Preparation of medium molecular weight heparin

Abstract

A method of synthesis of medium molecular weight heparin includes the steps of dissolving unfractionated heparin in an aqueous solvent to provide a first solution; adding an oxidising agent to the first solution to provide a second solution; and incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.

Description

PREPARATION OF MEDIUM MOLECULAR WEIGHT HEPARINFIELD OF THE INVENTION

[0001] The present invention relates to the synthesis of medium molecular weight heparins, preferably via room temperature periodate oxidation.BACKGROUND OF THE INVENTION

[0002] Heparin is a naturally occurring, highly sulphated polysaccharide characterised by a wide molecular weight range of polysaccharide chains. Heparin acts at a variety of different ligands with varied actions. Heparin is a member of the glycosaminoglycan carbohydrate family and consists of repeating disaccharide units of GlcApi-4GlcNAcal-4 with poly-disperse sulfation, N-acetylation and uronosyl epimerization. Heparin is highly heterogenous. Heparin isolated from natural sources contains polysaccharide chains with molecular weights ranging from about 3000 Da (g / mol) to about 30,000 Da (g / mol). This is known as unfractionated heparin (UFH or UF heparin). UFH can be enzymatically or chemically treated to deliver shorter polysaccharide chains. Heparinase I cleaves at the a-1,4 linkage between nonacetylated GlcNS6S and ldoA2S. The products of the chemically or enzymatically treated UFH can be affinity purified to yield fractionated heparin where the molecular weight of the polysaccharides in each fraction can be readily determined. Low molecular weight heparin (LMWH) contains polysaccharide chains in the range of about 4000 Da (g / mol) to about 8000 Da (g / mol).

[0003] There are few known methods of preparing medium molecular weight heparin. One example of a method of preparing medium molecular weight heparin is described in Poletti LF, Bird KE, Marques D, Harris RB, Suda Y, Sobel M. Structural aspects of heparin responsible for interactions with von Willebrand factor. Arterioscler Thromb Vase Biol. 1997 May;17(5):925- 31. This method requires incubation at 37 °C and produces a wide variety of products ranging in molecular weight from 10600 g / mol down to 1900 g / mol.

[0004] Accordingly, there is a need for a reliable method of preparing medium molecular weight heparin.SUMMARY OF THE INVENTION

[0005] In a first aspect, the invention provides a method of synthesis of medium molecular weight heparin, the method comprising the steps of: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; and (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.

[0006] The method may comprise the steps of: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; and (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.

[0007] The method may comprise the steps of: (a) dissolving unfractionated heparin in an aqueous solution or water to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; and (c) incubatingthe second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.

[0008] In a second aspect, the invention provides a kit suitable for preparing medium molecular weight heparin, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous solvent; (c) an oxidising agent; and (d) optionally, an inactivating agent.

[0009] The kit may comprise: (a) unfractionated heparin; (b) an aqueous solution; (c) an oxidising agent; and (d) optionally, an inactivating agent.

[0010] For the avoidance of doubt, embodiments related to each aspect of the invention apply mutatis mutandis to the other aspects of the invention. Further aspects and embodiments of the present invention will be evident from the discussion that follows below.BRIEF DESCRIPTION OF THE DRAWINGSFIG.l Ristocetin-induced platelet aggregation (RIPA) data 6 medium molecular weight heparin sample (ID23-007-1, ID24-002-1, ID24-009-1, ID24-005-4, ID24-005-1, ID24-005-6) and reduced mediummolecular weight heparin (MMWH-Red sample 1 and MMWH-Red sample 2) obtained from one blood donor. Average values determined from experiments carried out in at least technical duplicate.FIG.22H-NMR of MMWH-Red compared to MMWH.FIG. 3 HPLC chromatogram for unfractionated heparin.FIG. 4 HPLC chromatogram for unfractionated heparin treated with sodium periodate at 3°C overnight.FIG. 5 HPLC chromatogram for unfractionated heparin treated with sodium periodate at room temperature overnight.DETAILED DESCRIPTION OF THE INVENTION

[0011] Throughout this specification, one or more aspects of the invention may be combined with one or more features described in the specification to define distinct embodiments of the invention.

[0012] In the discussion that follows, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context expressly indicates to the contrary.

[0013] References herein to a singular of a noun encompass the plural of the noun, and vice-versa, unless the context implies otherwise.

[0014] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of any other element or integer, or group of elements or integers. The term "comprising" includes within its ambit the term "consisting" or "consisting essentially of".

[0015] The term "consisting" or variants thereof is to be understood to imply the inclusion of a stated element or integer, or group of elements or integers, and the exclusion of any other element or integer or group of elements or integers.

[0016] The term "consisting essentially of" or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and that further components may be present, but only those not materially affecting the essential characteristics of the formulation, composition, or compound.

[0017] The term "about" herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified.

[0018] The terms "treatment" and "therapy" define the therapeutic treatment of a patient, in order to reduce or halt the rate of progression of a disorder or condition, or to ameliorate or cure the disorder or condition. Prophylaxis of a disorder or condition as a result of treatment or therapy is also included.

[0019] As used herein, the term "patient" preferably refers to a mammal. Typically, the mammal is a human.

[0020] von Willebrand factor (VWF) is a blood glycoprotein involved in haemostasis. VWF is a large multimeric glycoprotein present in blood plasma and produced constitutively as ultra-large VWF in endothelium (in the Weibel-Palade bodies), megakaryocytes (a-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2050-amino acid protein.

[0021] A disaccharide is a sugar whose molecules contain two monosaccharide residues.

[0022] A low molecular weight heparin is defined herein as a heparin with an average molecular weight of from about 4000 Da (g / mol) to about 8000 Da (g / mol). A medium molecular weight heparin is defined herein as a heparin with an average molecular weight of from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).

[0023] Accordingly, in a first aspect, the invention provides a method of synthesis (or preparation) of medium molecular weight heparin (MMWH).

[0024] The medium molecular weight heparin prepared by the method of the first aspect of the invention may have an average molecular weight in the range of greater than about 8000 Da (g / mol)to about 13 500 Da (g / mol), preferably greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), more preferably about 10 000 Da (g / mol) to about 12 000 Da (g / mol).

