HEPARIN SODIUM
LAUNCHED 1937
9041-08-1 NA SALT
9005-49-6 (heparin)
Thromboliquine, Calciparine, Certoparin, Dalteparin, Fraxiparin, Heparinate, Multiparin, Novoheparin, Parnaparin
Unfractionated heparin (UH) is a heterogenous preparation of anionic, sulfated glycosaminoglycan polymers with weights ranging from 3000 to 30,000 Da. It is a naturally occurring anticoagulant released from mast cells. It binds reversibly to antithrombin III (ATIII) and greatly accelerates the rate at which ATIII inactivates coagulation enzymes thrombin (factor IIa) and factor Xa. UH is different from low molecular weight heparin (LMWH) in the following ways: the average molecular weight of LMWH is about 4.5 kDa whereas it is 15 kDa for UH; UH requires continuous infusions; activated partial prothrombin time (aPTT) monitoring is required when using UH; and UH has a higher risk of bleeding and higher risk of osteoporosis in long term use. Unfractionated heparin is more specific than LMWH for thrombin. Furthermore, the effects of UH can typically be reversed by using protamine sulfate.
Unfractionated heparin is indicated for prophylaxis and treatment of venous thrombosis and its extension, prevention of post-operative deep venous thrombosis and pulmonary embolism and prevention of clotting in arterial and cardiac surgery. In cardiology, it is used to prevent embolisms in patients with atrial fibrillation and as an adjunct antithrombin therapy in patients with unstable angina and/or non-Q wave myocardial infarctions (i.e. non-ST elevated acute coronary artery syndrome) who are on platelet glycoprotein (IIb/IIIa) receptor inhibitors. Additionally, it is used to prevent clotting during dialysis and surgical procedures, maintain the patency of intravenous injection devices and prevent in vitro coagulation of blood transfusions and in blood samples drawn for laboratory values.
Indication: For anticoagulant therapy in prophylaxis and treatment of venous thrombosis and its extension, for prevention of post-operative deep venous thrombosis and pulmonary embolism and for the prevention of clotting in arterial and cardiac surgery.
Mechanism of action: The mechanism of action of heparin is antithrombin-dependent. It acts mainly by accelerating the rate of the neutralization of certain activated coagulation factors by antithrombin, but other mechanisms may also be involved. The antithrombotic effect of heparin is well correlated to the inhibition of factor Xa. Heparin interacts with antithrombin III, prothrombin and factor X.
Heparin (from Ancient Greek ηπαρ (hepar), liver), also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule.[3] It can also be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines.
Although it is used principally in medicine for anticoagulation, its true physiological role in the body remains unclear, because blood anticoagulation is achieved mostly by heparan sulfate proteoglycans derived from endothelial cells.[4] Heparin is usually stored within the secretory granules of mast cells and released only into the vasculature at sites of tissue injury. It has been proposed that, rather than anticoagulation, the main purpose of heparin is defense at such sites against invading bacteria and other foreign materials.[5] In addition, it is conserved across a number of widely different species, including some invertebrates that do not have a similar blood coagulation system.
HEPARIN
Heparin structure
Native heparin is a polymer with a molecular weight ranging from 3 to 30 kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kDa.[6] Heparin is a member of the glycosaminoglycan family of carbohydrates (which includes the closely related molecule heparan sulfate) and consists of a variably sulfated repeating disaccharide unit.[7] The main disaccharide units that occur in heparin are shown below. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa.[8] Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free amine group (GlcNH3+). Under physiological conditions, the ester and amide sulfate groups are deprotonated and attract positively charged counterions to form a heparin salt. Heparin is usually administered in this form as an anticoagulant.
