Plasma Proteins For Therapeutic Use; An Introduction to Blood PLASMA & its Separation Methods

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Plasma Proteins
INTRODUCTIONTO PLASMA & its
Separation Methods

JensMartensson
Blood Plasma
pazirehmaryam@gmail.com

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What is Plasma?
Blood plasma is a straw colored fluid portion of blood that normally holds the blood cells (RBC,WBC and Platelets)
in whole blood in suspension form; making up for about 55% of the body's total blood volume.
What Does Plasma Do?
Plasma is designed to carry nutrients, hormones, and proteins to the different parts of the body. It also carries away
the waste products of cell metabolism from various tissues to the organs responsible for detoxifying and/or excreting
them. In addition, plasma is the vehicle for the transport of the blood cells through the blood vessels.
The Elements of the Blood

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Separation of Blood Plasma

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Blood Plasma Composition
- Plasma Proteins: constitute about 7% (7 gr/dl) by weight of the plasma. Most of these proteins which are of many types
are synthesized in the liver.
- Organic Molecules: include glucose, fats, phospholipids, amino acids and lactic acids. Lactic acid is produced in muscles as a
result of Glycolysis, and is transported by blood to liver where it is oxidized to release energy. Most of it is converted
into glycogen. Cholesterol is an important constituent, also…
- Inorganic Molecules: Together the inorganic ions and salts make 0.9 percent of the plasma, of humans, by weight;
more than two third of this amount is sodium chloride, the ordinary table salt. Even if the total concentrations of dissolved
substances remain the same, shifts in the concentration of particular ion can create serious disturbances. They help to
regulate osmotic pressure of the blood.
- Gases (dissolved): such as O2 and CO2
- Nitrogenous Waste Products: as a result of cellular metabolism such as Urea and Uric Acid
- Hormones
- Water
91%
- Solids
9%

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Plasma Proteins
For Therapeutic
Use

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General characteristics of Plasma Proteins
 Plasma proteins are large molecules with molecular weights ranging ٠٠٠,۵٠ - ٠٠٠,٣٠٠ Daltons.
 With the notable exception of albumin, nearly all plasma proteins are Glycoproteins, containing
oligosaccharides .
 Like most other proteins, their charged residues tend to be located on the surface.
 Many plasma proteins exhibit polymorphism.
 Due to the large size of the protein molecules, they can be separated from the plasma by
ultracentrifugation (unlike electrolytes or other smaller molecules).
 They are unable to pass across the capillary membrane and consequently exert an oncotic pressure of
about ٢۵ mm Hg.

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General characteristics of Plasma Proteins
 Owing to their size and particularly their shape, they greatly contribute to blood viscosity. The plasma
protein fibrinogen is a significant contributor to blood viscosity. Due to the presence of polar residues
on their surfaces,
 The protein molecules are soluble in water.
 The molecules show electrophoretic mobility.
 The molecules are amphoteric. This is because the polar residues comprise both NH2 and COOH
groups.
 They act as efficient buffers by virtue of their amphoteric nature.
 They easily bind with metallic ions and steroids .

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Classification of Plasma Proteins (normal pattern of plasma proteins electrophoresis)
- ALBUMIN
- GLOBULIN
- α1
- α2
- β
- γ
- FIBRINOGEN
two-dimensional gel electrophoresis

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Classification of Plasma Proteins
Protein
Percentage
of total
Origin Function
Albumin 55% Liver
Helps maintain blood osmotic pressure,
Transport insoluble molecules
Globulin 38%
Alpha Globulins Liver Transport lipids & fat-soluble vitamins
Beta Globulins Liver Transport lipids & fat-soluble vitamins
Gamma Globulins Lymphatic tissues Participate in Immune system
Fibrinogen 7% Liver Essential for Blood Clotting
Regulatory Proteins <1% Liver Regulation of gene expression
Clotting Factors
<1%
Liver Conversion of fibrinogen into fibrin

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Albumin
Albumin (molecular weight of 69 kDa), single polypeptide chain having 585 amino acids with 17 disulfide
bonds, is the most abundant protein (60%) in the blood plasma. (3.5-5.0 gr/dl)
Synthesis of albumin:
- Liver produced about 12gr albumin per day
- half-life: 20 days
For this reason, measurement of serum albumin concentration is used to assays liver function test.
Albumin is used to replace blood volume loss resulting
from trauma such as a severe burns or an injury that causes blood loss.