[0025] The medium molecular weight heparin prepared by the method of the first aspect of the invention may comprise polysaccharide chains with an average molecular weight in the range of greater than about 8000 Da (g / mol) to about 13 500 Da (g / mol ), more preferably greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), preferably about 10 000 Da (g / mol) to about 12 000 Da (g / mol).

[0026] The molecular weight of the medium molecular weight heparin prepared by the method of the first aspect of the invention may be determined by size exclusion chromatography as described herein.

[0027] The medium molecular weight heparin prepared by the method of the first aspect may comprise at least three units of a GlcNS6S-ldoA2S (or I d oA2S-G IcNSSS) disaccharide. The GlcNS6S and ldoA2S monosaccharides are linked by an al-4 linkage between GlcNS6S and ldoA2S, i.e. GlcNS6Sal- 4ldoA2S. For example, the medium molecular weight heparin prepared by the method of the first aspect may comprise at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of a GlcNS6S-ldoA2S disaccharide. The medium molecular weight heparin prepared by the method of the first aspect may comprise less than or equal to 25 units of the GlcNS6S-ldoA2S disaccharide, for example less than or equal to 20 units. The presence of the units of the GlcNS6S-ldoA2S disaccharide may be determined by an antibody, mass spectrometry, or inferred from chemical and enzymatical studies. The GlcNS6S-ldoA2S units may be ordered in succession.

[0028] "IdoA" is a-L-iduronic acid. "ldoA2S" is IdoA modified by the addition of an O-sulfate group at carbon position 2 to form 2-O-sulfo-a-L-iduronic acid. "GIcNS" is 2-deoxy-2-sulfamido-a-D- glucopyranosyl. "GlcNS6S" is 2-deoxy-2-sulfamido-a-D-glucopyranosyl-6-0-sulfate. An al-4 linkage is an alpha glycosidic bond between carbon-1 on one monosaccharide and carbon-4 on a second monosaccharide. A pi-4 linkage is beta glycosidic bond between carbon-1 on one monosaccharide and carbon-4 on a second monosaccharide.

[0029] The medium molecular weight heparin prepared by the method of the first aspect may comprise UA2S-GlcNS6S, UA2S-GlcNS, UA-GIcNAc, wherein U may be iduronic acid (IdoA) or glucuronicacid (GlcA). The medium molecular weight heparin prepared by the method of the first aspect may comprise at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GIcNAc. The medium molecular weight heparin prepared by the method of the first aspect may comprise at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% UA2S- GlcNS6S. The medium molecular weight heparin prepared by the method of the first aspect may comprise up to about 60%, preferably up to about 70%, preferably up to about 85% UA2S-GlcNS6S. The medium molecular weight heparin prepared by the method of the first aspect may comprise at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% UA2S-GlcNS. The medium molecular weight heparin prepared by the method of the first aspect may comprise up to about 15%, preferably up to about 20% UA2S-GlcNS. The medium molecular weight heparin prepared by the method of the first aspect may comprise at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% UA-GIcNAc. The medium molecular weight heparin prepared by the method of the first aspect may comprise up to about 15%, preferably up to about 20% UA-GIcNAc. In some embodiments the medium molecular weight heparin prepared by the method of the first aspect may comprise at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN and 5.4% UA-GIcNAc. In some embodiments the medium molecular weight heparin prepared by the method of the first aspect may comprise at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS and 9% UA- GIcNAc. The percentage composition of UA-GIcNAc comprised in the medium molecular weight heparin may be enriched compared to unfractionated heparin.

[0030] "UA" is a uronicacid, which is a hexose with a negatively charged carboxylate at the 6-position. The uronic acid may independently be glucuronic acid or iduronic acid. "UA2S" is UA modified by the addition of an O-sulfate group at carbon position 2 to form 2-O-sulfo-uronic acid. "GlcA" is -D- glucuronic acid "GIcNAc" is 2-deoxy-2-acetamido-a-D-glucopyranosyl.

[0031] The method of the first aspect comprises the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; and (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.

[0032] UF heparin may be obtained from bovine or porcine tissue, for example porcine intestine or bovine lung.

[0033] The aqueous solvent is typically water. The aqueous solvent may consist of or consist essentially of water. Step (a) may comprise dissolving the unfractionated heparin (U F) in water. The first solution may consist of or consist essentially of unfractionated heparin and water. The second solution may consist of or consist essentially of unfractionated heparin, water and the oxidising agent. Preferably, the reaction is carried out in water. Preferably, the aqueous solvent comprises water. Preferably, the aqueous solvent consists of water.

[0034] The aqueous solvent may be an aqueous solution. The aqueous solution may be an aqueous buffer solution. When the aqueous solution is an aqueous buffer solution, in step (a) unfractionated heparin is dissolved in an aqueous buffer solution adjusted to between about pH 4.0 and pH 9.0 to provide a first solution.

[0035] The aqueous solution is typically water. The aqueous solution may consist of or consist essentially of water. Step (a) may comprise dissolving the unfractionated heparin (U F) in water. The first solution may consist of or consist essentially of unfractionated heparin and water. The second solution may consist of or consist essentially of unfractionated heparin, water and the oxidising agent. Preferably, the reaction is carried out in water. Preferably, the aqueous solution comprises water.

[0036] "Buffer" refers to a chemical which, in a solution, resists a change in pH when acid or alkali is added to the solution. Typically, a buffer solution (or buffer system) comprises a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0037] Typically, a suitable buffer comprises an acid with a pKa value that lies within +1 of the desired pH of the formulation. For example, if the desired pH of the formulation is about 7.0, a suitable buffer comprises a weak acid with a pKa value of from about 6.0 to about 8.0. If the acid of a buffer has more than one pKa value (i.e. each molecule of the acid is able to donate more than one proton), in order for the buffer to be suitable, at least one of the pKa values should lie within the desired pH range.

[0038] The weak acid and conjugate base (or weak base and conjugate acid) of the buffer are in equilibrium with one another. In accordance with Le Chatelier's principle (if a constraint, such as a change in concentration of a reactant, is applied to a system in equilibrium, the equilibrium will shift so as to counteract the effect of the constraint), addition of acid or base to the solution shifts the position of equilibrium in favour of the conjugate base or weak acid, respectively. Consequently, the concentration of free protons in the formulation (and thus the pH) is relatively unchanged.