One unit of heparin (the “Howell unit”) is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 ml of cat’s blood fluid for 24 hours at 0°C.[9]
GlcA-GlcNAc
GlcA-GlcNS
IdoA-GlcNS
IdoA(2S)-GlcNS
IdoA-GlcNS(6S)
IdoA(2S)-GlcNS(6S)
Abbreviations
GlcA = β-D-glucuronic acid
IdoA = α-L-iduronic acid
IdoA(2S) = 2-O-sulfo-α-L-iduronic acid
GlcNAc = 2-deoxy-2-acetamido-α-D-glucopyranosyl
GlcNS = 2-deoxy-2-sulfamido-α-D-glucopyranosyl
GlcNS(6S) = 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate
Three-dimensional structure
The three-dimensional structure of heparin is complicated because iduronic acid may be present in either of two low-energy conformations when internally positioned within an oligosaccharide. The conformational equilibrium is influenced by sulfation state of adjacent glucosamine sugars.[10] Nevertheless, the solution structure of a heparin dodecasaccharide composed solely of six GlcNS(6S)-IdoA(2S) repeat units has been determined using a combination of NMR spectroscopy and molecular modeling techniques.[11] Two models were constructed, one in which all IdoA(2S) were in the 2S0 conformation (A and B below), and one in which they are in the 1C4 conformation (C and D below). However, no evidence suggests that changes between these conformations occur in a concerted fashion. These models correspond to the protein data bank code 1HPN.
In the image above:
A = 1HPN (all IdoA(2S) residues in 2S0 conformation) Jmol viewer
B = van der Waals radius space filling model of A
C = 1HPN (all IdoA(2S) residues in 1C4 conformation) Jmol viewer
D = van der Waals radius space filling model of C
In these models, heparin adopts a helical conformation, the rotation of which places clusters of sulfate groups at regular intervals of about 17 angstroms (1.7 nm) on either side of the helical axis.
Medical use
A sample of Heparin Sodium for injection
Heparin is a naturally occurring anticoagulant produced by basophils and mast cells.[12] Heparin acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the blood. While heparin does not break down clots that have already formed (unlike tissue plasminogen activator), it allows the body’s natural clot lysis mechanisms to work normally to break down clots that have formed. Heparin is generally used for anticoagulation for the following conditions:
Acute coronary syndrome, e.g., NSTEMI
Atrial fibrillation
Deep-vein thrombosis and pulmonary embolism
Cardiopulmonary bypass for heart surgery
ECMO circuit for extracorporeal life support
Hemofiltration
Indwelling central or peripheral venous catheters
Mechanism of action
Heparin and its low-molecular-weight derivatives (e.g., enoxaparin, dalteparin, tinzaparin) are effective at preventing deep vein thromboses and pulmonary emboli in patients at risk,[13][14] but no evidence indicates any one is more effective than the other in preventing mortality.[15] Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop.[16] The activated AT then inactivates thrombin and other proteases involved in blood clotting, most notably factor Xa. The rate of inactivation of these proteases by AT can increase by up to 1000-fold due to the binding of heparin.[17]
AT binds to a specific pentasaccharide sulfation sequence contained within the heparin polymer:
GlcNAc/NS(6S)-GlcA-GlcNS(3S,6S)-IdoA(2S)-GlcNS(6S)
The conformational change in AT on heparin-binding mediates its inhibition of factor Xa. For thrombin inhibition, however, thrombin must also bind to the heparin polymer at a site proximal to the pentasaccharide. The highly negative charge density of heparin contributes to its very strong electrostatic interaction with thrombin.[3] The formation of a ternary complex between AT, thrombin, and heparin results in the inactivation of thrombin. For this reason, heparin’s activity against thrombin is size-dependent, with the ternary complex requiring at least 18 saccharide units for efficient formation.[18] In contrast, antifactor Xa activity requires only the pentasaccharide binding site.
Chemical structure of fondaparinux
This size difference has led to the development of low-molecular-weight heparins (LMWHs) and, more recently, to fondaparinux as pharmaceutical anticoagulants. LMWHs and fondaparinux target antifactor Xa activity rather than antithrombin activity, with the aim of facilitating a more subtle regulation of coagulation and an improved therapeutic index. The chemical structure of fondaparinux is shown above. It is a synthetic pentasaccharide, whose chemical structure is almost identical to the AT binding pentasaccharide sequence that can be found within polymeric heparin and heparan sulfate.