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Albumin Functions
Albumin helps the body maintain intravascular colloid
osmotic pressure, neutralize toxins, and transport
therapeutic agents.
Transport: It can bind and transport many diverse
molecules and serve as low-specificity transport protein,
which include:
• Metal ions: such as calcium and copper.
• Free fatty acid
• Bilirubin
• Bile acid
• Hormones: such as thyroid hormones and the steroid
hormones.
• Esterase activity regulation

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Albumin Functions
Colloid osmotic pressure, is a form of osmotic pressure exerted by proteins in blood plasma that usually tends
to pull water into the circulatory system (holds water inside blood vessels and draws it from tissues);
• Because large plasma proteins cannot easily cross through the capillary walls.
• In conditions where plasma proteins are reduced, e.g. from being lost in the urine (proteinuria) or from
malnutrition, there will be a reduction in osmotic pressure, leading to enhanced fluid retention in tissue spaces
(edema)

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Gamma Globulins (Immunoglobulin)
Immunoglobulins, also known as antibodies, are glycoprotein molecules produced by plasma cells
(white blood cells).They act as a critical part of the immune response by specifically recognizing and
binding to particular antigens, such as bacteria or viruses, and aiding in their destruction.
General Functions
of Immunoglobulin
Antigen (Ag) binding:
Ig binds to a specific
antigenic determinant.
Effector functions
- Complement activation
- Binding to various cells

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Basic Structure of Immunoglobulin
- Four chains (H2L2):Y shape (4 polypeptides)
two identical light chains (L): 23 kDa
two identical heavy chains (H): 53-75 kDa
- Disulfide bonds
-Variable region (V):VL&VH
- Constant region (C): CL&CH
- Hinge region: flexibility
Light Chain:
–VL (110 aa) + CL (110 aa)
Heavy Chain:
–VH (110 aa) + CH1-CH3 (or CH4) (330-440 aa)

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Structural Regions of Immunoglobulin

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In terms of the
differences in
aminoacid sequence of
constant region of heavy
chain,
immunoglobulin
molecules are divided
into 5 classes;

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Fibrinogen
Fibrinogen (factor I) is a soluble plasma glycoprotein with a molecular weight of about 340 kDa. It is a
physiological substrate for three enzymes: plasmin, factor XIIIa and thrombin.
Fibrinogen is essential for blood clotting and produced in liver.
During tissue and vascular injury it is converted, enzymatically by thrombin, to fibrin and subsequently to
a fibrin-based blood clot.
Fibrinogen functions primarily to occlude blood vessels and thereby stop excessive bleeding.

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Blood Clotting Cascade:

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Blood Coagulation Activation:
FactorVIII (FVIII) is an essential blood clotting
protein, also known as anti-hemophilic factor
(AHF). Defects in F8 gene (factorVIII) result in
hemophilia A.This protein circulates in the bloodstream
in an inactive form, bound to another molecule
calledVon-willebrand factor, until an injury that
damages blood vessels occurs. In response to injury,
coagulation factorVIII is activated and separates from
vonWillebrand factor.The active protein (sometimes
written as coagulation factorVIIIa) interacts with
another coagulation factor called factor IX.This
interaction sets off a chain of additional chemical
reactions that form a blood clot.
Factor IX (or Christmas factor) is one of the serine
proteases of the coagulation system. It is used for
hemophilia B patients.

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Plasma Proteins
Methods of Separation

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Plasma Management

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Methods of Plasma Proteins Separation
A Mixture of Proteins Can Be Separated based on:
– solubility
– charge
– size
– specific binding activity (affinity for a ligand) and polarity
Many of the methods used in isolating molecules from cells involve some form of chromatography.

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Precipitation by Salting out
Salting out is a purification method that utilizes the reduced solubility of certain molecules in a solution
of very high ionic strength. Salting out is typically, but not limited to, the precipitation of large
biomolecules such as proteins.
 Most proteins precipitate out of solution at high
salt concentrations, an effect called salting out .
 The salt concentration at which a protein
precipitates differs from one protein to another.
 Many proteins lose their activity in the presence
of high salt.

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- At high salt concentrations, the solubility of proteins tends to decrease as water molecules become
bound to salt ions thereby allowing protein solutes to aggregate and precipitate. Centrifugation is used
to “pellet” precipitated protein.
- Different proteins precipitate at different concentrations of salt based on the inherent
polarity/hydrophobicity of the specific protein.
(a) Add a salt concentration just below the
precipitation point of your protein (green
dots). Centrifuge and remove the salted-out
contaminants (red dots in panel b)
(b) Add a salt concentration just above the
precipitation point of your protein. Centrifuge
the salted-out protein, keep the pellet, and
discard any other contaminants still in the
supernatant (grey dots in panel c).
(c) Resuspend the partially purified protein pellet
in a buffer to continue purification.

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By sodium sulphate solution
By ammonium sulphate solution
• By Howe (1922)
• Three major fractions
• Fibrinogen – removed with clot converting plasma into serum.
• Globulins – by 22% Na2SO4
• Albumin – remains in serum.
• Albumin precipitated by full saturation.
• Globulin – by half saturation.
• Fibrinogen by one fifth saturation.

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Dialysis
Dialysis is a removal of small molecules
from a solution of a macromolecule by
their diffusion through a semipermeable
membrane into a buffered solution
•Salt can be removed from the protein solution
by dialysis
•Protein molecules (red) are retained within the
dialysis bag, whereas small molecules (blue)
diffuse into the surrounding medium.

