Blood plasma fractionation

Last updated May 31, 2024

Blood plasma fractionation are the general processes separating the various components of blood plasma, which in turn is a component of blood obtained through blood fractionation. Plasma-derived immunoglobulins are giving a new narrative to healthcare across a wide range of autoimmune inflammatory diseases.

Blood plasma

Blood plasma is the liquid component of whole blood, and makes up approximately 55% of the total blood volume. It is composed primarily of water with small amounts of minerals, salts, ions, nutrients, and proteins in solution. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma.^{[ citation needed ]}

Plasma proteins

Plasma contains a large variety of proteins including albumin, immunoglobulins, and clotting proteins such as fibrinogen.^[1] Albumin constitutes about 60% of the total protein in plasma and is present at concentrations between 35 and 55 mg/mL.^[2] It is the main contributor to osmotic pressure of the blood and it functions as a carrier molecule for molecules with low water solubility such as lipid-soluble hormones, enzymes, fatty acids, metal ions, and pharmaceutical compounds.^[3] Albumin is structurally stable due to its seventeen disulfide bonds and unique in that it has the highest water solubility and the lowest isoelectric point (pI) of the plasma proteins. Due to the structural integrity of albumin it remains stable under conditions where most other proteins denature.^{[ citation needed ]}

Plasma proteins for clinical use

Many of the proteins in plasma have important therapeutic uses.^[1] Albumin is commonly used to replenish and maintain blood volume after traumatic injury, during surgery, and during plasma exchange.^[3] Since albumin is the most abundant protein in the plasma its use may be the most well known, but many other proteins, although present in low concentrations, can have important clinical uses.^[1] See table below.^[1]

Examples of Plasma Components for Clinical Use
Plasma Component	Reasons for Use
factor VIII	hemophilia A
factor IX	hemophilia B
Factor X	congenital deficiency
factor XIII	congenital deficiency
PCC complex	anticoagulant overdose factor II and factor X if Factor X not available deficiencies liver disease
immunoglobulin	passive prophylaxis immune deficiency disorders some types of immune thrombocytopenic purpura Guillain–Barré syndrome Polyneuropathies
antithrombin III	congenital deficiency disseminated intravascular coagulation
fibrinogen	congenital deficiency massive haemorrhage
C1 inhibitor	hereditary angioedema
albumin	hypoalbuminemia Ascites Restoring of blood volume in trauma, burns and surgery patients
alpha-I-antitrypsin	hereditary deficiencies emphysema and COPD cirrhosis

Plasma processing

When the ultimate goal of plasma processing is a purified plasma component for injection or transfusion, the plasma component must be highly pure. The first practical large-scale method of blood plasma fractionation was developed by Edwin J. Cohn during World War II. It is known as the Cohn process (or Cohn method). This process is also known as cold ethanol fractionation as it involves gradually increasing the concentration of ethanol in the solution at 5 °C and 3 °C.^[3] The Cohn Process exploits differences in properties of the various plasma proteins, specifically, the high solubility and low pI of albumin. As the ethanol concentration is increased in stages from 0% to 40% the [pH] is lowered from neutral (pH ~ 7) to about 4.8, which is near the pI of albumin.^[3] At each stage certain proteins are precipitated out of the solution and removed. The final precipitate is purified albumin. Several variations to this process exist, including an adapted method by Nitschmann and Kistler that uses fewer steps and replaces centrifugation and bulk freezing with filtration and diafiltration.^[1]^[3]

Some newer methods of albumin purification add additional purification steps to the Cohn Process and its variations, while others incorporate chromatography, with some methods being purely chromatographic.^[3] Chromatographic albumin processing as an alternative to the Cohn Process emerged in the early 1980s, however, it was not widely adopted until later due to the inadequate availability of large scale chromatography equipment.^[3] Methods incorporating chromatography generally begin with cryodepleted plasma undergoing buffer exchange via either diafiltration or buffer exchange chromatography, to prepare the plasma for following ion exchange chromatography steps.^[3] After ion exchange there are generally further chromatographic purification steps and buffer exchange.^[3]

For further information see chromatography in blood processing.