With LMWH and fondaparinux, the risk of osteoporosis and heparin-induced thrombocytopenia (HIT) is reduced. Monitoring of the activated partial thromboplastin time is also not required and does not reflect the anticoagulant effect, as APTT is insensitive to alterations in factor Xa.
Danaparoid, a mixture of heparan sulfate, dermatan sulfate, and chondroitin sulfate can be used as an anticoagulant in patients having developed HIT. Because danaparoid does not contain heparin or heparin fragments, cross-reactivity of danaparoid with heparin-induced antibodies is reported as less than 10%.[19]
The effects of heparin are measured in the lab by the partial thromboplastin time (aPTT), one of the measures of the time it takes the blood plasma to clot. Partial thromboplastin time should not be confused with prothrombin time, or PT, which measures blood clotting time through a different pathway of the coagulation cascade.
Administration
Heparin is given parenterally because it is not absorbed from the gut, due to its high negative charge and large size. It can be injected intravenously or subcutaneously (under the skin); intramuscular injections (into muscle) are avoided because of the potential for forming hematomas. Because of its short biologic half-life of about one hour, heparin must be given frequently or as a continuous infusion. Unfractionated heparin has a half-life of about one to two hours after infusion, [20] whereas LMWH has a half-life of four to five hours.[21] The use of LMWH has allowed once-daily dosing, thus not requiring a continuous infusion of the drug. If long-term anticoagulation is required, heparin is often used only to commence anticoagulation therapy until an oral anticoagulant e.g. warfarin takes effect.
Details of administration are available in clinical practice guidelines by the American College of Chest Physicians:[22]
Non-weight-based heparin dose adjustment
Weight-based-heparin dose adjustment
Production
Pharmaceutical-grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine (pig) intestines or bovine (cattle) lungs.[23] Advances to produce heparin synthetically have been made in 2003 and 2008.[24]
Protamine sulfate (1 mg per 100 units of heparin that had been given over the past four hours) has been given to counteract the anticoagulant effect of heparin.[26]
Heparin is one of the oldest drugs currently in widespread clinical use. Its discovery in 1916 predates the establishment of the Food and Drug Administration of the United States, although it did not enter clinical trials until 1935.[27] It was originally isolated from canine liver cells, hence its name (hepar or “ήπαρ” is Greek for “liver”). Heparin’s discovery can be attributed to the research activities of Jay McLean and William Henry Howell.
In 1916, McLean, a second-year medical student at Johns Hopkins University, was working under the guidance of Howell investigating procoagulant preparations, when he isolated a fat-soluble phosphatide anticoagulant in canine liver tissue. In 1918, Howell coined the term ‘heparin’ for this type of fat-soluble anticoagulant. In the early 1920s, Howell isolated a water-solublepolysaccharide anticoagulant, which was also termed ‘heparin’, although it was distinct from the phosphatide preparations previously isolated. McLean’s work as a surgeon probably changed the focus of the Howell group to look for anticoagulants, which eventually led to the polysaccharide discovery.
In the 1930s, several researchers were investigating heparin. Erik Jorpes at Karolinska Institutet published his research on the structure of heparin in 1935,[28] which made it possible for the Swedish company Vitrum AB to launch the first heparin product for intravenous use in 1936. Between 1933 and 1936, Connaught Medical Research Laboratories, then a part of the University of Toronto, perfected a technique for producing safe, nontoxic heparin that could be administered to patients in a salt solution. The first human trials of heparin began in May 1935, and, by 1937, it was clear that Connaught’s heparin was a safe, easily available, and effective blood anticoagulant. Prior to 1933, heparin was available, but in small amounts, and was extremely expensive, toxic, and, as a consequence, of no medical value.[29]
A posthumous attempt to nominate McLean for a Nobel Prize failed
Heparin Sodium Injection, USP is a sterile, nonpyrogenic solution of heparin sodium (derived from porcine intestinal mucosa) in water for injection. Each container contains 10000, 12500, 20000 or 25,000 USP Heparin Units; 40 or 80 mg sodium chloride added to render isotonic (see HOW SUPPLIEDsection for various sizes and strength). May contain sodium hydroxide and/or hydrochloric acid for pH adjustment. pH 6.0 (5.0 to 7.5).
