Albumins: Functions and Applications

Functions:

Albumins serve important functions in the maintenance of the blood plasma system and is an essential component of regulation due to its functions of transporting physiological substances and binding toxic ones. It is the main regulator of the colloid-osmatic pressure of plasma. Albumin constitutes about 4% of the total plasma proteins. Human serum albumin (HSA) is administered to patients in various clinical situations including shock or burn patients for restoring normal blood volume and thereby alleviating certain trauma associated symptoms.

Serum albumin is the most abundant protein in mammalian sera (50 g/l, about 0.7 mM in human) and one of its functions is to bind molecules such as lipids and bilirubin. The half-life of serum albumin is directly proportional to the size of the animal where for example human serum albumin (HSA) has a half life of 19 days and rabbit serum albumin has a half-life of about 5 days.  Serum albumin is devoid of any enzymatic or immunological function and thus should not exhibit undersired side effects upon coupling to a bioactive polypeptide.. HSA is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules. Several strategies have been reported to either covalently couple protiens directly to serum albumins or to a peptide or portein that will allow in vivo assocaition to serum albumins. (Ekblad (US14/427102). 

Structure:

Albumin is a single chain protein with low MW of 66.5 kDa which cotnains 585 amino acids. It is a simple protein, non-glycosylated polypeptide, hydrophobic pathces/cavities, and it lacks prosthetic groups. Human albumin gene is located on chromaosome 4 q adn mutation of this gene will end in anomalous protein. ((Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016). 

Synthesis:

Albumin syntehsis occurs in hepatocyte cells, but it is not stored by the liver. Once produced, it is secreted into the protal cirulation. The noraml concentraiton of albumin is 3.5-5 g/dl in healthy adults. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Applications

Serum albumin is utilized udner various clinical conditions. Restoration of blood volumn emergency treatment of shock, acute management of burns, and other situations assocaited with hypovolemia are some of the clinical applicaiton sof albumin. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Albumin Binding Proteins

Streptococca. protein G has three Ig binding motifs and three serum albumin binding domains. The structure of one of the 3 serum alumin binding domains whos a three helix bundle domain , named “ABD (albumin binidng domain) and is 46 amino acid residues in size. It has been designated as “G148-GA3”. Other bacterial albumin binding proteins other that protin G has been identified. (Ekblad (US14/427102)

Methods of Albumin Purification

Procedures using Ethanol Fractionation

–Cohn Method: The traditional method for the purification of albumin for therapeutic use has been cold ethanol fracitonation, as described by Cohn in 1946 and its later variants. Albumin has some unique properties that allow relatively simple and effective purifcation by precipitaiton methods. It has the highest solubility and the lowest isoelectric point (the pH at which it bears no net charge). Adjustments to pH, temperature, ionic strenght, ethanol concentration and protein concentration thus allow the separation of albumin from the other plasma proteins (Matejtschuk, British J. of Anaesthesia, 85(6): 887-95 (2000). The processes for obtaining albumin from human plasma normally starts with fraction V (FrV) of the alcoholic fractionation of plasma according to the Cohn method. Although less frequent, other starting materials can also be used such as supernatants (S/N) of this Cohn fracitonal, such as the S/N of Fraction IV or S/N of Fraction II+III, including an additional stage of purificaiton such as IEX (Jorquera, US 2011/0137283). 

–Kistler and Nitschmann process: The Kistler and Nitschmann process uses fewer protein precipitation steps and hence less thanol that the Cohn method. With either method, an initial low ethanol precipitaiton stage removeds fibringoen fromt he source plasma. Subsquently, by raising the ethanol concentraiton to 25% at pH 6.9 ofr the Cohn method or 19% at pH 5.85 for the Kistler and Nitchmann method, the immunogloubulins are precipitated while the albumin remains in solution. Albumin is then isolated from the majority of the other plasma contaminants (mainly alpha and beta globulins) which are precipitated by furterh addition of thanol to a final ethanol concentraiton of 40%. This is carried out in two stages in the Cohn process but as a single step in the Kistler and Nitschmann method. In a final step, the albumin is itself precipitated near its isoelectric point. The precipitate paste (fraction V) can be held frozen before futher prcoessing. 

Hao (Vox Sang, 36: 313-320 (1979) diclses a two purificaiton step method for purifying human serum alumin with a higher yield than that of the conventional ethanol prcoedure. Threefold diluted plasma is placed in a water bath at –5C and pH adjusted to 5.6 using acetate buffer. After stirring for 1 hr, precooled -5C 95% ETOH is added to a final concentraiton of 42% (v/v) with stirring. After the pH becomes 5.75, the mixture was continuously sittred for at least 1 phour prior to centifugation. The pH of the peurnatant is adjusted to 4.8 using acetate buffer. After stirring for 1 hr, the mixture was allowed to age without stirring for at least 3 h prior to centrifugation. The precipitate, similar to Cohn fraction V was albumin paste to be used for furtehr processing, whereas the supernatant, which is analogous to Cohn fraction V supernatant could be used for preparing alpha-1-acid glycoprotein as a by product. 

Procedures involving Ion Exchange

IEX is widely used for albumin production. Among them AEX has the most usage. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

–Plasma preparation -AEX:

Curling, Vox Ang, 33, 97-107 (1977) discloses algumin that is obtainable from human blood plasma by IEX in a yeild of about 95% and purity well above Pharmacopoeia requirements. Cryosupernatant, factor IX depleted plasma is preciptiated with 12-25 w/v polyethylene glycol 4000. The second precipitate is dissolved to 8% w/v protein and applied to a DEAE-Sephadex A-50 or a DEAE-Sepharose CL-6B column. Albumin is futher purified by chromatography on SP-Sephadex C-50. 

–Cohn II+III — Remove lipoprotein (e.g., SiO2) — CEX or AEX:

Fisher (US 4,228,154) discloses subjecting a cryosupernatant plasma (Cohn Rfaction II+III or equilvanet) to finely divided lipoprotein extractant, adjusting pH to 4.5-4.9 and contacting with CEX to remov albumin contaminating proteinaceous material.  Alternatively, pH is adjusted to about 5.1-5.5 and contacted with AEX to remove the proteinaceous material having and isoelectric point aove that of albumin. 

Multi-column Chromatography

–IGselect (4 columns)-HIC

Bataille (US 15/125483, published as US 2017/0073396) discloses using a an IgSelect gel from GE Healthcare (ligand si from CAC, BioAffinityCompany) which specifically binds the Fc fragments of human IgGs and  Capture Select FcXL affinity gel from Life Technologies which specifically binds the CH3 domain of the 4 human IgG subclasses on 4 columns, controlled sequentially an an automated system followed by diafiltration concentraiton of the eluate. Elution with the IgSelect was carreid out with 0.1 M glycine solution, pH 3. The IgG depleted plasma is collected (albumin not being bound by the affinity gel) and a salt tolerant mixedmode gel HEA Hypercell is used to capture albumin. 

–Simulated moving bed chromatography (SMB):

This is a multi-column method based on reversed-phase chromatography, in which the sorbent and solvent consumption is decreased. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Expanded bed adsoprtion chromatography:

Expanded bed absorption uses high-density modified agarose/tungsten carbide beads has been used to purify alumin. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Affinity chromatography

There are different types of affinity ligands such as prtoein, enzyme, antibody, antigen, hormone, dyes (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Removal of Albumin associated proteins and metabolites: 

As the principal transporting molcule in the blood, serum albumin has specific binding sites for lipophilic substances such as fatty acids, bilrubin, etc. The majority of albumin ligands bind to one of the two binding sties I or II. Free fatty acids, metallic ions such as copper and bilirubin bind selectively to specific binding domains. (Jorquera, US 2011/0137283). 

–Removal of Lipids: Serum albumin preparations contain variable amounts of lipid impurity. Chen describes treatment of such samples with charcoal at low pH resulted in virtually complete removal of fatty acids (Cehn, J. Biological Chemistry, 212(2), 1967, pp. 178-181). Such impurities present in commerical albumin preparations are known to influence in vitro drug binding parameters. Nakano (analytical Biochemistry 129, 64-71 (1983)) describe activated carbon beads embedded in agarose to produce column beads effective for removal of long chain fatty acids from human serum albumin. 

–Removal of Billirubin: Bilirubin which is a metabolite also has high affinity for serum albumin. The formation of this albumin-bilirubin complex is of great biological importance since a free bilirubin fraction suppresses several major enzymes and metabolic systems of the brain, causing severe toxicity. Nikolaev (International J. Artificial Organs, 14(3), 1991, pp. 179-185) discloses that high activated carbons can be promising not only for plasma adsorption but also for direct contact with whole blood (i.e., hemocarboperfusion). 

Christensen (WO/2007/063129) discloses a large scale process for the isolation of recombinant human serum albumin (rHSA) by contacting a protein solution comprising the rHSA to an adsorbent having a functionalized matrix polymer carrying a plurality of covlantly attached functional groups comprising an aromatic or heteroaromatic ring system and one or more acidic groups or the adsorbent comprises a particle with at least one high density non-porous core, surround by a porous material, optionally washing the adsorbent and obtaining the rHSA from the adsorbent.

Most plasma proteins have their isoelectric point below pH7 so that their solubility will decrease with acidification.

