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.