration of 30 mM. The pH of the mixture was 7.3. The flocculated culture was incubated while mixing and then treated through centrifuge  and the antibody recovered in the supernatant to separate the solid from the antibody target. Coffman discloses that examples of first cations inclue calium (Ca2+), magnesium (Mg2+), aluminum (AL3+), copper (Cu(I) or Cu(II), etc) and examples of first anion include those preferred ligands to the metal ion used such as fluoride, phosphate, carbonate, sulfite, etc. 

Divalent Cation/Alcohol – Alchohol/Divalent Cation

Jungbauer (US14/423802, published as US 2015/0225473) discloses a method for isolating a protein such as an antibody by combining a cell culture supernatant with a divalent cation salt to precipitate impurities and then combining the supernatant with an aliphatic alcohol such as ethanol to precipitate the antibody, resuspending the antibody containing precipitate in a buffer comprising a divalent cation salt suitable for the precipitation of impurities therefrom to produce a protein contianing solution and combining the protein containing solution with an aliphatic alcohol such as ethanol to precipitate the antibody/protein and isolating the protein containing precipitate. In another embodiment the aliphatic alcohol is added first to form the protein containing precipitate  which is resuspended in a buffer comprising the divalent cation salt to precipitate impurities and the antibody containing solution is combined with the aliphatic alcohol to form an antibody precipitate.

Other Precipitating Agents

Allantoin (2,5-dioxo 4-imidazolidinyl urea):

Allantoin cyrstalling pwoder can be added to a protein solution at suerpstarated conentraitons, endotoins bind and undissolved cyrsals with bound endotoxins are removed by filtration or centrifugation. The method has been shown to remove an average of 99.98% endotoxin for 20 test proteins (Vagenende, J Chromatography A, 2013, 15-20). 

DNA intercalating Agents

–Ethacridine (7-ethaoxyacridine-3,9-diamine): 

Gan (J of Chromatography A, 1291 (2013) 33-40) discloses that IgM mAbs and aggregates in mammalian cell culture supernatants are associated with contaminants such as nucleosomes, DNA and histone proteins drived form nucleic of host cells and that for this purpose adding the DNA intercalating agent ethacrine and allantoin and then flowing the supernatant through a column of mixedmode particles bearing metall affinity, AEX and CEX functionalities was effective for removing the contaminants. 

Ethacridine (2-ethoxy-6.9-diaminoacridine lactate)

Ethacridine is one of several organic bses used for commecial fractionation of plasma proteins and the only one that has been adapted systematically for purificaiton of mAbs. It is strongly hydrophobic, positively charged and used at alkaline pH. This might lead one to believe that the precipitaiton mechaism is similar to octanoic acid but in fact the complexant is applied at much lower ionic strenght than octanoic acid and it precipitates contaminants by forming stable insoluble ionic complexes. Functionally ethacridine operates like a liqui phage anion exchanger, with selectivity to match. (Gagnon, “Purificaiton Tools for Monoclonal antibodies” Vaoidated Biosystems, 1996, pp. 1-269). 

Organic Solvents

Organic solvents are often used for fractional precipitation of proteins. One of the largest scheme for sepration of therpaeutic proteins from human plasma is based on a series of precipitation steps based on changes in temperature and addition of ethanol. In general, however, protein separation by precipitation lacks specificity which has led in the late 1970s to the development of affinity precipitation (see below).(Hibrig, J. Chromatography B, 790 (2003) 79-90).

Addition of a mild organix solvent to an aqueous protein solution reduces the solvent dielectric constant, thereby indcing protein precipitation. The solvent must be completely miscibe with water (e.g., ethanol and acetone). Solvent precipitation is typically performed at low temperature (<10C) because conformational regidity then prevents irreversible denaturation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Ethanol: Fractionation of antibodies by precipitation using ethanol has been in practice for many years. In plasma fradtionation, precipitaiton is still the method choice. In the case of the Cohn fractionation, immunoglobulins are precipitated in the so-called precipitate II+III with 25% ethanol, at pH=6.8, and the termpature of -5C (Josic, Food Technol. Biotechnol. 39(3) 215-226 (2001).