Major Human Plasma Proteins (67% of total plasma protein mass)

Albumin: serves important functions in the maintenance of the blood plasma system and is an essential component of regulation due to its functions of transporting physiological substances and binding toxic ones. It is the main regulator of the colloid-osmatic pressure of plasma. Albumin constitutes about 4% of the total plasma proteins. Huan serum albumin (HSA) is administered to patients in various clinical situations including shock or burn patients for restoring normal blood volume and thereby alleviating certain trauma associated symptoms. 

The traditional method for the purification of albumin for therapeutic use has been cold ethanol fracitonation, as described by Cohn in 1946 and its later variants. Albumin has some unique properties that allow relatively simple and effective purifcation by precipitaiton methods. It has the highest solubility and the lowest isoelectric point (the pH at which it bears no net charge). Adjustments to pH, temperature, ionic strenght, ethanol concentration and protein concentration thus allow the separation of albumin from the other plasma proteins (Matejtschuk, British J. of Anaesthesia, 85(6): 887-95 (2000). The processes for obtaining albumin from human plasma normally starts with fraction V (FrV) of the alcoholic fractionation of plasma according to the Cohn method. Although less frequent, other starting materials can also be used such as supernatants (S/N) of this Cohn fracitonal, such as the S/N of Fraction IV or S/N of Fraction II+III, including an additional stage of purificaiton such as IEX (Jorquera, US 2011/0137283). 

–Removal of Albumin associated proteins and metabolites: 

As the principal transporting molcule in the blood, serum albumin has specific binding sites for lipophilic substances such as fatty acids, bilrubin, etc. The majority of albumin ligands bind to one of the two binding sties I or II. Free fatty acids, metallic ions such as copper and bilirubin bind selectively to specific binding domains. (Jorquera, US 2011/0137283). 

     –Removal of Lipids: Serum albumin preparations contain variable amounts of lipid impurity. Chen describes treatment of such samples with charcoal at low pH resulted in virtually complete removal of fatty acids (Cehn, J. Biological Chemistry, 212(2), 1967, pp. 178-181). Such impurities present in commerical albumin preparations are known to influence in vitro drug binding parameters. Nakano (analytical Biochemistry 129, 64-71 (1983)) describe activated carbon beads embedded in agarose to produce column beads effective for removal of long chain fatty acids from human serum albumin. 

–Removal of Billirubin: Bilirubin which is a metabolite also has high affinity for serum albumin. The formation of this albumin-bilirubin complex is of great biological importance since a free bilirubin fraction suppresses several major enzymes and metabolic systems of the brain, causing severe toxicity. Nikolaev (International J. Artificial Organs, 14(3), 1991, pp. 179-185) discloses that high activated carbons can be promising not only for plasma adsorption but also for direct contact with whole blood (i.e., hemocarboperfusion). 

Christensen (WO/2007/063129) discloses a large scale process for the isolation of recombinant human serum albumin (rHSA) by contacting a protein solution comprising the rHSA to an adsorbent having a functionalized matrix polymer carrying a plurality of covlantly attached functional groups comprising an aromatic or heteroaromatic ring system and one or more acidic groups or the adsorbent comprises a particle with at least one high density non-porous core, surround by a porous material, optionally washing the adsorbent and obtaining the rHSA from the adsorbent.

Globulins: 

Globulins are a class of proteins.

Alpha globulins: are a group of globular proteins in plasma such as alpha-antitrypsin and alpha 1-lipoprotein. 

Beta-globulins: are a group of globular proteins which include proteins like properdin, transferrin, angiostatins. 

Gamma-globulins (AKA “immune serum globulin”, “IgG” and “immunoglobulin G”): Among the globulins are gamma globulins, a group of plasma globulins which have sites of antibody activity. The gama globulins are also known as immunoglobulins, of which immunogllobulin G (IgG) is a major constituent  (Hou, US 4,639,513).

      –IgA: IgA is has been prepared from fraction III by a variety of techniques, the most suitable being the extraction of the paste with distilled water, precipitation of the bulk of proteins with caprylic acid and batch adsorption of IgA on DEAE-cellulose at pH 5.7 ().015M acetate buffer), elution being acheived with 0.9 M acetate buffer. The purified protein contains no IgM, but some IgG (about 2 %). Pejaurdier, Vox Sang, 23, 165-175 (1972). 

Fibrinogen: 

Fibrinogen is the main component of the blood clotting system and represents about 4% of the total plasma proteins. Fibrinogen and prothrombin complex concentrates are used for congential and acquired deficiency of individual factors.

Fibrinogen is a long, slender, thread-shaped protein having a lenght of about 60 nm, which is polymerized upon bleeding and this is useful for hemostasis. It differs significantly from say an  monoclonal antiboy that has a spherical shape having a diameter of about 15 m and which has physicochemical properties (e.g., isoelectric point and hydrophilicity) that differs significanlty form fibrinogen. (Hongo, US 13/260419). 

Fibrinogen has a MW of 340,000 and isoelectric point 5.5. In Cohn fractionation, fibrinogen is precipitated out at 8% ethanol, 10.14 ionic strenght, pH 7.2 at -3C. (Hou, US 4,639,513). 

Typically, proteins such as fibrinogen are removed by an initial precipitation step (Fraction I precipitation) performed at high pH (7.2) and low ethanol cocentration (8-10% v/v). However, Bruckschwaiger (US13/776448) discloses an initial purificaiton step that copresicipates IgG and A1PI followed by a solubization step of this precipitate which leaves fibrogen in the insoluble portion and the immunoglobulins in the soluble portion. As compared to a Cohn Fraction II+III precipitate or Kistler-Nitschman Precipitate A, the initial precipitate formed provides substantially higher levels of fibrinogen. Nearly 100% of the fibrinogen content of a starting Cohn pool is co-precipitated with immunoglobulins in the intial low pH (pH 5-6), high alcohol (20-30%) precipitation reaction. This is in contrast to the Cohn-Oncley and Kistle-Nitschman purificaiton schemes where the bulk of the fibrinogen is removed in an anitial low alcohol precipitation step (Fraction I precipitation).

Bataille (US 15/125483, published as US 2017/0073396) discloses using a an IgSelect gel from GE Healthcare (ligand si from CAC, BioAffinityCompany) which specifically binds the Fc fragments of human IgGs and  Capture Select FcXL affinity gel from Life Technologies which specifically binds the CH3 domain of the 4 human IgG subclasses on 4 columns, controlled sequentially an an automated system followed by diafiltration concentraiton of the eluate. Elution with the IgSelect was carreid out with 0.1 M glycine solution, pH 3. The IgG depleted plasma is collected (albumin not being bound by the affinity gel) and a salt tolerant mixedmode gel HEA Hypercell is used to capture albumin. The IgG and albumin depleted plasma collected for purifying albumin is then used for purfying fibrinogen after having been frzone and thawed using a CaptureSelect Fibrinogen affinity gel. 

salt precipitation

Buettner (US 15/770,111, published as US 2018/0305401) discloses a method for proudcing a composition having a high concentraiton of fibrinogen (Fg) from whole palsma by salt precipitation. First, the termpature of pooed plasma is dropped to 20C and Potassium acetate is added to a concentration  of 1.31M. The precipitation is allowed to compelte by stirring the suspension and the suspension is centrifuged. The clairifed solution is decanted (it can be used separated for harvesting proteins like IgG) and the past harvested. The past is disolved , enveloped viruses are denatured by a S/D treatment. Plasminogen is displaced off the Fg by addition of episolon-amine caproic acid, the solution temperature is dropped to 1C and then Potassium aceitate is added for a second Fg precipitation. The past from the second KAcet prepication is harvested form the centrfuge and is dissolved in a Tris/NaCl/Citrate/His buffer. A stabilizer can be added. 

–-salt + Silica:

(Kotitschke (US 4272523) discloses  a method for making figrinogen, a prothrombin complex containing the coagulation factors II, VII, IX and X that can contain antithrombin III, antithrombin III and a solution of stable serum proteins form blood plasma stabilized with citrate by adsorption of the fibrogen on colloidal silica of a specific surface of 50-400 m2/g and a concentraiton of 40-400 mg per g plasma protein. The silica and absorbed fibrinogen is separated form the residual protein sulution. The residual protein solution is subjected to UF or dialysis to remove citrate and calcium ions and then contacted with an adsorbent for the prothrombin complex and antighrombin III and sseparating the absorbent with material adsorbed thereon form the residual solution of stable serum proteins. 

Lipids: 

Human serum or plasma contains considerable amounts of lipids. Most of the fatty acid molecules are bound to albumin while the other lipids are combined with other proteins in complexes called lipoproteins. This combination promotes solubility of the lipids in an aqeous mdium. Lipids can be removed by solubilzing in ethanol combined with salt precipitation in Cohn fractionation (Hou, 4,639,513). 

Other Plasma Proteins 

The remaining 33% of total plasma protein mass consists of well over a hundred different minor protien species, most of which are known or suspected to be carriers of important biological activities. Many are enzymes or other biological products playing a role in the process of coagulation. The isolation and characterization of all thes minor components has proven to be a task of awesome complexity (Schultz, H.E. “Molecular Biology of Human Proteins” Volume 1: Nature and Metabolism of Extracellular Proteins. 1966, Elsevier Publishing Company, pp. 236-317.