[0039] Suitable buffer systems comprise an acetate salt and acetic acid ( pKa = 4.75), a citrate salt and citric acid (pKa = 3.13, 4.76 and 6.40), and phosphoric acid (pKa = 2.14, 7.20 and 12.37), or mixtures thereof. Phosphate buffered saline may also be used. The pKa values cited herein are those reported at 25 °C in water. Typically, the buffer comprises only one of the pairs listed above, i.e. one acid and its conjugate base. The buffer may comprise an acetate salt and acetic acid, a citrate salt and citric acid, or a phosphate salt and phosphoric acid.

[0040] The pH may be adjusted to ensure that the pH of the first solution is between from about pH 4.0 to about pH 9.0 because dissolving unfractionated heparin may result in the pH of the aqueous buffer solution changing. However, it has been surprisingly found that the reaction can be carried out in water and a buffer is not needed.

[0041] Optionally, in step (a), the aqueous buffer solution is adjusted to between about pH 5.0 and about pH 8.0.

[0042] Typically, the temperature of the aqueous solvent in step (a) is from about 15 °C to about 30 °C. Preferably, the temperature of the aqueous solvent in step (a) is from about 16 °C to about 25 °C, preferably from about 17 °C to about 22 °C, preferably from about 18 °C to about 21 °C, preferably about 19 °C to about 20 °C. Preferably, the temperature of the aqueous solvent in step (a) is room temperature. By performing the reaction at or around room temperature, costs to produce the product are reduced due to shorter reaction times.

[0043] Typically, the temperature of the aqueous solution in step (a) is from about 15 °C to about 30 °C. Preferably, the temperature of the aqueous solution in step (a) is from about 16 °C to about 25 °C, preferably from about 17 °C to about 22 °C, preferably from about 18 °C to about 21 °C, preferably about 19 °C to about 20 °C. Preferably, the temperature of the aqueous solution in step (a) is room temperature. By performing the reaction at or around room temperature, costs to produce the product are reduced due to shorter reaction times.

[0044] Typically, the aqueous buffer solution is a phosphate buffer, a citrate buffer or an acetate buffer, i.e. the buffering system is phosphate, citrate or acetate. Preferably, the aqueous buffer solution is a phosphate buffer, more preferably the aqueous buffer solution is a sodium phosphatebuffer or a potassium phosphate buffer, i.e. the buffering system is sodium phosphate or potassium phosphate.

[0045] The buffering system in the aqueous buffer solution may be present at a concentration of from about 10 mM to about 100 mM, more preferably from about 20 mM to about 90 mM, more preferably from about 30 mM to about 80 mM, more preferably from about 40 mM to about 70 mM, more preferably from about 50 mM to about 60 mM. The buffering system in the aqueous buffer solution may be present at a concentration of about 50 mM.

[0046] The concentration of UF heparin in the aqueous solution may be from about 0.5 mg / mL to about 100 mg / mL, more preferably from about 1 mg / mL to about 85 mg / mL, more preferably from about 1.5 mg / mLto about 80 mg / mL, more preferably from about 2 mg / mLto about 75 mg / mL, more preferably from about 2 mg / mLto about 70 mg / mL, more preferably from about 10 mg / L to about 65 mg / mL, more preferably from about 25 mg / mL to about 65 mg / mL, most preferably from about 30 mg / mL to about 60 mg / mL.

[0047] The concentration of UF heparin may be from about 0.5 mg / mL to about 100 mg / mL, more preferably from about 1 mg / mLto about 85 mg / mL, more preferably from about 1.5 mg / mLto about 80 mg / mL, more preferably from about 2 mg / mL to about 75 mg / mL, more preferably from about 2 mg / mL to about 70 mg / mL, more preferably from about 10 mg / L to about 65 mg / mL, more preferably from about 25 mg / mL to about 65 mg / mL, most preferably from about 30 mg / mLto about 60 mg / mL.

[0048] The concentration of UF heparin in the aqueous solution may be about 1.5 mg / mL, more preferably about 1.8 mg / mL, more preferably about 2 mg / mL, more preferably about 2.5 mg / mL, more preferably about 2.7 mg / mL, more preferably about 3 mg / mL, more preferably 8 mg / mL, more preferably about 16 mg / ml, more preferably about 30 mg / mL and most preferably about 60 mg / mL. The concentration of UF heparin in the aqueous solution is most preferably about 60 mg / mL.

[0049] The concentration of UF heparin may be about 1.5 mg / mL, more preferably about 1.8 mg / mL, more preferably about 2 mg / mL, more preferably about 2.5 mg / mL, more preferably about 2.7 mg / mL, more preferably about 3 mg / mL, more preferably 8 mg / mL, more preferably about 16 mg / ml, more preferably about 30 mg / mL and most preferably about 60 mg / mL. The concentration of UF heparin is most preferably about 60 mg / mL.

[0050] The oxidising agent may be a periodate, for example sodium periodate or potassium periodate. Preferably, the oxidising agent is sodium periodate. Alternatively, or additionally, the oxidising agent may be a perchlorate, for example sodium perchlorate. Preferably, the oxidising agent does not comprise a perchlorate. Use of a perchlorate in combination with a periodate as the oxidising agent results in an increased level of sample breakdown to smaller molecular weight species.

[0051] The concentration of the oxidising agent in the aqueous solution may be from about 1 g / L to about 40 g / L. Preferably, the concentration of the oxidising agent may be from about 2g / L to about 35 g / L, more preferably from about 4 g / L to about 30 g / L, more preferably from about 20 g / L to about 25 g / L. The concentration of the oxidising agent in the aqueous solution may be about 23 g / L or greater. Preferably the concentration of oxidising agent is about 23 g / L. Preferably the concentration of the oxidising agent is about 23 g / L and the oxidising agent is sodium periodate.

[0052] The concentration of the oxidising agent may be from about 1 g / Lto about 40 g / L. Preferably, the concentration of the oxidising agent may be from about 2g / L to about 35 g / L, more preferably from about 4 g / L to about 30 g / L, more preferably from about 20 g / L to about 25 g / L. The concentration of the oxidising agent may be about 23 g / L or greater. Preferably the concentration of oxidising agent is about 23 g / L. Preferably the concentration of the oxidising agent is about 23 g / L and the oxidising agent is sodium periodate.