Plasma for analytical uses

In addition to the clinical uses of a variety of plasma proteins, plasma has many analytical uses. Plasma contains many biomarkers that can play a role in clinical diagnosis of diseases, and separation of plasma is a necessary step in the expansion of the human plasma proteome.^{[ citation needed ]}

Plasma in clinical diagnosis

Plasma contains an abundance of proteins many of which can be used as biomarkers, indicating the presence of certain diseases in an individual. Currently, 2D Electrophoresis is the primary method for discovery and detection of biomarkers in plasma. This involves the separation of plasma proteins on a gel by exploiting differences in their size and pI. Potential disease biomarkers may be present in plasma at very low concentrations, so, plasma samples must undergo preparation procedures for accurate results to be obtained using 2D Electrophoresis. These preparation procedures aim to remove contaminants that may interfere with detection of biomarkers, solubilize the proteins so they are able to undergo 2D Electrophoresis analysis, and prepare plasma with minimal loss of low concentration proteins, but optimal removal of high abundance proteins.^{[ citation needed ]}

The future of laboratory diagnostics are headed toward lab-on-a-chip technology, which will bring the laboratory to the point-of-care. This involves integration of all of the steps in the analytical process, from the initial removal of plasma from whole blood to the final analytical result, on a small microfluidic device. This is advantageous because it reduces turn around time, allows for the control of variables by automation, and removes the labor-intensive and sample wasting steps in current diagnostic processes.^{[ citation needed ]}

Expansion of the human plasma proteome

The human plasma proteome may contain thousands of proteins, however, identifying them presents challenges due to the wide range of concentrations present. Some low abundance proteins may be present in picogram (pg/mL) quantities, while high abundance proteins can be present in milligram (mg/mL) quantities. Many efforts to expand the human plasma proteome overcome this difficulty by coupling some type of high performance liquid chromatography (HPLC) or reverse phase liquid chromatography (RPLC) with high efficiency cation exchange chromatography and subsequent tandem mass spectrometry for protein identification.^[2]^[4]

Related Research Articles

Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.

Serum is the fluid and solvent component of blood which does not play a role in clotting. It may be defined as blood plasma without the clotting factors, or as blood with all cells and clotting factors removed. Serum contains all proteins except clotting factors, including all electrolytes, antibodies, antigens, hormones; and any exogenous substances. Serum also does not contain all the formed elements of blood, which include blood cells, white blood cells, red blood cells (erythrocytes), and platelets.

The globulins are a family of globular proteins that have higher molecular weights than albumins and are insoluble in pure water but dissolve in dilute salt solutions. Some globulins are produced in the liver, while others are made by the immune system. Globulins, albumins, and fibrinogen are the major blood proteins. The normal concentration of globulins in human blood is about 2.6-3.5 g/dL.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

Plasma proteins, sometimes referred to as blood proteins, are proteins present in blood plasma. They serve many different functions, including transport of lipids, hormones, vitamins and minerals in activity and functioning of the immune system. Other blood proteins act as enzymes, complement components, protease inhibitors or kinin precursors. Contrary to popular belief, haemoglobin is not a blood protein, as it is carried within red blood cells, rather than in the blood serum.

Snake venom is a highly toxic saliva containing zootoxins that facilitates in the immobilization and digestion of prey. This also provides defense against threats. Snake venom is injected by unique fangs during a bite, whereas some species are also able to spit venom.

Ammonium sulfate precipitation is one of the most commonly used methods for large and laboratory scale protein purification and fractionation that can be used to separate proteins by altering their solubility in the presence of a high salt concentration.

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

Bovine serum albumin is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments.

Fast protein liquid chromatography (FPLC) is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid and a porous solid. In FPLC the mobile phase is an aqueous buffer solution. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

$<span class="mw-page-title-main">Blood fractionation</span> Separation of blood into its components$

Blood fractionation is the process of fractionating whole blood, or separating it into its component parts. This is typically done by centrifuging the blood.

QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision electrophoresis technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point and by continuous elution from a gel column.

The Cohn process, developed by Edwin J. Cohn, is a series of purification steps with the purpose of extracting albumin from blood plasma. The process is based on the differential solubility of albumin and other plasma proteins based on pH, ethanol concentration, temperature, ionic strength, and protein concentration. Albumin has the highest solubility and lowest isoelectric point of all the major plasma proteins. This makes it the final product to be precipitated, or separated from its solution in a solid form. Albumin was an excellent substitute for human plasma in World War Two. When administered to wounded soldiers or other patients with blood loss, it helped expand the volume of blood and led to speedier recovery. Cohn's method was gentle enough that isolated albumin protein retained its biological activity.