Alpha2-macroglobulin: is important in a variety of enzymatic processes as an inhiibtor against plasmin, against thrombin and against kallikrein activity. In addition, it has been shown to possess radioprotective activity which might be utilized to increase the resistance of patients against damaging levels of radiation exposure during, for example, cancer therapy. (Wickerhauser, Vox Sang, 23: 119-125 (1972).

Antihemophilic factor (AHF): Cryoprecipitation as described by Pool (Nature, 203, 312 (1964) has been a principlal method for the isolation and partial purificaiton of AHF. In order to obtain more highly purified concentrates of AHF, the cryoprecipitation procedures have been combined with fractionation methods that employ various chemical agents such as amonium sulfate, PEG (US3,631,018). Various patents described increasing the yield of AHF from cyroprecipitate. For example, Shambrom describes that fibrinogen and its denatured products which are the primary contaminating proteins of cryoprecipitation are thermoreactive and can be selectively7 thermally removed from the cryoprecipitate without the substantial loss of AHF (US4,305,871).

Carrier proteins: including proteins which transport metal ions such as the iron-binding protein, transferrin and the copper binding protein ceruloplasmin. Prealbumin and the throxin-binding globulin transport the thyroid hormone and transcortin transports the steroid hormones. Hemoglobin is eliminated form the circulation by haptoglobin, and heme is bound to hemopexin. The retinol-binding globulin binds vitamin A. The transcobalamins I, II, and III bind vitamin B12. Gc-globulin binds vitamins D2 and D3.

Clotting or Coagulation Factors: are proteins that cause the clotting of blood to stop bleeding. Blood clots are formed by an enzymatic cascade, with the activated form of one factor catalyzing the activaiton of the next factor which results in a large amplification. Examples of inactivated and activated clotting factors include prothrombin and thrombin, fibrinogen and fibrin. Individuals with hemophilia have genetic abnormalities in which the blood fails to clot normally. This condition has been treated by adminsitering concentrates of the missing or defective proteins. The basic methods for preparing clotting factor concentrates typically involved a cryoprecipitation step whiere plasma is frozen and then thawed. During the freezing process certain proteins precipitate to form a cryoprecipitate. Various additives such as ethanol and/or polyethylene glycol are often added to enhance the efficiency of the cryoprecipitation step. Following cryoprecipitation cryoprecipitation, the partially purified factors may be further purified by additional precipitation steps or by chromatographic methods and even using monoclonal antibodies (US 3,560,475, 3631,018, 3682881, 4069216, 4305871, 5770704, US 2003/012967A1).

–Factor VIII (Blood coagulation factor VIII; factor VIII:3, antihemophilic factor (AHF) or antihemophilic globulin (AHG) or hemophilic factor A or platelet cofactor or thromboplastinogen or thrombocytolysin): is a blood plasma protein that is defective or absent in Hemophilia A disease. The disease is a hereditary bleeding disorder affecting about 1 in 20,000 males. Factor VIII consists of two polypeptides with light chain MW of 80k daltons and a heavy chain MW variable from 90 to 220k. It is considered as one of the key cofactors in the coagulation pathway necessary for the conversion of Factor X into its active form Factor Xa. Factor VIII ciruclates in plasma as a non-covalent complex with von Willebrand Factor (also known as FVIII:RP).

Traditionally, osolatuion and purification of Factor VIII has been from a plasma derived source (cryoprecipitate). Purification proecdures from plasma servie sources include those exploring the use of immunoaffinity purification using polyclonal and monoclonal antibodies. However, Ljungqvist (US6,602,977) discloses using modified polypeptide derivative of the B or Z domain of staphylococcal protein A (SPA) which results in the ability of said domain to interact with at least one domain of a human Facotr VIII protein. 

Bang (WO2009/007451) discloses purifying Factor VIII in a single step chromatographic process using a mixed mode resin containing ligands which comprise a hydrophobic part and a negatively charge part such as Capto MMC. In one embodiment, an elution buffer containing at least 1.5M salt and at least 40% (w/v) of ehtylene glycol, propylene glycol or a mixture thereof and calcium ions is used.

Borgvall (WO2009/156430) also discloses purifying FVIII using a multimodal resin such as Capto MMC, washing, and eluting with a buffer that contains at least one amino acid which is positivley charged at pH 6 to 8 such as arginine lysin and/or histidine. Additionally, the elution buffer can contain at least one hydroxyl group containing organic comound such as an alcohol selected from methanol, propanol and ethylene glycol.

In general, factor VIII is isolated in the form of a complex, the complex being formed with von Willebrand Factor (vWF). One method of purification of factor VIII is adsorption onto and elution from a separation medium having an affinity for factor VIII such as an inert substrate such as an agarose (e.g., sepharose, carrying functional groupings having an affinity for factor VIII, notably aminoalkylgroups (WO 90/14886). 

–Factor VII (FVII): is an important protein in the blood coagulation cascade, is a vitamin K dependent plasma protein synthesized in the liver and secreted into the blood as a single chain glycoprotein with a MW of 53 kDa. The FVII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site resulting in two chains linked by a single disulfide bride. Recombinant human FVIIa is commercially available from Novo Nordisk and is used for the treatment of bleeding episodes (e.g., hemophilia or trauma).

Purification of recombinant Factor VII (rFVII) or recombiantn activated Factor VII (rFVIIa) is geenrally carried out using a combination of ion exchange and immuno-affinity chromatography based on Ca2+ dependent recognition of the Gla region of FVII. However, Jensen (US2009/0047723) disclose a method for purifying rFVII or rFVIIa comprising subjecting the protein to liquid chromatogrpahy on a hydroxyapatite (HAP) column).  

Rock (US4,289,691) dicloses a method of obtaining Factor VII includes adding heparin to blood plasma collected into a calcium chelating anticoagulant or collecting blood plasma by plasma pheresis using heaprin and a calcium chelating anticoagulant, freezing the plasma, resolubilizing the plasma, isolating a cryoprecipitate from the plasma, resolubilizing the cryoprecipitate, adding a citrate slaid heparin buffer to the resoluilized cryoprecipitate. 

Factor IX concentration (Factor II, VII,  IX and X): Mitra (US4,404,132) discloses methods for isolating blood coaglation complexes comprising Factor IX concentrates by applying a blood plasma fraction containing thesse coagulation factors to an anion (basic ion) exchange to absorb the coagulation Factors, washing with a non-vaolatile sale and citrate ions having an ionic strenght sufficinet to remove less strongly bound blood plasma proteins and then treating with a non-vaoltile salt and citrate ion having an ionic strengh sufficinet to elue the absorbed coagulation factors. The eluate is then treated to reduce its water content and non-valitile sale as by a combination of ultrafiltration and diafiltration.

Cold-insoluble globulin (CIg or fibronectin): is an opsonic plasma factor now identified as alpha2-surface binding globulin.

Complement components: include more than 15 proteins including c1, c1q. 

Enzymes/pro-eznymes or enzyme inhibitors: Enzymes such as proteinases include cholinesterase, ceruloplasmin, plasminogen, protein C. Pro-enzymes (i.e., zymogens) are converted to enzymes by the action of specific enzymes. Protease inhibitors: include alpha-1 antitrypsin and the antithrombins such as antithrombin III which prvent the effects of thrombin. Another proteinase inhibitor is C1-esterase inhibitor which reduces or eliminates the activity of C1-esterase, which is the activated first ocmponent of complement, C1.

–Inter-alpha-Inhibitor Proteins (IaIp):  see outline

—-Single initial precipitation step (Fraction I+II+III+IV-1 preciptiation or Fraction I-IV-1 precipitation or initial low pH, high alcohol precipitation):

Bruckschwaiger (US13/776448) discloses an initial purificaiton step that copresicipates IgG and A1PI followed by a solubization step of this precipitate which leaves inter-alpha-trypsin inhibitor (IalIp) in the insoluble portion and the immunoglobulins in the soluble portion. The method includes the steps of precipitating immunoglobulins, A1PI, Factor H and IaIp in a first precipitation step by adding ethanol to a Cohn pool to 20-30% at a pH 5-6 to form a first precipitate and first supernatant, separating the first precipitate (containing the immunoglobulins, A1PI, IaIp, albumin, Factor H) from the supernatant. Next the first precipitate is suspended to form a first supension which is then treated as with silicon dioxide. The soluble porition of the supsnsion contains immunoglobulins while the insoluble porition contains A1pI, fibrinogen, Factor H and IaIP.

Lipoproteins: 

Lipoproteins include 3 classases, 1. alpha1-lipoprotein, pre-beta-liporotein and beta1-lipoprotein.

Plasminogen: is contained in blood. Plasminogen is the inactive precursor of the potent proteolytic enzyme plasmin. Plasminogen may be converted to the fibrinolytic enzyme, plasmin, in many ways. Activators derived from sources extrinsic to the circulation or from circualting blood cells have been described. Activation of plasmiogen can also occur through surface mediated pathways involving only factors endogenous to human plasma. Under ordinanary conditions such as those involved in the prepraration of plasma from citrated whole blood, plasminogen is not activated in any detectable quantities since there are inhibitors in plasma which can act to bloc activation by kinases as well as inhibitors that can bloc the action of the enzyme plasmin. However, on long term storage of plasma there is some activation of plasminogen to plasmin and the subsequent proteolysic of fibrinogen with development of toxid side products as well as proteolysis of immunoglobulins (Cohn, 3,998,946) (see also prepration of IVIG and silicon dioxide treatment to remove plasminogen).