[0053] The molar ratio of UF heparin to oxidising agent may be from about 1:1 to about 1:200, more preferably from about 1:2 to about 1:150, more preferably from about 1:10 to about 1:100, more preferably from about 1:20 to about 1:50, more preferably from about 1:20 to about 1:30, most preferably from about 1:25-1:30. Typically, the molar ratio of UF heparin to oxidising agent may be about 1:27.

[0054] The molar ratio of UF heparin to oxidising agent may be determined based on the estimated molecular weight of the canonical heparin disaccharide as opposed to the molecular weight of heparin. The estimated molecular weight of the canonical heparin disaccharide is 593.45 g / mol. This is used to approximate the number of moles of this disaccharide and therefore the uronicacid residues present in the sample.

[0055] For example, if it is desired to add 1 equivalent of NalO4based on uronic acid and 3 grams of heparin is used, the number of moles NalO4to add is 0.005 moles i.e. 3 divided by 593.45.

[0056] Thus, the ratio of UF heparin determined based on the estimated molecular weight of the canonical heparin disaccharide may be from about 1:0.5 to about 1:10, preferably from about 1:0.7 to about 1:8, more preferably from about 1:0.9 to about 1:5, more preferably from about 1:1 to about 1:2, more preferably from about 1:1.1 to about 1:2. Preferably, the ratio of moles of uronic acid present in the UF heparin to moles of oxidising agent may be 1:1.1.

[0057] Typically, the incubation temperature in step (c) is carried out at room temperature. The incubation in step (c) may be carried out at from about 15 °C to about 30 °C, more preferably from about 16 °C to about 28 °C, more preferably from about 17 °C to about 1 °C, more preferably from about 18 °C to about 25 °C. Preferably, the incubation temperature in step (c) is about 18 °C to about 20 °C. Typically, the incubating step (c) is carried out for from about 0.5 hour to about 40 hours, more preferably from about 1 hours to about 24 hours, more preferably from about 2 hours to about 20 hours, more preferably from about 3 hours to about 18 hours,. Preferably, the incubating step may be carried out for about 3 hours.

[0058] Incubation step (c) of the first aspect of the invention may be carried out on a laboratory bench at ambient room temperature.

[0059] In a preferred embodiment, the method of the first aspect further comprises purifying the medium molecular weight heparin from the medium molecular weight heparin solution.

[0060] Medium molecular weight heparin that has been purified from the medium molecular weight heparin solution may be in a suitable form for biological testing. To be suitable for biological testing, the medium molecular weight heparin may be free or substantially free of organic solvent.

[0061] Purifying the medium molecular weight heparin may comprise precipitating and filtering the medium molecular weight heparin, dialysis, ion exchange, lyophilization, size exclusion chromatography, ultra filtration, or a combination thereof. For example, the medium molecular weight heparin may be purified from the medium molecular weight heparin solution by dialysis and lyophilization.

[0062] The medium molecular weight heparin may be precipitated from the medium molecular weight heparin solution by the addition of a precipitation agent. The precipitation agent may comprisea precipitation agent as described herein. The precipitation agent may comprise or consist of an alcohol or acetone. The precipitation agent may also quench the oxidising agent.

[0063] In a preferred embodiment, the method of the first aspect further comprises preparing a composition for biological testing comprising the medium molecular weight heparin prepared in step c. To be suitable for biological testing, the medium molecular weight heparin composition may be free or substantially free of organic solvent.

[0064] Preparing a composition of the medium molecular weight heparin for biological testing may comprise any of the purification methods described herein. For example, it may comprise precipitating and filtering the medium molecular weight heparin, dialysis, ion exchange, lyophilization, size exclusion chromatography, ultra filtration, or a combination thereof.

[0065] For example, the medium molecular weight heparin solution prepared in step c may be dialysed against a suitable solvent such as water or buffer, for example phosphate buffered saline, to prepare a composition for biological testing.

[0066] If the medium molecular weight heparin has been purified to a solid form, it may be dissolved in a suitable solvent, such as water or aqueous buffer, for example phosphate buffered saline, for biological testing. In some instances, a solid form of medium molecular weight heparin maybe suitable for biological testing, for example in the form of a tablet.

[0067] In a preferred embodiment, the method of the first aspect further comprises (i) quenching the oxidising agent to provide a quenched solution, (ii) precipitating the medium molecularweight heparin to provide a medium molecularweight heparin precipitate in a precipitation mixture, or (iii) quenching the oxidising agent and precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture.

[0068] Quenching the oxidising agent to provide a quenched solution may comprise adding a quenching agent to the medium molecular weight heparin solution to provide a quenched solution.

[0069] Precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture may comprise adding a precipitating agent to themedium molecular weight heparin solution to provide a medium molecular weight heparin precipitate in a precipitation mixture.

[0070] Quenching the oxidising agent and precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture may comprise adding a quenching agent and precipitating agent to the medium molecular weight heparin solution to provide a medium molecular weight heparin precipitate in a precipitation mixture.

[0071] Preferably, the quenching agent and the precipitating agent are the same material, i.e. the quenching agent and the precipitating agent are comprised in a quenching and precipitating agent. The quenching agent, precipitating agent or quenching and precipitating agent may comprise or consist of an alcohol or acetone.

[0072] Surprisingly, the alcohol precipitates the medium molecular weight heparin, as well as quenches the excess oxidising agent. The precipitated medium molecular weight heparin may then be isolated from the precipitation mixture, for example by filtration. Thus, the method of the first aspect may further comprise the step of isolating the precipitated medium molecular weight heparin from the precipitation mixture.

[0073] This quenching and / or precipitation procedure advantageously results in a more efficient and high purity method for the production and purification of medium molecular weight heparin than known methods. This results in highly pure material that does not need to be purified, for example, via a column.

[0074] Preferably, the quenching and precipitating agent is added to the medium molecular weight heparin solution to give a final concentration of the quenching and precipitating agent in the precipitation mixture of from about 50% v / v to about 90% v / v, more preferably from about 55% v / v to about 80% v / v, and even more preferably from about 60% v / v to about 75% v / v. Preferably the final concentration of the quenching and precipitating agent in the precipitation mixture is about 73% v / v.