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Column Chromatography
Column chromatography allows separation of a
mixture of proteins over a solid phase (porous
matrix) using a liquid phase to mobilize the
proteins.
•Proteins with a lower affinity for the solid phase will
wash off first; proteins with higher affinity will retain on
the column longer and wash off later.

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Separation by Charge;
Ion Exchange

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Separation by Size;
Size Exclusion

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Separation by Binding;
Affinity

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Electrophoresis for Protein Analysis
• It is the most common method to separate plasma proteins.
• Proteins move in the electric field.Their relative speed depends on the charge (isoelectric point: pI),
shape, and size of the protein.
•The migration of proteins is affected by multiple factors involving their structural organization.
•Proteins can carry either a net positive, net negative, or neutral charge depending on the combination
of amino acids they contain.
•To make protein migration rates a function of molecular weight, it is necessary to impose a uniform
shape and charge on all of the proteins in the mixture. So, using detergents like SDS (Sodium dodecyl
sulphate) and PAGE (Polyacrylamide Gel Electrophoresis) will provide this purpose.

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• Using cellulose acetate electrophoresis, plasma proteins can be separated into six groups.
Albumin α1globulin α2globulin βglobulin fibrinogen γglobulin
anode cathode

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Separation in analytical scale is commonly done
by electrophoresis.
–The electric field pulls proteins according to
their charge.
–The gel matrix hinders mobility of proteins
according to their size and shape.
–The gel is commonly polyacrylamide, so
separation of proteins via electrophoresis is
often called polyacrylamide gel electrophoresis,
or PAGE.

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It separate plasma proteins into different fractions due to electric charges of each fractions.
Clinical use
• To know presence of abnormal proteins
• Absence of normal proteins
• Change in relative conc. Of different proteins
Techniques
• Free boundary electrophoresis.
• Paper electrophoresis.
• Agar gel electrophoresis.
• Cellulose strip electrophoresis.

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Ultracentrifugation
Plasma proteins in buffered solutions are
centrifuged at high speeds.
Separated due to sedimentation constant by
property of weight, shape & density.
Rate of particle sedimentation depends mainly
on its size and the applied g-force. Most
commonly used method for isolation of
intracellular organelles from tissue homogenates
because of its relative ease, convenience and
time economy.
Poor resolution and recovery because of
•Particle size heterogeneity.
•Preparations obtained are never pure.

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Ultracentrifugation
Density gradient Centrifugation carried out
in suspending medium such as sucrose or
cesium chloride having density gradients.
This involves careful layering of a thin layer of the sample solution on top of a preformed liquid
density gradient whose density continuously increases towards the bottom of the sample tube.
Components of mixture sediments/separates according to shape, size and density. Useful for
separating particles which differ in size but not in density

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Cohn Fractionation Method;
Industrial Method
The process is based on the differential precipitation of
plasma proteins by manipulation of
- ethanol concentration
- pH of a Low ionic strength concentration
- maintained at subzero temperature
• Each Cohn fraction is an enriched source of various
plasma protein need further purification to generate a
therapeutic product.
A wide range of cleaning options, from mobile,
independent cleaning systems to diverse clean-in-place
(CIP) satellites fed with conditioned cleaning solutions is
proposed.

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Cohn Fractionation Method;
EVOLUTION OF PLASMA FRACTIONATION

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Plasma Fractionation
based on Cohn’s Method

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Plasma Fractionation Processes

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Step Target Protein(s)
Cryoprecipitation FactorVIII, vWF, Fibrinogen
Chromatography Coagulation factors, Protease inhibitors,Anticoagulants
Ethanol fractionation Albumin, IgG,Alpha 1-AT
Plasma Fractionation Processes

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Cohn’s Fractional Precipitation Method;

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Cohn’s Method;
Max Plasma Protein
Yields
per Liter of Plasma
Plasma Proteins
25-28 grams
Albumin
Shock, Burns,Adult Respiratory Distress
Syndrome, Cardiopulmonary Bypass
Surgery
3.5-4 grams
 IVIG (Intravenous Immunoglobulin)
Primary Immunodeficiency Diseases,
Autoimmune Diseases, Chronic
Inflammatory Demyelinating
Polyneuropathy, Idiopathic
Thrombocytopenic Purpura
0.15 to 0.30 grams
I Antitrypsin-Alpha
Alpha-I Antitrypsin Deficiency (Genetic
COPD)
FactorVIII: 300 to 450 IUs
Factor IX: 180 to 200 IUs
Coagulation Factors
Hemophilia A & B, von Willebrand Diseases,
Bleeding Disorders

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Maryam Pazireh

Plasma Proteins For Therapeutic Use; An Introduction to Blood PLASMA & its Separation Methods

Plasma Proteins For Therapeutic Use; An Introduction to Blood PLASMA & its Separation Methods

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Plasma Proteins For Therapeutic Use; An Introduction to Blood PLASMA & its Separation Methods