Transferrin (Tf): is a glycoprotein (79 kDa) that is primarily involved in iron transport. It is relatively abundant in blood plasma (2.0-3.0 g/l) Teach lobe of the protein contains a metal-binding site enabling a maxium of two iron molecules to be transported by a single Tf molecule. Specific interactions with Tf receptors expressed on cell surfaces ensure that Tf delivers iron to growing cells. This transport role is important, because free iron readily catalyses oxidation reactions, leading to tissue damage. Additionally, free iron promotes pathogen growth. (McCann, Biotechnol. Appl. Biocehm (2005) 42, 211-217). 

Purification methods of Tf in blood plasma include thanol precipitation, ammonium sulphage precipitation and IEX. (McCann, Biotechnol. Appl. Biocehm (2005) 42, 211-217).

Von willebrand factor Fraction: The von Willebrand factor is a multimetric blood protein with molecular weights within about 200 kDa and of about 20,000 kDa and even more. This protein, that is synthetised by blood platelets and endothelial cells, plays a key role in the struggle against bleeding insofar as the FvW acts as a gelifying plug which spreads over a vascular breach providing for the adhesion of platelets in order to carry out the first phase of the hemostasis, namely the formation of “platelet-thrombus) (thrombus). FvW are used for the treatment of von Willebrand disease. Various processes for the preparation of FvW concentrates are typically associating steps of precipitation of a plasma fraction to remove the undersirable proteins (fibrinogen, fibronectin, etc) and or chromatography aiming at obtaining very high purity concentrates. Martel (US 2006/0036081) discloses a process for the preparation of vWf with anion exchange using a vinyl polymer support of weak base type.

Condie, (US 3,998,946) teaches treating blood plasma products with fumed colloidal silica to remove fibrinogen and plasminogen. According to the procedure, blood is centrifuged to separate out the precipitate, the supernantant (plasma) is then centrifuged again and then the supernatant is treated with dry silicon dioxide to separate out the precipitate. The procedure offers the advantage over the classic Cohn alcohol fractionation procedure where plasminogen is concentrated and freed of its inhibitors resulting in greater quantities of plasminogen than are present initially in plasma which can lead to the degradation of fibrinogen to form toxic split products and can also lead to degradation of immunoglobulins (column 2, lines 44-54). Condi (4,296,027, issued 10/20/1981) also teaches plasma stabilization by treatment with fumed colloidal silica.

Condi (US 4136.094) also teaches isolation of IgG for intravenous administration and albumin from plasma by initial stabilization of plasma by treatment with silica. IgG and albumin are then isolated form the stabilized plasma by chromatography.

Bertolini (US 6,093,324) teaches a method for the purification of immunoglobulins from plasma which includes a pretreatment step with finely divided silicon dioxide in order to remove lipoproteins (column 3, lines 49-65). Bertolini teaches that Fraction II+III is already lipoprotein depleted at this point which would obviate the need for application of silicon dioxide and that such a starting material can thus simply be passed through the anion exchange resin (column 6, lines 1-8).

Hirao (US 6159,471) discloses an immunoglobulin preparation formed by treating an immunoglobulin containing fraction with a low concentraiton of PREG, (b) concentrating at acidic pH, (c) anion exchange (e) filtration, (e) treatment with colloidal silica such as silica gel, light silicic anhydride, diatomaceous earth, acid clay, bentonite, kaolin and magnesium silicate aluminate which reduces the amount of serum albumin.

Radowitz (US 4,216,205, IDS Ref. of 10/16/2012) teaches that it was known for the removal of lipoproteins to apply colloidal silicic acid to the serum.

ETOH and Silicon Dioxide

Stephan (US 4,318,902) teaches preparation of an immunoglobulin solution containing IgM comprising treating an IgM containing fraction obtained by conventional fractionation from blood plasma. Advantageously, the IgM containing protein fraction is freed of lipids by treatment with colloidal silica gel (abstract). Stephan exemplifies application of the on Cohn fraction III of human plasma to free lipids (see example 2 of Stephan) (see Buchacher, Biotechnol. J. 2006, 1, 148-163 for a scheme of plasma fractionation according to Oncley’s separation method 9).

Teschner (US2010/0330071) teaches a method for preparing concentrated IgG from plasma by (1) separating liuqid and precipitate from plasma by centrifugation; (2) mixing pre-cooled ethanol with the liquid from (1) to form a mixture, (3) separating liquid and cprecipitate from the mixture of (2) by centrifugation; (4) adjusting pH and ethaol concentration of the liquid from (3) to about 7.0 and 20-25% respectively, thereboy forming a mixture, (5) separating liquid and cprecipitate form the mixture of (4) by centrifugation; (5) resuspending the precipitate of (5) with a buffer, (7) mixing silicon dioxide (SiO2) with the suspension from (6) and obtaining a filtrate by filtration, (8) mixing a detergent and cold alcohol with the filtrate of (7) and obtaing a precipitate by centrifugation, (9) dissovling the precipitate in an aqueous solution comprising a solvent or detergent and maintaining the solution for at least 60 minutes, (10) passing the solution after (9) through a cation exchange column and eluting proteins absorbed on the column in an eluate, (11) passing the eluate form (1) through an anion exchange chromatography column to generate an effluent, (12) passing the effluent through a nanofilter to generate a nanofiltrate, (13) passing the nanofiltrate through an ultrafiltration membrane to generate an ultrafiltrate, (14) diafiltrating the ultrafiltrat.

Teschner also teaches a methdo for reducing the amount of a serine proteasesuch as Factor IIa, Factor XIIa, Factor XI, Factor XII  in a plasma derived target protein (e.g., Factor H, IaI, composition by (a) enriching a plasma derived target protein in a first target protein enricnemnt step comprising alcohol fractionation of a Cohn pool, thereby forming a first enriched plasma derived target protein composition (b) contacting the first enriched plasma derived target protein with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease, the conditions comprising a conductivity of from 0.1 mS/cm to 3.0 mS/cm and a PH 4-7.

Silicon Dioxide + Salts

Zurlo (US 17/225,934, published as US 2021/0317163) discloses adding silica granules or poweder to a blood product with an organic salt such as citrate or acetate salt, resulting in precipitate and supernatant fractions. The supernatant fraction can be applied directly to a chromatogrpahic separation step. In a separate embodiment, the blood product is contacted with an organic salt at a first concentration which is sufficient to precipitate non-protein of interest for example from about 5-10% by weight to product a first precipitate which can include the non-prtoein of interest and a first supernatant followed by adding a finely divided silica (e.g., fumed silica) and an additional amount of the organic salt to the frist supernatant to procide a second concentration of the organic salt. This generates a second preciptiate which includes protein of interest such as IgG and the finely divided silica and a second sueprnatant which can include non-protein of intrest. The second preciptiate is resuspended and dissolved which can be applied to a separation column and the IgG containing fraction is collected. 

For inactivation of coagulation factors: 

Kaar (US 2018/0118812) discloses that contacting a solution such as a Fraction I+II+III from plasma fractionation or comparable fractions containing a complex mixture of coagulation factors such as factors VII, FVII, FVIIa, FIX, FIXa, FX, FXI and FXIa with an organic acid or its salt, in particular the sodium salt, inactivated or removed said coagulation factors . It was additionally observed that factor XI-antigen values were reduced to below 5 mlU/mg IgG, when the solution was contacted with caprylic acid  ot its salt. A filtraiton aid such as silicates such as diatomaceous earth, fumed silica, perlites or zeolithes may optionally be present during the stirring and removed together with devleping precipitate afte the contacting. The supernatant can be further processed as by anion chromatogrpahy. 

Human blood plasma is conveniently fractionated by the cold ethanol precipitation method. For this method the plasma is treated with ethanol at below 5C and by increasing the concentration of ethanol and reducing the pH, a succession of fractions are precipitated. The supernatant liquor after reach precipitation is subjected to the next. It is based on the methods described by E.J. Cohn et al., J. Amer. Chem. Soc. 68, 459-475 (1946), but the original methods have been modified over the years. (EP0440509A2). The Cohn process is essnetially a batch process where the various proteins are precipitated from the solution sequentially. 

The major components (US4,540,573)

In the Cohn fractionation method, the frist fractionation step results in fraction I which comprises mainly fibrinogen and fibronectin. The sueprnatant from this step is futher processed to precipitate out fraction II+III and then fractions III and II. Typically, fraction II+III contains about 60% IgG, together with impurities such as fibrinogen, IgM and IgA. Most of these impurities are then removed in fraction III, which is considered a waste fraction. The supernatant is then treated to preciptiate out the main IgG containing fraction, fraction II, which can contain greater than 90% IgG. 