[0075] The alcohol may be a Ci to C3alcohol. For example, the alcohol may be methanol, ethanol, propanol, or isopropanol. Preferably the alcohol is methanol. Preferably, the alcohol is added to the medium molecular weight heparin solution to give a final concentration of the alcohol in theprecipitation mixture of from about 50% v / v to about 90% v / v, more preferably from about 55% v / v to about 80% v / v, and even more preferably from about 60% v / v to about 75% v / v. Preferably the final alcohol concentration in the precipitation mixture is about 73% v / v. For example, addition of 73 m L of an alcohol, e.g. methanol, to a 27 mL medium molecular weight heparin solution provides a final alcohol concentration of 73% v / v MeOH in the precipitation mixture.

[0076] Without wishing to be bound by theory, it is believed that when the alcohol comprises methanol the remaining l\lalO4is converted to NalO3by reaction with the methanol and this remains in the mother liquor / supernatant. The formic acid and formaldehyde resulting from the reaction of NalO4with the methanol also remains in the liquor / supernatant. The precipitate obtained is a handleable, filtrable powder which can readily be isolated by filtration and subsequently dried under vacuum. It will be readily appreciated that if, for example, ethanol is used as the alcohol, acetic acid will be formed instead of formic acid and acetaldehyde will be formed instead of formaldehyde. In this instance, these byproducts will also remain in the mother liquor / supernatant. The skilled person will readily understand that this also applies to other alcohols.

[0077] During the addition of the alcohol, the temperature of the solution may be kept between about 0 °C and about 15 °C, preferably between about 5 °C and about 10 °C.

[0078] In some embodiments, a salt is added to the medium molecular weight heparin solution prior to quenching the oxidising agent and precipitating the medium molecular weight heparin. The step of quenching the oxidising agent and / or precipitating the medium molecular weight heparin may comprise adding a salt to the medium molecular weight heparin solution followed by addition of the quenching agent, precipitating agent, or quenching and precipitating agent. As described above, the quenching agent, the precipitating agent and the quenching and precipitating agent may consist of, consists essentially of or comprises an alcohol.

[0079] The salt may be a lithium salt, a sodium salt, or a potassium salt. Preferably, the salt is a sodium salt. The salt may be lithium chloride, sodium chloride, potassium chloride. Preferably the salt is sodium chloride.

[0080] The mass of salt added to the medium molecular weight heparin solution is selected to result in a final salt concentration in the medium molecular weight heparin solution of from about 0.5% m / v to about 3% m / v, preferably from about 1% m / v to about 2.5% m / v, and even more preferably fromabout 1.5% to about 2% m / v. Preferably the concentration of the salt is about 1.8% m / v. For example, addition of addition of 0.9 g solid NaCI to a 50 mL medium molecular weight heparin solution provides a NaCI concentration of 1.8% m / v. Most preferably, the salt solution comprises sodium chloride at a concentration of 1.8% m / v.

[0081] Following addition of the salt, the resulting solution may be cooled to from about 5 °C to about 20 °C , preferably from about 10 °C to about 15 °C . Preferably, the resulting solution may be cooled to about 15 °C. The quenching agent, precipitating agent or quenching and precipitating agent may then be added.

[0082] In an alternative embodiment, the method of the first aspect may further comprises the step of inactivating the oxidising agent in the medium molecular weight heparin solution. The oxidising agent may be inactivated by the addition of an inactivating agent selected from the group consisting of: methanol, D-mannitol, glycerol, A / -acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred inactivating agent is methanol or D-mannitol.

[0083] The molar ratio of the oxidisin agent to the inactivating agent may be from about 1:1 to about 1:10, more preferably from about 1:2 to about 1:8, more preferably from about 1:3 to about 1:6, more preferably from about 1:4 to about 1:5. Typically, the molar ratio of the oxidising agent to the inactivating agent may be about 1:2 or about 1:4.

[0084] In this embodiment, the method of the first aspect may further comprise the step of dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample.

[0085] "Dialysis" or "dialysing" means the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, for example dialysis tubing. The sample for dialysis and a dialysate (or buffer) are placed on opposite sides of the semipermeable membrane. Target sample molecules (e.g. proteins, DNA or polysaccharides) that are larger than the membrane pores remain on the sample side of the membrane. Contaminants such as small molecules and salts can pass through the membrane into the dialysate, thereby reducing the concentration of contaminants in the sample to low levels. Changing the dialysate for fresh dialysate removes the contaminants that have passed from the sample to the dialysate. This allows more contaminants to diffuse from the sample into the dialysate.

[0086] Dialysis can separate small molecules such as salts, reducing agents, or dyes, from larger macromolecules such as proteins, DNA or polysaccharides. Dialysis can also be used to separate polysaccharides by molecular weight. The semipermeable membrane is typically made of a film of regenerated cellulose or cellulose esters.

[0087] Dialysis may be carried out by placing a dialysis tubing containing a sample in a dialysate. A "dialysate" is the fluid into which material passes from the dialysis tubing. The dialysate may be refreshed as often as necessary to achieve optimum separation. The dialysis may be carried out over a period of about 1 day to about 14 days, preferably about 5 days to about 10 days, preferably about 7 days. The dialysate may be refreshed about 1 time a day to about 10 times a day, preferably about 2 times a day to about 5 times a day, preferably about 3 times a day. Typically, the dialysate is many times the volume of the sample, for example from about 2 to about 500 times the volume of the sample. The dialysate may be about 4 times the volume of the sample.

[0088] Typically, the dialysate is water. Optionally, the dialysate may comprise electrolytes such as sodium, potassium, magnesium, calcium, chloride, bicarbonate, lactate, glucose, amino acids, or combinations thereof.

[0089] The dialysing step may be carried out in 2 kD cut off tubing, for example provided by Spectra / Por®. The skilled person is aware of the appropriate tubing cut off size for different purposes. Alternatively, the dialysing step may be carried out in a dialysis device or dialyzer. Suitable dialyzers may be the Slide-A-Lyzer™, the Float-A-Lyzer, the Pur-A-lyzer, the D-Tube, and GeBAflex Dialyzers product lines.

[0090] In this embodiment, the method of the first aspect may further comprise the step of isolating the medium molecular weight heparin from the dialysed heparin sample. The medium molecular weight heparin may be isolated from the dialysed heparin sample by freeze-drying, centrifuging, or filtration. Preferably, the medium molecular weight heparin is isolated from the dialysed heparin sample by freeze-drying.