20% ETOH — 25% ETOH

(Methods in Immunogly and Immunochemistry, Curtis Williams and Merrill W. Chase, Volume 1) discloses a rapid approach where serum is diluted with distilled water, pH is adjustd to 7.7 and sufficient ethanol slowly added with vigrous stirring to give a final concentraiton of 20%. The insoluble proteins designated as precipitate A are removed by centrifugation. This precipitate A is suspended in cold NaCl, the pH lowered so that yG immunoglobulins remain in the supernatant while precipitating yA and yM immunoglobulins are in the precipitate (precipitate B). The supernatant is raised to about pH 7.4 and thanol added to concentraiton of 25% to precipitate yG immunoglobulins. 

Phillips (J Immunol Methods, 1984, 30; 74(2): 385-93) discloses using the method of Coh (1946) as modified by Deutsch (1967) for purifcation of IgG by ethanol precipitation. Antserum is deluted in deionised water and pH adjusted to 7.5, cooled to 0C and sufficient ethanol added to final concentraiton of 20% with vigorous stirring adn same time reducing temperature to about 0%c. The resulting recipitate is removed by centrifugation at 0% and resuspended in 15 mM NaCL, pH lowered to 5.2. The precipitated material contains IgA and IgM which can be removed by centifiguation. The pH of the suerpnatant is brought to 7.4 and 95% ethanol added to overall concentraito of 25% to precipitate IgG which can be removed by centrifugation. 

Polymer precipitation: (See outline)

Salting-Out Procedures

Salting out procedures have certain advantages over ethanol fractionation for preliminary concentration and partial purification of immunoglobulins. Among these are simplicity and relatively slight danger of denaturation. (Methods in Immunogly and Immunochemistry, Curtis Williams and Merrill W. Chase, Volume 1). 

Salting-out invoves the precipitation of a protein by the addition of high concentrations of neutral salts or amino acids under appropriate conditions of pH, total prtoein concentration and temperature. (Rothstein, “Differential Precipitation fo proteins” in Dekker, Bioprocess technology, 1994, 115-160).

Ammonium sulfate (NH4)2SO4 precipitation 

Ammonium sulfate fractionation, is generally employed as the initial step in the isolation of crude antibodies from serum or ascitic fluid. Lorette C. Javoid “Immunocytochemical Methdos and Protocols” Second Edition, volumen 115 in “methods in Molecular Biology”, 1999.

Ammonium sulfate precipitation also known as “salting out” is still widely used for protein separation based on the fact that the solubility of most proteins decreases at high electroyte concentration. Sulfate is used because multivalent ions are more effective than monovalent ions. This procedure is usually carried out in the cold with control of pH close to neutrality. In sequential combination with caprylic acid, precipitation can even achieve a crude antibody purification by precipitation alone.eted polypeptide.

Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins form targeted polypeptid(s). The preferred salt is ammonium sulfate. It precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discared (unless the targeted catalytic polyptide is hydrophobic) and ammonium sulfate is added to the supernantant to a concentration known to precipitate the target protection. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other method that rely on solubility of proteins, such as cold ethanol precipitation, are well known in the art and can also be used to fractionate complex protein mixtures (US 20060088883).

 By Lowering the pH: 

Proteins are soluble in water due to the interaction of their charged groups with ionized water molecuels. Adjustment of the pH to the isoelectric point (pI) yields minium solubility, since the net charge of the protein is eliminated. Most proteins have a pI<7, and the relatively low cost of acids make pH adjustment with acid a popular method of protein preciptiation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Binder (WO2011/110598) teaches a method for purifying cell cultivation supernatants by adjusting the pH value in the acid range so that nucleic acid and host cell protein is precipitated by the targeted polypeptide remains in solution.

Liddel (US 13/122676) disclsoes a process of purifying a fragment antibody from a culture by separating the fragment antibody from the medium by lowering the pH so that the fragment antibody is souble, but one or mroe of the impurities are insoluble. 

Lydersen (“Acid precipitation of mammalian cell fermentation broth” Annals New York Academy of Sciences, 745, 222-231, 1994) discloses a process for the purificaito of an antibody from impurities in a cell culture medium by reducing pH. Accordingly to the procedure, the antibody remains soluble and the impurities are precipitated. The precipitate is then removed during centriguation or microfiltration. 

Takeda (EP1561756A1) also discloses a method for removing impurities in a sample by converting the sample into an acidic aqueous solution of low conductivity at a pH below the isoelectric point of the active protein which can be an antibody and then removing the resulting particles. 

Lower pH + Divalent Cations:

Romero (WO 2008/127305) discloses a method of isolation a macromolecule such as an antibody by lowering the pH of the composition, adding a divalent cation such as Ca2+ and then separating the antibody from the impurity.