In the Kistler & Nitschmann method, fraciton I is equivalent to fraction I of the Cohn method. The next preciptiate/fraction is referred to as precipitate A (fraction A). This preciptiate is broadly equivalent, alhtough not identical to Cohn fraciton II+III. The preciptiate is then redissolved and conditions adjusted to preciptiate out preciptiate B (fraction B) which is equivalent to Cohn fraciton III. Again, this is considered to be a waste fraciton. The preciptiate B supernatant is then processed further to produced preciptiate II, which coreesponds to Cohn fraciton II. 

Plasma is cooled to about 1C and then centrifuged to seaprate a cold insoluble precipitate from a supernatant. The supernatant is further fractionated to yeild Precipitate I and Supernatant I. Preciptiate I which consists principally of fibrinogen is discarded. Supernatant I is fruther fracitoanted to yeild Supernatant II+IIII and Precipitate II+III. Superantant II+III, which is discarrded, contains alpha and beta globulin and lipids. Precipitate II+III consists principally of beta and gamma globulins and isoagglutins, but also contains prothrombin, plasminogen, cholesterol and other lipdis.  Precipitate II+III upon further fractionation yields Supernatant II+III W and Precipitate II+IIIW. The beta globulin, cholesterol and other lipids are largely removed in Supernatant II+IIIW which is discard. Precipitate II+IIIW consists principally of gamma goblulins, isoagglutins, plasminogen and prothrombind and some beta goblulin, chloesterol and other lipids. (Van Holten, EP 1247818). Upon further fractionation, Precipitate II+IIIW yeilds Supernatant III and Precipitate III. Precipitate III, which is discarded, contains isoagglutinins, plasminogen and prothrombin. Supernatant III consists principally of gamma globulins and minor amounts of fibrinogen and lipids. The final step of teh fractionation yields Precipitate II which is essentially pure gamma G globulin almost completely free of 19S globulin, plasminogen and lipides. (Van Holten, EP 1247818)

 Cohn Fraction I (AKA Fraction I Precipitation or Kistler-Nitschman Fraciton I)

First fraction precipitate (Fraction I): contains fibrinogen and fibronectin (EP0440509A2),

Fraction I is the starting material for the preparation of fibrinogen which represents 50-60% of its total protein. The isolation of purified fribrinogen can be undertaken either by using ethanol at various pH values, or with the aid of ammonium sulfate, after a preliminary treatment with barium sulfate.

To form a Fraction I precipitate, the cryopoor plasma solution is cooled to below about 6C (typically to about 1C) and the pH adjusted to about 7-75, Pre-cooled alcohol (typicaly ethanol) is then added to a concentraiton of fromb aobut 6-10% typically while stirring the solution. After completion of the precipitation reaction, the supernatant (e.g., Supernatnat I) is then separated from the precipitate (Fraction I precipitate) by centrifugation, filtration or other suitable means. (Hofbauer et al. (US 2014/0271669). 

Cohn Fractions II+III (AKA Fraction II+III Precipitation)

To form a Fraction II+III precipitate (e.g., a Cohn Fraction II_III precipitate or Kistler-Nitschmann Precipitate A), the fraction I supernatant from above is cooled to below about 0C and the pH adjsuted to about 5.5-7.0. Precooled alcohol is added to a target concentraiton of from about 20-25. At the same time the temperature is further lowered typically between -9–5. Typically, the precipitation reaction includes an incubation time of at least about 1 hour. After completion of the precipitaiton reaction, the supernatant (e.g., Supernatant II+III) is then separated form the precipitation (e.g., Fraction II+II” precipitate) by centrifugation, filtration or other suitable means. (Hofbauer et al. (US 2014/0271669). 

Cohn Fraction II+III contains premodminantly IgG, IgA and IgM and is commonly prepared accoridng to Cohn’s method 6. 

The second (Fraction II) contains gamma-globulins (EP0440509A2),. Gamma globulins are blood protteins produced by lymphocytes and plasma cells of the immune system. Almost all gamma globulins are antibodies. The three main types are IgM, IgG and IgA. 

third (Fraction III) contains beta-globulins (EP0440509A2). Beta globulins are blood proteins. There are beta-1 and beta-2 globulins. They are simliar to alpha globulins which are produced by the liver. Beta globulins have similar functions and carry lipids, hormones and cholesterol through the bloodstream. Beta globulins also assist immune cells in mounting an immune response. 

Cohn Fraction IV

fourth (fraction IV) alpha-globulins (can be recovered under different conditions to give different products as for example IV(1) and IV(4), the latter containing more transferrin and albumins) (EP0440509A2). 

globulins were described (Cohn, J. Am. Chem. Soc., 1946, 68(3):459-475); US 2,390,074, US 6893639).

Several years later, Oncley (J. Am. Chem Soc., 1949, 71(2): 541-550) expanded upon the Cohn methods by publishing a method (method 9) that resulted in further division of these major fractions into subfractions. Fraction IV can be collected as two cuts, knwon as IV(1) and IV(4), the lattter containing more transferring and albumins.

Cohn Fraction V

Fifth (Fraction V) contains albumins (this is a large volume product collected at a 40% ethanol concentration and pH5.2 and represents a commercially valuable product). Each fraction is heavily contamined by compoents of other factions and other materials (EP0440509A2). 

The first fraction is a large volume collected at a 40% ethanol concentraiton and pH 5.2 and represents a commercially valuable product.

See also Antibody precipitation under Antibody purification   See also IVIG for use of ethanol as fractionation agent

The main fractionation agent used to isolate immunoglobulins from donor plasma is ethanol fractionation based on the Cohn procedure and covered in a separate section. However, even with the Cohn procedure, it is necessary to further process the immunoglobulins due to the anti-complementary activity caused by IgG aggregates formed during the fracxtionation process. Four basic procedures to do this include 1. enzymatic degradation by plasmin or pepsin, 2. chemical modification of the IgG by beta-propiolactone or by cleavage of the interchain disulfide bridges by sulfonation or reduction and alkylation, 3. selective elimination of aggregates by precipitation with PEG ands hydroxyethyl starge (HES) or by treatment at pH with traces of pepsin and 4. adsorption of aggregates by DEAE gels such as Sephadex C50® (Hou, US 4,639,513). 

Acidic/pH Fractionation/Preciption

Low pH followed by pH shift:

Menyawi (US 14/900499 published as US 2016-0368970) disclsoes a purificaiton process from a solution comprising IgG such as paslam-derived antibodies which includes (a) providing an acidic solution of the IgG solution at pH 3.5-5.2 and a total protein concentraiton of at least 10 g/l, (b) adjusting the pH to 5.2-6.2 while minatinaing conductivity below 1.5 mS/cm, (c) incubating and (d) removing precipitate such as by depth filtration. The adjustment of pH is advantageously done using a multi-hydroxylated amine compound with or without carboxyl groups such as with Tris. In one exemplification, pH was lowered to pH 4 and then adjusted ot pH 5.8 using 1M Tris buffer. The precipitate foremd was then removed by filtraiton. 

Takeda (US2006/0142549) discloses a method for remvoing impurities from a protein/antibody containing smaple which includes low conductivity at a pH below the isoelectric point of the antibody and then removing the resulting particles. In one embodiment elution fractions from Protein A column were adjusted to pH 3.2 with hydrochlorid acid for 30 inutes and each fraction was subsequently adjusted to pH 7.2 with a 300 mmol/L Tris solution.

–Low pH – Caprylic Acid -pH shift  (for precipitation with caprylic acid see outline)

Lebing (US2002/0177693) discloses a process for the purificaiton of antibodies form human plasma whihc includes suspension at pH 3.8-4.5 followed by addition of caprylic acid and a pH shift to pH 5.0-5.2. The precipitate of contaminating proteins, lipids and caprylate forms are removed while the majority of anitobdies remain in solution. 

Caprylate acid see outline 

Polyacids:

Polyacid + Salt: 

Van Alstein (US 2014/0343253) discloses providing a sample of blood plasma and adding a polyacid and a slat cuasing a first protein precipitate and first supernatant and adding a polyacid and/or a salt to said first supernatant causing formation of a second protein precipitate and a second supernatant. Thyl cellulose. The salt is slected form sodium pohsophate, potassium phosphates, ammonium phosphates, sodium citrates, potassium citrates, ammonium citrates, sodium sulphates, potassium sulphates, ammonium sulphates, sodium acetate, potassium acetate, ammonium acetate. rene-sulfonic acid, carboxymethl-dextran and carboxymethe first protein is fibrinogen and the second protin is an immunoglobulin such as IgG. Polyacids are slected from polyacrylic acid, poly-methacrylic acid, polyvinylsulfonic acid, polysty

Ethacridine lactate: 

Ethacridine lactate is a highly aromatic cationic dye shown to recover antibodies.

Glycine: 

Levy (WO/2003/034982) discloses a method of purification of immune gloubilins from human plasma from an immune globulin source such as Cohn’s fraction using glycine extraction.

Organic solvents (other than Ethanol): 

Organic solvents include emthanol which has the disadavantage of toxicity, acetone which has not been shown to be of value as a fractionating agent for proteins, ether which has been employed in England for extraction and separation of human plasma proteins,

Metal Ions:

Zinc ions have been used in the fractionation of plasma proteins.

Polyanions: 

Nucleic acids, when used at weakly acid pH, will precipitate proteins wihtout denaturing them. Polyacrylic acid is suitable for the fractionation of human serum.