The solution contains no bacteriostat, antimicrobial agent or added buffer and is intended for use only as a single-dose injection. When smaller doses are required, the unused portion should be discarded.
Heparin sodium in the ADD-Vantage™ system is intended for intravenous administration only after dilution.
Heparin Sodium, USP is a heterogenous group of straight-chain anionic mucopolysaccharides, called glycosamino-glycans having anticoagulantproperties. Although others may be present, the main sugars occurring in heparin are: (1) α- L-iduronic acid 2-sulfate, (2) 2-deoxy-2-sulfamino-α-D-glucose-6-sulfate, (3) β-D-glucuronic acid, (4) 2-acetamido-2-deoxy-α-D-glucose, and (5) α-L-iduronic acid. These sugars are present in decreasing amounts, usually in the order (2) > (1) > (4) > (3) > (5), and are joined by glycosidic linkages, forming polymers of varying sizes. Heparin is strongly acidic because of its content of covalently linked sulfate and carboxylic acid groups. In heparin sodium, the acidic protons of the sulfate units are partially replaced by sodium ions. The potency is determined by a biological assay using a USP reference standard based on units of heparin activity per milligram.
Structure of Heparin Sodium (representative subunits):
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http://www.medgadget.com/2008/08/on_the_road_to_a_fully_synthetic_heparin.html
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The chemoenzymatic synthesis of heparin from E. coli’s carbohydrate coat
Now, Linhardt’s team – who were also the first to identify the contaminant in the tainted batches as oversulfated chondroitin sulfate – have come up with a potentially safer way to produce heparin. The researchers grew flasks of the gut bacteria E. coli, then converted its naturally produced carbohydrate coat to heparin in just a few steps using enzymes and chemical treatment.
Linhardt says the key to the procedure was starting with the carbohydrate capsule that E coli produces to hide itself from the human immune system. The capsule is made from heparosan – a polysachharide that is already quite similar to heparin.
The team first chemically removed acetyl groups from the heparosan with sodium hydroxide and added a sulfate group using sulfur trioxide trimethylamine. Then, using four enzymes found in all mammals that produce heparin, they introduced further modifications, including the addition of three more sulfates at different positions on the molecule to get to heparin.
The checked the structure of the compound using NMR and showed that the synthetic compound could stop blood clotting as well as heparin derived from animals. To date, however, the team have only made a total of around 100mg of pure heparin – barely enough for a single dose. That is still a million times more than produced by a 2003 total synthesis of heparin, from researchers at the Massachusetts Institute of Technology, US.
Heparin Sodium injection
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15383472
9-1-2004
Heparin and low-molecular-weight heparin: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy.
Chest
10201371
4-1-1999
Synthesis of thrombin-inhibiting heparin mimetics without side effects.
Nature
10549711
1-1-1999
Production and chemical processing of low molecular weight heparins.
Seminars in thrombosis and hemostasis
8352752
8-1-1993
N.m.r. and molecular-modelling studies of the solution conformation of heparin.
The Biochemical journal
2331699
1-15-1990
Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences.