[0091] "Freeze drying" (also known as lyophilisation or cryodesiccation) is a drying process carried out at low temperature. Freeze drying generally involves reducing temperature and pressure to below the substance's triple point and removing the frozen solvent (e.g. water ice) by sublimation. Foraqueous compositions, such as those disclosed herein, freeze drying may be carried out at temperatures of from about -20 °C to about -80 °C, preferably about -40 °C, and pressures of from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0092] The MMWH may be purified by any suitable method known to the skilled person. Thus, in this embodiment, the method may further comprise the step of purifying the medium molecular weight heparin. For example, the MMWH may be purified by exhaustive dialysis using phosphate buffer (pH = 7.0) or saline, a desalting column (e.g. Sephadex G-25 with phosphate buffer (pH = 7.0) or saline used as the mobile phase) or precipitation of the M MWH.

[0093] In general, the method of the first aspect may include an alkaline elimination step. Alternatively, the method of the first aspect may not include an alkaline elimination step. Alkaline elimination may be carried out using an alkali salt such as, for example, sodium hydroxide, potassium hydroxide or lithium hydroxide. The alkaline elimination step may be carried out by addition of an alkali salt to increase the pH of the medium molecular weight heparin solution to about pH 10 to about pH 14, preferably about pH 12, for about 10 minutes to about 3 hours, preferably about 30 minutes, at about room temperature.

[0094] Preferably, the method does not include an alkaline elimination step. Preferably, the method does not include the addition of an alkali metal salt, for example an alkali metal salt such as NaOH, KOH or LiOH. Preferably, the method does not include the addition of NaOH, KOH or LiOH. Surprisingly, a method that does not include an alkaline elimination step or the addition of an alkali metal salt produces medium molecular weight heparin as defined herein that displays very low activity against Factor Ila and / or Xa as compared to UF heparin and low molecular weight heparin.

[0095] Preferably, the method of the first aspect does not include an acid hydrolysis step. An acid hydrolysis step may be performed to further cleave the heparin polysaccharide chain. For example, following step c the method does not include an acid hydrolysis step. Surprisingly, a method that does not include an acid hydrolysis step and particularly an acid hydrolysis following step c produces a medium molecular weight heparin as defined herein that displays very low activity against Factor Ila and / or Xa as compared to UF heparin and low molecular weight heparin.

[0096] The method described herein may not include the addition of acid, particularly the method described herein may not include the addition of an acid following step c.

[0097] MMWH may be characterised by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), Factor X analysis. The MMWH may comprise a peak in the NMR spectrum corresponding to an aldehyde. The MMWH may comprise a peak in the NMR spectrum in region of 9- 10 ppm. The MMWH may comprise a peak in the NMR spectrum in region of 9.20-9.30 ppm. The MMWH may have activity in a ristocetin-induced platelet aggregation assay comparable to that of UF heparin and little or no activity in Factor X analysis. The MMWH may reduce the amplitude observed in a RIPA assay compared to control and have less than 5 lU / mg activity in a Factor X analysis. The MMWH may have less than 5 lU / mg activity in a Factor X analysis.

[0098] The MMWH may be characterised by a molecular weight analysis. This may be performed by high pressure size exclusion chromatography (HP-SEC). The MMWH may have a ratio of Msooo-isooo to MI6OOO-24OOO greater than 2 and less than 10. The MMWH may be comprised of less than 10%, preferably less than 8%, polysaccharide chains with a mass greater than or equal to 24000 Da (g / mol). The MMWH may be comprised of between 2% and 8% polysaccharide chains with a mass greater than or equal to 24000 Da (g / mol). The MMWH may be comprised of 21% or less polysaccharide chains with a mass between 16000 Da (g / mol) and 24000 Da (g / mol). The MMWH may be comprised of between 14% and 21% polysaccharide chains with a mass between 16000 Da (g / mol) and 24000 Da (g / mol). The MMWH may be comprised of between 12% and 31% polysaccharide chains with a mass equal to or less than 8000 Da (g / mol).

[0099] This method is suitable for preparing medium molecular weight heparin on the milligram, gram or kilogram scale.

[0100] Advantageously, the method of the first aspect provides medium molecular weight heparin reliably, in good purity, and with reduced degradation.

[0101] When heparin is exposed to an oxidant (eg. IO4', periodate ion), the diol portion of the glucoronic acid is broken and two aldehyde groups are formed which as described herein results in MMWH. The result is a significant reduction in the activity as performed by the anti-Xa and anti-lla tests routinely performed on heparin. It is proposed that this oxidation of the glucuronic acid changes the binding capability of heparin to antithrombin III (ATI 11). Ristocetin-induced platelet aggregation (RIPA) tests of MMWH show an increased activity of MMWH, indicating that the von Willebrand Factor interaction of the heparin polysaccharide remains intact.

[0102] The two aldehyde groups on the glucuronic acid of the MMWH produced during preparation may in some circumstances be reactive. This reactivity can potentially decrease the stability of the oxidized polysaccharide over time. Additionally, it may interact with free amines on proteins when injected into the body. The latter would be through a Schiff base reaction with the free amines in the same way as glucose reacts with hemoglobin to form the well-known glycated hemoglobin commonly known as A1C.

[0103] Thus, if mitigation of these potential side reactions is required, the method of the first aspect may also go on to remove these aldehyde groups.

[0104] Thus, the method may further comprise the step of incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red). Preferably, the reducing agent is a mild reducing agent. The reducing agent may be sodium borohydride (NaBH , sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3) or potassium borohydride (KBH4).

[0105] The term "reduced" in the context of reduced medium molecular weight heparin means that the aldehyde groups formed in MMWH by oxidation of one or more of (or a plurality of, or substantially all of, or all of) the glucuronic acid moieties (in step (c) of the method) are reduced to hydroxyl groups. The one or more (or a plurality, or substantially all, or all) glucuronic acid derived moieties in MMWH-Red have the following structure:

[0106] The one or more (or a plurality, or substantially all, or all) glucuronic acid derived moieties in MMWH have the following structure:Preferably, substantially all or all of the glucuronic acid moieties in MMWH are have this structure.