Polymers: 

Polson et al have proposed to use linear polymers of high molecular weight for the fractionation of complex protein mixtures. Among the agents used are polyetheylene glycol, dextran and polyvinylpyrrolidone. In contrast with organic solvents, the agents do not cause denaturation of the proteins when employed at ordinary room termpature, nor is the selectivity of their action influenced by changes in ionic strenght of the mediu. A disadvantage of these methods is that the PEG must be removed form the end products such as through a column of DEAE or CM cellulose.

polyethylene glycol: 

Polyols, particularly polyethylene glycol (PEG) have for many years attracted the itnerest of the blood derivative industry. Curling (1980) presented a plasma fractionation method using PEG as a precipitating agent and concluded that PEG has considerable advantage of use compared to ethanol. PEG has also been used in human plasma fractionation for the production of a crude fraction of immunoglobuilin and then treated with two stage IEX by Teshner in 2007. (Lucena, “A new methodology for polyvalent intravenous immunoglobuilin solution production with a two-stage process of vrial inactivation” Brazilian J. Pharm. Sciences, 46(4), 2010, 777-783)

The fractionation with polyethyelne glycol or pluronics is believed to be the most desirable means to recover immunoglobulin. The procedure was disclosed by Polson and Coval (JP46,814/1975, 91,321/1976 and 20,415/1978).  Lars-Gunnar (Swededn 1344340) also disclose a method of fractionating plasma proteins, by removing blood corpuscles and cell fragments form the plasma in the blood, precipitating the globulins in the plasma with polyethylene glycol of average MW 6000, centriguging out all the precipitate, dissolving the precipitate, adsorbing the globulins on a cation exhcanger and eluting, precipitating with polyetheylene glycol and absorbing the globulins on an anion exchanger.  

Numerous variations have been disclosed where improvements consistent, as for example, improved yield. For example Uemura (US4371520) described using a starting material consiting of Cohn’s plasma fractions I+II+III, II+III, II, and III obtained by precipitation with cold ethanol and subjecting the starting material with an acid and adding to the resulting mateiral an alkyl-ene oxide polymer or copolymer haivng a MW of 2k-20k. (see also US4276283, ammonium sulfate and polyethyleneglycol), (Polyethylene glycol associated to single stage precipitation by ethanol rather than cohn-Oncley process which employs cold alcohol as the precipitating agent in a 3 stage process Lucena, “A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation” Brazilian J. P.S. 2103.1, 46(4), 777-78) (Fractionation with ethanol, PEG, anion resin, ultrafiltration, US 6875848).

(Falksveden “Ion exchange and Polyethylene Glycol Precipitaiton of Immunoglobulin G” , from “methods of plasma Protein Fractionation” by J.M. Curling, Academic Press 1980) discloses precipitating the main part of the globulins at neutral pH with PEG 600 under conditions that ost of the albumin remains in solution. The precipitate is separated and the IgG fraction extracted at pH 5.8 in such a way that most of the IgM, fibrinogen and plasminogen are left as an unextracted residue. Positively charged IgG, at pH 5.8, is then bound ato a CEX. 

 –Ethanol Precipitation to Fraction II/III – PEG:

Radowitz (US 4,216,205, IDS Ref. of 10/16/2012) teaches that it was known to apply PEG on the precipitate gamma-globulin fraction II/III of a standard Cohn procedure. According to an embodiment, (1) a first preciptiation step where crude fibringoen is removed from plasma by adding ETOH up to 8%, temperature -2.5 to -3C. S1 is further prcoessed (2) in 2nd precipitation with 18-25% ETOH, temperature -5C, pH 5.8. P2 (fraction II/III or gamma-globulin fraction) is removed by centrifuging. Now, Radowitz does different fractionations (see diagrams on right hand column, 1st diagram) where to obtain immunoglobulin, the Fraction II/III P2 is resuspended and 3% of PEG 4,000 or 2.5% of PEG 6,000 are added. Upon agitation for up to 4 hours the mixture is centrifuged whereby supernatant S5 and fraction III-1 (precipitate P5) are formed. Supernatant S5 is against reacted with PEG and fractionated. Fraction III-2 (precipitate P6) and the supernatant S6 are fored. S6 contains primarily IgG.

The S2 from the above can also be further processed (3) in a 3rd precipitation step with ETOH 40%, temp -7C and pH 5.8. P3 (fraction IV) contains alpha and beta globulines), S3 is further processed (4) in 4th preciptiation step with 40% ETOH. P4 (crude albumin).  The gama-globulin fraction II/III is further process by the following steps: (1) P2 is supended in a citrate phosphate buffer at pH of from 7-7.4, temp 15C, 3% of PEG are added. Supernatant S5 and fraction III-1 )precipitate P5) are formed. S5 is against reated with PEG and fractionated. Fraction III-2 (precipitate P 6) and S6 which contains IgG are formed. The globulins of fractions III-1 and III-2 are enriched or concentrated with the immune globulins IgG, IgA and IgM. Further, alpha1-antitripsin, alpha2-haptoglobulin, coeruloplasmin, transferrin and haemopexin are present in concentrated form.

 –PEG + caprylic acid: 

Debart (US 15/276544) discloses a method for prefaring immunogobulins from an initial solution such as a fraction II+III by adding caprylic acid to a solution having a polyether or polymer of glycol such as polyetheyle glycol (PEG), incubating the solution for a time to inactivate enveloped ciruses and then performing a step of UF/DF. In some embodiments the caprylic acid is added to a concentration of 9-15 mM.  In some embodiments the UF/DF is carried out using a membrane of 100k and is carried out in two phases; a first phase in which pH is adjsuted to 5-6 or irder to reduce or eliminate most of the caprylate and a 2nd pahse in which the pH is adjsuted to less than 5 in order to reduce or eliminate most of the polyether or polymer of glycol. 

Hansen (US 4,164,495) teaches that immunoglobulins or gammaglobulins can be recovered by fractionated precipitation of blood plasma with a polycondensed di or polyol, such as PEG, in the present of a mono or polyalkanoic acid have 4-12 carbon atoms such as caprylic acid.

Lucena, (“A new methodology for polyvalent intravenous immunoglobuilin solution production with a two-stage process of vrial inactivation” Brazilian J. Pharm. Sciences, 46(4), 2010, 777-783) discloses starting with fresh frozen plasma, thawing at 4C, clarification by centrifugation at 12,000 rpm. The cryoprecipitate paste recovered is then discarded and the sueprnatant treated with PEG, pH 4.1. After 6 hours under slow homogenization, the precipitate was discrarded and the supernatant suchjected to a 2nd precipitation with PEG 12%, pH 7.4. The sueprnatant was discarded and the paste was dissolved in water and then subjected to carpylic acid as the first viral inactivation step. PEG 3% was added and the supertant subjected again to PEG 25%, pH 7.4-7.6. 

Parkkinen (WO 2005/073252) disclose a process for preparing immunoglobulin by subjected a crude immunogloublin solution to caprylic acid treatment, remove the precipitatnt and subject to the supernatant to PEG precipitation, then AEX and vinally virus removal. 

 –PEG + Ethanol: 

Lucena (“A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation” Brazilian J. of Pharmaceutical Sciences, 46(4), 2010) discloses use of PEG for crude purifciation followed by ethanol in 25% concentration to produce IgG which had 95% purity.

–Silicon dioxide: see outline

Salts: 

The oldest method employed for precipitating plasma proteins is the use of neutral salts, especially ammonium sulfate. The salting out processs is a system comprising for variables: salt concentration, protein concentration, pH and temperature. Included in the salt precipitation are large amounts of neutral salts which must be removed by dialysis (Falksveden, US3,869,436). 

Ammonium sulphate: has a long history as a useful precipitatnt for the production of crude protein fractions.

Eibl (US 4,276,283) discloses a method of preparing an intravenously adminsitrable immune globulin preparation containing antibodies in yethylene glycol. which the immune globulin containing fraction is subjected in a first purificaiton step prior to polyethylene glycol precipitation to a treamtent with an aqeuous solution of a salt of an inorganic acid, particular ammonium sulphate. Suitably, the immune globulin containig fraction is gained form human blood plasma according to the Cohn method. First, the immunoglobulin fraction is subjected to a treatment with an ammonium sulfate solution. The precipitate is separated and rejection and in a subsequent precipitaotn step a precipitate containing immune globulin is precipitated from the remaining solution by treatment with an ammonium sulfate solution.  Then the rmaining immune globulin containing solution in a secon purificaiton step is subjected to treatment with pol

Sodium citrate: 

Bruckschwaiger (WO 2013/126904) discloses an initial low pH, high alcohol precipitation of blood plasma for production of IVIG and A1PI. However, they also disclose that that the precipitation can be carried out by salting out using ammonium phosphate, ammonium sulfate or sodium citrate. Next, the first precipitate is suspending to form a first suspension, removing the A1Pi from the suspension and recovering the soluble fraction thereby forming an enriched immunoglobulin composition. 