Depyrogenation refers to the removal of pyrogens from solutions, most commonly from injectable pharmaceuticals.

Protein precipitation is widely used in downstream processing of biological products in order to concentrate proteins and purify them from various contaminants. For example, in the biotechnology industry protein precipitation is used to eliminate contaminants commonly contained in blood. The underlying mechanism of precipitation is to alter the solvation potential of the solvent, more specifically, by lowering the solubility of the solute by addition of a reagent.

Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.

Surface-enhanced laser desorption/ionization (SELDI) is a soft ionization method in mass spectrometry (MS) used for the analysis of protein mixtures. It is a variation of matrix-assisted laser desorption/ionization (MALDI). In MALDI, the sample is mixed with a matrix material and applied to a metal plate before irradiation by a laser, whereas in SELDI, proteins of interest in a sample become bound to a surface before MS analysis. The sample surface is a key component in the purification, desorption, and ionization of the sample. SELDI is typically used with time-of-flight (TOF) mass spectrometers and is used to detect proteins in tissue samples, blood, urine, or other clinical samples, however, SELDI technology can potentially be used in any application by simply modifying the sample surface.

Multicolumn countercurrent solvent gradient purification (MCSGP) is a form of chromatography that is used to separate or purify biomolecules from complex mixtures. It was developed at the Swiss Federal Institute of Technology Zürich by Aumann and Morbidelli. The process consists of two to six chromatographic columns which are connected to one another in such a way that as the mixture moves through the columns the compound is purified into several fractions.

Top-down proteomics is a method of protein identification that either uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry (MS/MS) analysis or other protein purification methods such as two-dimensional gel electrophoresis in conjunction with MS/MS. Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. The name is derived from the similar approach to DNA sequencing. During mass spectrometry intact proteins are typically ionized by electrospray ionization and trapped in a Fourier transform ion cyclotron resonance, quadrupole ion trap or Orbitrap mass spectrometer. Fragmentation for tandem mass spectrometry is accomplished by electron-capture dissociation or electron-transfer dissociation. Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry (MS). Top-down MS (non-gel) proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.

Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column and is then displaced by a solute that is more strongly sorbed than the components of the original mixture. The result is that the components are resolved into consecutive "rectangular" zones of highly concentrated pure substances rather than solvent-separated "peaks". It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.

References

1 2 3 4 5 Brodniewicz-Proba, T. 1991. "Human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-derived Products". Blood Reviews. Vol. 5. pp. 245–57.
1 2 Shen, Y., Jacobs, J. M., et al. 2004. "Ultra-High-Efficiency Strong Cation Exchange LC/RPLC/MS/MS for High Dynamic Range Characterization of the Human Plasma Proteome". Anal Chem. Vol. 76. pp. 1134–44.
1 2 3 4 5 6 7 8 9 Matejtschuk, P., Dash, C.H., and Gascoigne, E.W. 2000. "Production of human albumin solution: a continually developing colloid". British Journal of Anaesthesia. Vol 85. pp. 887–95.
↑ Wu, S., Choudhary, G., et al. 2003. "Evaluation of Shotgun Sequencing for Proteomic Analysis of Human Plasma Using HPLC coupled with Either Ion Trap or Fourier Transform Mass Spectrometry". Journal of Proteome Research. Vol. 2. pp. 383–93.

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[Brod-1] 1 2 3 4 5 Brodniewicz-Proba, T. 1991. "Human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-derived Products". Blood Reviews. Vol. 5. pp. 245–57.

[Shen-2] 1 2 Shen, Y., Jacobs, J. M., et al. 2004. "Ultra-High-Efficiency Strong Cation Exchange LC/RPLC/MS/MS for High Dynamic Range Characterization of the Human Plasma Proteome". Anal Chem. Vol. 76. pp. 1134–44.

[Mate-3] 1 2 3 4 5 6 7 8 9 Matejtschuk, P., Dash, C.H., and Gascoigne, E.W. 2000. "Production of human albumin solution: a continually developing colloid". British Journal of Anaesthesia. Vol 85. pp. 887–95.

[Wu-4] Wu, S., Choudhary, G., et al. 2003. "Evaluation of Shotgun Sequencing for Proteomic Analysis of Human Plasma Using HPLC coupled with Either Ion Trap or Fourier Transform Mass Spectrometry". Journal of Proteome Research. Vol. 2. pp. 383–93.

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