Carbohydrate research
Heparin, a highly sulfated glycosaminoglycan (GAG), is used extensively as an anticoagulant. It consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. Heparin, and its analogues, are used during surgery and dialysis, and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Administered parenterally, often continuously due to its short half-life, over 0.5 billion doses are required per year. Currently obtained from mucosal tissue of meat animals, mainly porcine intestine, and to a lesser extent bovine lung, its early stage production is poorly controlled, due to the source of the material (Figure 1). This problem came into sharp focus in 2008 when the presence of contaminating over-sulfated chondroitin sulfate in heparin, sourced from pigs, resulted in almost 100 deaths in the USA. This, coupled with the fact that only two doses are obtained per animal means that the demand for alternative and more controlled sources of heparin is high.
Figure.1. Heparin ProductionIn an attempt to mimic the natural processes, which occur in mammalian synthesis of heparan sulfate, a less sulfated form of heparin was first examined (Figures 1 & 2).
Figure 2. Biosynthetic pathway of HS: The biosynthetic pathway includes the biosynthesis of polysaccharide backbone as well as the modification steps. The synthesis is initiated with a tetrasaccharide linkage region that contains xylose-galactose-galactose-glucuronic acid. The backbone is synthesized by HS polymerase. The backbone polysaccharide is then modified via five enzymatic modification steps. The modification site at each step is highlighted in a blue box2Based on this pathway, a biosynthetic pathway was designed that began with bacterial synthesis of the backbone structure. Escherichia coli K5 synthesizes a polysaccharide capsule consisting of repeating units of [(→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→)]n,1 known as heparosan (Figures 2 & 3). Since heparosan is a precursor of heparin and heparan sulfate in eukaryotes, this provides an ideal starting point of the bioengineered heparin production process.
Figure.3. (A) The structure of heparosan disaccharide unit. (B) the structures of the major and minor variable repeating disaccharides comprising heparin where X = SO3- or H and Y = SO3- or COCH3.1
The fermentation process involves the use of glucose as the sole carbon source and ammonium chloride as the sole nitrogen source. Initially developed and optimized on a 500mL scale, the process was then scaled up to a 3L fermentation and is currently at the 15L scale. Using a fed-batch fermentation, with exponential feeding, a growth rate of 0.12h-1 was achieved with a yield of 15gL-1 heparosan.
Figure.4. Time course of dry cell weight (g/L) and heparosan concentration in the fermentation supernatant (g/L) during the fermentation in a 20 L fermentor1
Once extracted from the cell and purified the heparosan polysaccharide undergoes a series of chemoenzymatic modifications to make heparin. The first step is performed chemically and all other steps in the process use the enzymes listed in Figure 2 (above), which have been cloned and overexpressed in E. coli.The heparosan polysaccharide generated through this process is analyzed by polyacrylamide gel electrophoresis (PAGE), 1D- and 2D-NMR and disaccharide analysis is done using LC-MS of heparinase-digested polysaccharide. At each point in the process the polysaccharide is analyzed by a combination of these methods.References1. Z.Wang, J.S.Dordick, & R.J.Linhardt, “Escherichia coli K5 heparosan fermentation and improvement by genetic engineering.“ Bioengineered Bugs 2, 1-5 (2011)2. R. Liu, Y.Xu, M.Chen, M.Weiwer, X.Zhou, A.S.Bridges, P.L.DeAngelis, Q.Zhang, R.J.Linhardt, J.Liu, ”Chempenzymatic design of heparan sulfate oligosaccharides” , Journal of Biological Chemistry, 285, 34240-342
3. Z.Wang, M.Ly, F.Zhang, W. Zhong, A.Suen, A.M.Hickey, J.S.Dordick, R.J.Linhardt,”E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor“, Biotechnol. Bioeng. 107, 964-973 (2010).
M.Ly, Z.Wang, T.N.Laremore, F.Zhang, W.Zhong, D.Pu, D.V.Zagorevski, J.S.Dordick, R.J.Linhardt, “Analysis of E. coli K5 capsular polysaccharide heparosan.” Analytical and Bioanalytical Chemistry 399, 737-745 (2011).
Filed under: GENERIC DRUG Tagged: HEPARIN