Zurlo (US 2007/0049733) discloses large scale alcohol free plasma fractionation which produces human IgG product. The process employs sodium citrate in two initial fractionation steps, followed by diafiltration to remove the sodium citrate. For the first fractionation, sodium citrate is added to plasma by gentle stiring such that the supernatant contains virtually of the IgG. For the second fractionation, additional soidum citrate is added to the supernatant. The resulting supernatant contains virtually no IgG and virtually all the IG and other serum proteins are now found in the paste. Next remal of sodium citrate is done by diafiltration, viral inactivation is accomplished such as by solvent/detergent (S/D). Finally purificaiton is done by chromatographic purificaiton techniques.

Zurlo (US 2019/0055282) also discloses methods in which one can produce a protein product from a blood based material which includes the steps of adding a salt to the material to produce a first sueprnatant and a first past, adding a salt at higher concentration to the first supernatant to rpodcue a second sueprnatant and second paste and subjecting the second supernatant to a suitable chromatogrpahy method such as ion exchange. With respect to the salt added to the blood based material, a side range of salts can be used including for example citrates, acetates, gluconates and/or caprylates. 

Sodium chloride: was once believed to precipitate gloublins as well as fibrinogen but most of the gloublin faction actually remained in solution even at the saturation point.

Magnesium sulfate: is probably the oldest of all protein precipitatns of the neutral salt class. At full startation it precipitates only the gloublin fraction, while the albumins remain in solution.

Blood: Blood cells make up about 45% of adult blood. These cells include oxygen carrying erythrocytes (red blood cells), immune cells caled leukocytes (white blood cells) and thrombocytes (platelets). Whole blood can be serapted by centrifugation into three layers: (a) the upper plasma layer; (b) the “buffy coat” layer which contains the leukocytes and thrombocytes and (c) the lower layer which contains the erythrocytes. 

Chelators: One hazard in the fractionatation of protein mixtures  is the activation or proteolytic enzymes, whose inactive precursors may be present in the fractions. One means of conteracting such effects is the addition of small amount of chelators such as sodium citrate or ethylene-diaminetetraacetate (EDTA). EDTA also prevents the metal catalyzed oxidation of the lipid moiety of lipoproteins.

pH: Most plasma proteins have their isoelectric point below pH7 so that their solubility will decrease with acidification.

Plasma: makes up 55% of the blood (the other 45% are blood cells), is a straw coloured fluid which contains water, blood plasma proteins (including clotting factors), minerals and dissolved nutrients (such as glucose, amino acids, and fatty acids) and waste produces (such as urea and lactic acid). 

Protein concentration: Another factor to be kept under strict control is the protein concentraiton of the medium. As a rule, the resolving power can be improved by lowering the protein content of the mixture. Some proteins, however, tend to become unstable at very low concentration but this can be countered by the use of stabilizers such as glycine. (Schultz, H.E. “Molecular Biology of Human Proteins” Volume 1: Nature and Metabolism of Extracellular Proteins. 1966, Elsevier Publishing Company, pp. 236-317.

Serum: is the name given to plasma which has had the clotting factors removed. 

Siliceous materials: One technical problem in the isolation of plasma proteins is the sizeable amount of lipoprotiens (8% of the total plasma). It is of advantage to remove the lipoproteins as a preliminary step to any kind of fractionation. For this purpose extensive use is made of the absorbant power of finely idspersed siliceous materials such as glass powder, bentonite and aerosil as well as of calcium phosphate.

Temperature: Proper control of termperature is a critical factor. With fractionation systems employing ethanol, the temperature should not be allowed to rsie above 0, if irreversible denaturation is to be avoided.

Ultrafiltration/Diafiltration:

UF and DF of protein solutions are extensively performed in the course of a manufacturing prcoess for palsma prtoeins, often being sued to concentrate protein solutions or in the exchange of buffers. This may be required to prepare for a subsequent purificaiton step or to adjust protein concentraiton and salt content to the specificaitons required for formulation. Membranes with MWCO of between 10 and 11 kD are widely used. ((Bertolini, “Production of Plasma Proteins for Therapeutic Use” ISBN: 978-0-470-92431-), 512 pages. ). 

For more details on the Cohn Alcohol Fractionation Procedure with respect to purifying immunoglobulin (IVIG)  see “alcohol precipitation” right hand panel.

Common plasma fractionation processes are the Cohn fractionation method (Cohn, 1946, J Am Chem Soc. 68: 459) and its modification (e.g., Kistler and Nitschmann, 1962, Vox Sang, 7: 414-2424). This process begins with cryoprecipitation to remove some of the coagulation factors. The resultant cyroprecipitate-depleted plasma pool is treated to precipitate IgG fraction (Fraction 1 accordingly to the Kistler and Nitschmanna method at 19% ETOH, pH 5.85 and -5C or the equivlaent Fraction II+III according to the Cohn method at 25% ETOH, pH 6.9 and -5C). The remaining impurities are removed by precipitation of Fraction IV at 40% ETOH, pH 5.85 and -5C, according to the Kistler and Nitschman method or a two step process accoding to Cohn (Fr IV-1 at 18% ETOH, pH 5.2, -5C, followed by Fr IV-4 at 40% ETOH, pH 5.8, -5C). Lowering the pH of the fraction IV supernatant to 4.8 and then dropping the temperature from -5C to -10C while maintaining the ETOH concentration at 50% causes the rpeciptiation of Cohn fraciton V. 

Fractionation Procedure for Plasma Proteins

1. collection of blood and separation of plasma: The preparation of the concentrates bigins with the combining or “pooling” of individual plasma donations. Following the collection, the blood is centrifuged to cause the blood cells to settle and separate from the plasma. Interactions between blood cells and plasma proteins are a potential source of trouble during the fractionation of blood. The plasma should thus be separated form the cell as soon as possible. Optionally, platelets may be harvested at this stage by a second centrifugation. The plasma is then frozen, either by refrigeration or by immersion in a mixture of dry ice and ethanol or a similar solvent. After this, the frozen plasma is thawed and then centrifuged once again to settle solid material in the cold thawed plasma. This solid material is known as a cryoprecipitate which contains the coagulation factors FVIII and von Willebrand factor in enriched form but may also serve as a source for the recovery of fibrinogen and other plasma proteins, such as fibronectin.(Kumpalume, US 2009/0292114). As a result of the slow, controlled de-freezing procedure, a cryoprecipitate is formed comprising Factor VIII (complexed with the von willebrand factor, vWF) and fibrinogen and a supernatant comprising the builk of the proteins remains in the plasma. The cryoprecipitate fraction may be resolubilised to form a protein solution and the proteins may be isolated by contacting it with an adsorbent, In particular, Factor VIII, vWF and fibrinogen may be isolated from the resolubilised fraction. (called cryo-poor plasma). Lihme (US2007/0299251)

Subjecting plasma to controlled thawing at 2-3C is known as cryo-precipitation. The “cryosupernatant” (or “cryo-poor palsma”) may uundergo chromatographic adsorption to isolate the proteins of the prothrombin complex, antithrombin or C1-inhibitor. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

2. Fraction I precipitation (Removal of fibrionogen): The supernatant from (1) above, cryo-poor plasma may be supplemented with alchol and fraction I (a precipitate) and supernatant I are formed. Lihme (US2007/0299251)

The first precipitation step, referred to as Fraction I precipitation, is performed at high pH (7.2) and low ethanol concentration 8-10% w/v) to precipitate protein such as fibrinogen and Factor XIII away from IgG and A1PI, which remain in the supernatant.  (Bruckschwaiger US 13776448) 

After the conventional centrifugation, the plasma, which is now cryoprecipitate-poor is separated leaving behind the Factor VIII rich cryoprecipitate. The Factor VIII rich cryopricipitate is the source of an important therapeutic agent for hemophilia (i.e., AHF) and that cryoprecipitate cryo-poor plasma is also used for the preparation of the other concentrates. 

Radowitz (US4216205) dicloses removal of  fibrionogen from plasma by adding cold ethanol up to a concentration of 8%, at temperature maintained at form -2.5 to -3.0 degrees C. The precipitate (fibrinogen) is removed by centrifuging and the supernatant further processed.

3. Fraction II/III (gamma globulin fraction): Supernatant I may be supplmented with more alchol and precipitated fraction II+III and supernatant II+III are foremd. Freaction II+III may be separated and resolubilised to form a protein solution and proteins of interest may be isolated by contacting it with an adsorbent. Proteins such as immunglobuilins (subh as IgG) may be isolated form the protein solution obtained from fraction II+III. Supernatant II+III will mianly compirse albuman and alpha-1-proteinase inhibitor. Lihme (US2007/0299251)

IgG is next precipitated from the Fraction I supernatant above in a second precipitation reaction, refrrered to as a Fraction II+III precipitation, performed at moderate pH (6.8) and high ethanol concentration (20-25%). The bulk of the A1pI remains in the supernatant of the Fraction II+III precipitation reaction and is subsequently separated from albumin in a third initial precipitation reaciton, referred to as a Fraction I-IV-1 precipitation, performed at low pH (5.2) and moderate ethanol concentration (18%). (Bruckschwaiger US 13776448) 

Radowitz (US4216205) discloses further treatment of the supernatant from above with ethanol at temperature of -7 C and pH to 5.8. This fraction includes the immune globulins and other proteins. The precipitate is removed by centrifuging and the supernatant processed further.

a. Further processing of globulin fraction II/III

–PEG precipitation: For example, Radowitz (US4216205) discloses resuspending the precipitate above in a citrate -phosphate bufer at a pH of from about 7.0-7.4 at termpature held at 15C. 3% of polyethylene glycol (PEG) are added. Upon agitation and reaction time from 1/2 to 4 hours, the mixture is centrifuged and supernatant (S5) and fraction III-1 (precipitate P5) are formed. Supernatant S5 is against reated under modified conditions (pH 4.6, PEG 4k up to 5.5%, temperature 15C) and fractionated. Fraction III-2 (precipiate P6) and superantant (S6) are formed, the later containing primarily the IgG. 