[0107] The mild reducing agent may selectively reduce aldehydes and ketones to alcohols in the presence of esters. In other words, the mild reducing agent reduces aldehydes and ketones to alcohols at a faster rate than it reduces esters to alcohols. The mild reducing agent does not reduce carboxylic acids, nitriles, and amides under normal conditions. Preferably, the mild reducing agent may be sodium borohydride (Na BH4).

[0108] Typically the reducing agent is used at greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to medium molecular weight heparin. Typically the reducing agent is used at less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to medium molecular weight heparin. The reducing agent may be used in amount of between about 0.5 molar equivalents and about 15 molar equivalents, or between about 1 molar equivalents and about 10 molar equivalents, or between about 2 molar equivalents and about 6 molar equivalents.

[0109] Preferably, the reducing step is carried out at from about 0 °C to about 30 °C, preferably from about 5 °C to about 30 °C, preferably from about 10 °C to about 25 °C, preferably from about 10 °C to about 20 °C, preferably from about 15 °C to about 25 °C, preferably from about 20 °C to about 25 °C. Typically, the reduction step is carried out at about 20 °C. typically, the reduction step is carried out at room temperature. Typically, the reduction step is carried out for from about 1 hour to about 24 hours, preferably from about 2 hours to about 16 hours, more preferably from about 3 hours to about 12 hours, more preferably from about 6 hours to about 10 hours.

[0110] The solvent for this step may typically be selected from the group consisting of methanol, ethanol, water, THF, or combinations thereof. Preferably, the solvent is water.

[0111] The MMWH-Red may be purified by any suitable method known to the skilled person. Thus, the method may further comprise the step of purifying the MMWH-Red. For example, the MMWH- Red may be purified by exhaustive dialysis using phosphate buffer (pH = 7.0) or saline, a desalting column (e.g. Sephadex G-25 with phosphate buffer (pH = 7.0) or saline used as the mobile phase) or precipitation of the MMWH-Red.

[0112] MMWH-Red as described herein does not comprise an aldehyde or comprises fewer aldehydes than MMWH produced during the method of to the second aspect. The presence of an aldehyde or lack thereof may be determined using 2,4-Di n itrophenyl Hydrazine test. MMWH-Red may not produce an orange-yellow precipitate with reacted with 2,4-dinitrophenylhydrazine or less orange-yellow precipitate than a corresponding amount of MMWH. Alternatively, the reduction of MMWH to MMWH-Red may be monitored by infra-red spectroscopy or ^-NMR.

[0113] As described herein, reduction step (d) means that the cyclic ring of the glucuronic acid monosaccharide is not reformed and the binding of the MMWH-Red to ATIII would be to be unchanged. However, the reduced MMWH would still maintain the interaction with von Willebrand factor and be useful for the treatment of endotheliopathy.

[0114] The MMWH-Red prepared as described herein may have an average molecular weight in the range of greater than about 8000 Da (g / mol) to about 13 500 Da (g / mol), preferably greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), more preferably from about 10 000 Da (g / mol) to about 12 000 Da (g / mol).

[0115] The MMWH-Red prepared as described herein may comprise polysaccharide chains with an average molecular weight in the range of greater than about 8000 Da (g / mol) to about 13 500 Da (g / mol), more preferably greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12 000 Da (g / mol).

[0116] The molecular weight of the MMWH-Red prepared as described herein may be determined by size exclusion chromatography as described herein.

[0117] The MMWH-Red may comprise at least three units of a GlcNS6S-ldoA2S (or ldoA2S-GlcNS6S) disaccharide. The GlcNS6S and ldoA2S monosaccharides are linked by an al-4 linkage between GlcNS6S and ldoA2S, i.e. GlcNS6Sal-4ldoA2S. For example, the MMWH-Red may comprise at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of a GlcNS6S-ldoA2S disaccharide. The MMWH-Red may comprise less than or equal to 25 units of the GlcNS6S-ldoA2S disaccharide, for example less than or equal to 20 units. The presence of the units of the GlcNS6S-ldoA2S disaccharide may be determined by an antibody, mass spectrometry, or inferred from chemical and enzymatical studies. The GlcNS6S-ldoA2S units may be ordered in succession.

[0118] The MMWH-Red may comprise UA2S-GlcNS6S, UA2S-GlcNS, UA-GIcNAc, wherein U may be iduronic acid (IdoA) or glucuronic acid (GlcA). The MMWH-Red may comprise at least about 60% UA2S- GlcNS6S, UA2S-GlcNS, and UA-GIcNAc. The MMWH-Red may comprise at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% UA2S-GlcNS6S. The MMWH-Red may comprise up to about 60%, preferably up to about 70%, preferably up to about 85% UA2S-GlcNS6S. The MMWH-Red may comprise at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% UA2S-GlcNS. The MMWH-Red may comprise up to about 15%, preferably up to about 20% UA2S-GlcNS. The MMWH-Red may comprise at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% UA-GIcNAc. The MMWH-Red may comprise up to about 15%, preferably up to about 20% UA-GIcNAc. In some embodiments the MMWH-Red may comprise at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN and 5.4% UA-GIcNAc. In some embodiments the MMWH-Red may comprise at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS and 9% UA-GIcNAc. The percentage composition of UA-GIcNAc comprised in the MMWH- Red may be enriched compared to unfractionated heparin.

[0119] In a further aspect, the invention provides MMWH produced according to the method of the first aspect.

[0120] In an even further aspect, the invention provides MMWH-Red produced according to the method of the first aspect.

[0121] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubatingthe second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and (d) purifying the medium molecular weight heparin. Step (d) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0122] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and (d) purifying the medium molecular weight heparin. Step (d) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0123] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) purifying the medium molecular weight heparin; and (e) preparing a composition comprising the medium molecular weight heparin for biological testing. Step (d) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0124] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) purifying the medium molecular weight heparin; and (e) preparing a composition comprising the medium molecular weight heparin for biological testing. Step (d) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0125] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubatingthe second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) purifying the medium molecular weight heparin; and (e) preparing a composition comprising the medium molecular weight heparin for biological testing. Step (d) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0126] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) purifying the medium molecular weight heparin; and (e) preparing a composition comprising the medium molecular weight heparin for biological testing. Step (d) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0127] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; and (e) purifying the medium molecular weight heparin. Step (e) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0128] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; and (e) purifying the medium molecular weight heparin. Step (e) may comprise quenching the oxidising agent, precipitating the medium molecular weight heparin and isolating the medium molecular weight heparin.

