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.