4. Fraction IV: 

For example, Radowitz (US4216205) disclcoses further treating the supernatant form above with ethanol at concentration set to 40%, temerpature at -7C and pH 5.8. By centrifuging, the precipitate, Fraction IV is obtained which  contains the alpha and beta globulins. The suepernatant is further processed. 

–Fraction IV-1: The superantant II+III may be further supplemented with alcohol and forming supernatant IV-1 and precipitated fraction IV-1. Fraction IV-1 may be separated and resolubilised and form a protein solution and proteins may be isolated by contacting it with an adsorbent. In particular plasma proteins such as alpha-1-proteinase inhibitor, anti-thrombin III and Factor IX complex may be isolated form the protein solution obtained from fraction IV-1. 

–Fraction IV-4: The supernatant IV-1 may be supplemented with more alcohol to form precipitated fraction IV-4 and supernatant IV-4. Fraction Iv-4 may be separated and resolubilised to form a protein solution and the proteins of interest may be isolated by contacting it with an adsorbent. In particular Butyryleholinesterase may be isolated form the protein solution obtained from fraction IV-4. Lihme (US2007/0299251)

5. Fraction V: Supernatant VI-4 may be further supplmented with alochol to provide precipitated fraction V which may be separated and resolubilised to form a protein soluiton and proteins of interest may be isolated by contacting it with an adsorbent. In particular albumin may be isolated form the protein solution obtained from fraciton V. Lihme (US2007/0299251)

See also Precipitation of antibodies,  Fractionation of plasma and Recovery of concentrates and Types of Plasma Protein fractions 

Companies:   Takeda 

Collected human plasma may be used as a source material for the production of pharmaceutical fractionated products. This complex biologic material contains hundreds of proteins covering a myriad of physiological functions. Many components still have undiscovered roles. About 20 different plasma protein therapeutics are used for treating life threatening diseases associated to bleeding and thrombotic disorders to immuhological diseases.

The oldest method employed for precipitating plasma proteins is the use of neutral salts, especially ammonium sulfate. The salting out process is a system comprising four variables: salt concentration, protein concentration, pH and temperature. Cohn’s alcohol method is by far the most opular method, however, and was worked out in the 1940s. Ethanol is used as a precipitant and by varying the concentration of the ethanol at low ion strenght and low temperatures, the plasma proteins are subdivided into five main fractions. If ethanol is replacec by other organic solvents (e.g., ether or acetone) the possibilities are further increased of finding suitable precipitation conditions for the separation of complex protein mixtures. The advantage of using ethanol or other organic solvents inssead of neutral salts is that their volatility allows them to be easily removed by means of freeze vacuum drying.

Blood and Its Constituents

Human blood is made up of about 35% cellular components, including red cells, white cells and platelets with the resmaining 65% being a fluid called plasma. The plasma suspends the cells and platelets and comprises about 90% water, 7% protein and 3% various other organic and inorganic solutes. The protein protion of plasma consists of various different protein fractions including albumin, fibrinogen, gamma globulin, alpha and beta globulins, and others. (Chang US 4,624,780). Blood consists of suspended solid blood corpuscles in plasma. The blood corpuscles make up about 45% of the total volume while the rest, the so-called blood plasma, is constituted of about 90% water, 9% protein, 0.9% salts and lesser amounts of organic compounds.  (Lars-Gunnar, Sweden 1344340). The blood corpuslces in vertebrates are the red blood cells or erythrocytes, the white clood cells or luekocytes and the platelets which are small disc-shapped bodites. 

Cryo-poor plasma: refers to the supernatant created after the removal of cryo-precipitate formed by thawing plasma or pooled plasma at temperatures near freezing (e.g., below 10C). “Cryo-poor plasma” is the supernatant formed by cryo-precipitation of blood plasma. It is typically perforemd by thawing frozen plasma at a temperature near freezing (e.g., at a temperature below about 10C, preferably at about 6C. After thawing at low termperature, the solid cryo-precipitates are separated from the luiqd supernatant (i.e., the “croy-poor plasma”) by centrifugation or filtration. (Hofbauer, US2014/0271669)

Cohn pool: refers to the starting material used for the fractionation of a plasma sample or pool of plasma samples. Cohn pools include whole plasma, cryo-poor plasma samples and pools of cryo-poor plasma samples that may or may not have been subjected to a pre-processing step. For example, a Cohn pool might be a cryo-poor plasma sample from which one or more blood factors have been removed in a pre-processing step such as adsorption onto a solid phase (e.g., silicon dioxide or chromatographic step). 

Cryoprecipitate: refers to the precipitate obtained from the freezing and cold thawing of blood plasma and separated from the supernatant fraction of the plasma. The first step in the typical procedure for producing plasma cryoprecipitate is to centrifuge whole blood to separate the plasma from the red blood cells. Then, it is common to place the supernatant plasma into another blood bag where the plasma is rapidly freezed and then slowly thawed. When a protein solution is frozen, ice crystals form and protein molecules, which are excluded form the crystals become increasingly concentrated. Depending on the particular proteins, the proteins may actually fall out of solution (i.e., form a precipitate) if the protein more readily interacts with itself or with other proteins than with water. For example, clotting factors and other proteins form a cryoprecipitate which can be readily harvested by filtration or centrifugation. Cryoprecipitation is believed to result when the removal of water from the immediate vicinity of the protein molecules causes the proteins to preferentially associate with each other rather than the water. The process may be enhanced by using additive which “tie up” the water such as any number of hydrophilic materials such as ethanol, polyethylene glycol, heparin and various salts. The “salting out” of proteins form solution is a classical biochemical procedure. Citrate has also been used as an agent to enhance the preparation of cyroprecipitate proteins from plasma (US2003/0129167).

Cryoprecipitation: Thawing of whole plasma at +1C to +4C (Burnouf, Transfusion Medicine Reviews, 21(2), 2007, 101-117. 

Immunoglobulins (also known as “Gobulins”): are a class of proteins. Among the globulin are gamma globulins (plasma globulins which have sites of antibody activity). Immunoglobuilins are humoral glycoproteins which, in electrophoresis of plasma or serum proteins migrate with the so-called gamma-fraction and thus were formerly referred to as gamma-globulins. Immune serum globulins are classified with symbols representing the main class and pertinent polypeptide chains. gamma or Ig have been selected as suitable symbols for immune serum globulin. The sumbol is followed by a capital letter representing the main class, e.g., yG, yM or IgG, IgM.

Intravenous IgG (IVIG): refers generally to intravenous, subcutaneous or intramuscular admistration of a ocmposition of IgG immunoglublins. 

Plasma is blood minus the corpuscles. The corpuscles can easily be removed from whole blood to produce plasma by centrifugation, and the fibrinogen can be easily removed from plasma by coagulation (US 2,765,299, issued 10/2/56). Plasma comprises about 90% water. The remaining about 10% comprises a variety of substances dissolved in the water as solutes such as (1) inorgnanic ions, (2) plasma proteins, (3) organic nutrients, (4) nitrogenous waste products, (5) special products being transported and (6) dissolved gases (Hou, US 4,639,513). 

In medical diagnostics, the separation of plasma from whole blood is very important for analysing constituents present in the blood. Such analyses often take place with the aid of rapid diagnostic means such as substrates which comprise a separating matrix for separating plasma form whole blood and a test reagent. In this procedure, a drop of whole blood is applied to the substrate, lear plasma passes through the separating matrix and the blood corpuscles, such as erythrocytes and leucocytes, remaining behind in the matrix. De Rooij (US6,245,244). 

Serum is the fluid part of natural blood after it has gone through coagulation. In other words Serum = Plasma – Clotting Factor. The coagulation of plasma removes mainly the fibrinogen (US2,765,299). Serum is collected by centrifugation of whole blood samples in tubes that are free of anti-coagulant. The blood is permitted to clot prior to centrifugation. The yellowish-reddish fluid that is obtained by centrifugation is the serum. Compare the term “plasma” which is prepared by removing cells from blood without allowing clotting to occur.

Columns

Chang (US4,624,780) discloses withdrawing from a suspension tank a portion of the suspension contained therein to form a recicyle stream which consists of partciles of a selected protein fraction precipitated form a solution containing proteins. The process can incude stages in series with the supernatant from a preceding stage being sent to the next stage for recovery of a selected protein fraction remaining in the supernatant. If desired, a spray nozzle can be provided for spraying ethanol uniformly across the suspension surface. 

Nakashima (US4,384,954) discloses a column for adsorption of blood proteins having a blood inlet and blood outlet each with a filter and a porous material packed between both the filters. 

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.