[0129] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; and (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample.

[0130] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; and (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample.

[0131] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample; and (f) Isolating the medium molecular weight heparin from the dialysed heparin sample.

[0132] The method of synthesis of medium molecular weight heparin may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample; and (f) Isolating the medium molecular weight heparin from the dialysed heparin sample.

[0133] In a second aspect, the invention provides a kit suitable for preparing medium molecular weight heparin, wherein the kit comprises (a) unfractionated heparin; (b) an aqueous buffer solution adjusted to between about pH 4.0 and about pH 9.0; (c) an oxidising agent; and (d) optionally, a quenching agent, a precipitating agent or a quenching and precipitating agent. The quenching and precipitating agent may comprise an alcohol as described herein. The quenching and precipitating agent may comprise an alcohol and a salt as described herein.

[0134] MMWH produced by the method of the first aspect of the invention includes two aldehyde groups on the glucuronic acid. In some situations, the reactivity of the aldehyde groups may result in reaction of the aldehyde groups with amine residues on proteins within the body via a Schiff base reaction. The method of the first aspect may further comprise incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

[0135] In an third aspect, the invention provides a method of synthesis of reduced medium molecular weight heparin (MMWH-Red) comprising the steps of: (a) dissolving unfractionated (UF) heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

[0136] The method may comprise: (a) dissolving unfractionated (UF) heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and (d ) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

[0137] The reduced medium molecular weight heparin prepared by the method of the third aspect of the invention may comprise polysaccharide chains with an average molecular mass in the range of about greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), preferably about 10 000 Da (g / mol) to about 12 000 Da (g / mol). The reduced medium molecular weight heparin prepared by the method of the third aspect of the invention may comprise polysaccharide chains with an average molecular mass of about 11 000 Da (g / mol).

[0138] The molecular weight of the MMWH-Red prepared by the method of the third aspect of the invention may be determined by size exclusion chromatography as described herein.

[0139] The MMWH-Red prepared by the method of the first aspect may comprise at least three units of a GlcNS6S-ldoA2S (or ldoA2S-GlcNS6S) disaccharide. The GlcNS6S and ldoA2S monosaccharides are linked by an al-4 linkage between GlcNS6S and ldoA2S, i.e. GlcNS6Sal-4ldoA2S. For example, the MMWH-Red prepared by the method of the third aspect may comprise at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of a GlcNS6S-ldoA2S disaccharide. The MMWH-Red prepared by the method of the third aspect may comprise less than or equal to 25 units of the GlcNS6S-ldoA2S disaccharide, for example less than or equal to 20 units. The presence of the units of the GlcNS6S-ldoA2S disaccharide may be determined by an antibody, mass spectrometry, or inferred from chemical and enzymatical studies. The GlcNS6S- ldoA2S units may be ordered in succession.

[0140] The MMWH-Red prepared by the method of the first aspect may comprise UA2S-GlcNS6S, UA2S-GlcNS, UA-GIcNAc, wherein U may be iduronic acid (IdoA) or glucuronic acid (GlcA). The medium molecular weight heparin prepared by the method of the third aspect may comprise at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GIcNAc. The MMWH-Red prepared by the method of the third aspect may comprise at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% UA2S-GlcNS6S. The MMWH-Red prepared by the method of the third aspect may comprise up to about 60%, preferably up to about 70%, preferably up to about 85% UA2S-GlcNS6S. The MMWH-Red prepared by the method of the third aspect may comprise at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% UA2S-GlcNS. The MMWH-Red prepared by the method of the third aspect may comprise up to about 15%, preferably up to about 20% UA2S-GlcNS. The MMWH-Red prepared by the method of the third aspect may comprise at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% UA-GIcNAc. The MMWH-Red prepared by the method of the third aspect may comprise up to about 15%, preferably up to about 20% UA-GIcNAc. In some embodiments the MMWH-Red prepared by the method of the third aspect may comprise at least 49.2% UA2S-GlcNS6S, 5.4% UA2S- GlcN and 5.4% UA-GIcNAc. In some embodiments the MMWH-Red prepared by the method of the third aspect may comprise at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS and 9% UA-GIcNAc. The percentage composition of UA-GIcNAc comprised in the MMWH-Red may be enriched compared to unfractionated heparin.

[0141] The method of the first aspect may further comprise the step of inactivating the oxidising agent in the MMWH-Red solution. The oxidising agent may be inactivated by the addition of an inactivating agent selected from the group consisting of: D-mannitol, glycerol, / -acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred inactivating agent is D-mannitol.

[0142] The molar ratio of the oxidising agent to the inactivating agent may be from about 1:1 to about 1:10, more preferably from about 1:2 to about 1:8, more preferably from about 1:3 to about 1:6, more preferably from about 1:4 to about 1:5. Typically, the molar ratio of the oxidising agent to the inactivating agent may be about 1:2 or about 1:4.

[0143] The MMWH-Red may be purified by any suitable method known to the skilled person. Thus, the method may further comprise the step of purifying the medium molecular weight heparin. For example, the MMWH-Red may be purified by exhaustive dialysis using phosphate buffer (pH = 7.0) or saline, a desalting column (e.g. Sephadex G-25 with phosphate buffer (pH = 7.0) or saline used as the mobile phase) or precipitation of the MMWH-Red.

[0144] For example, the method may further comprise dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample prior to the reducing step.

[0145] This method is suitable for preparing reduced medium molecular weight heparin on the milligram, gram or kilogram scale.

[0146] Advantageously, the method of the third aspect provides reduced medium molecular weight heparin reliably, in good purity, and with reduced degradation.

[0147] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated (UF) heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (e) purifying the MMWH-Red.

[0148] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated (UF) heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (e) purifying the MMWH-Red.

[0149] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (f) purifying the MMWH-Red.

[0150] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (f) purifying the MMWH-Red.

[0151] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample; (f) incubating the dialysed medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (g) purifying the MMWH-Red.

[0152] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample; (f) incubating the dialysed medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (g) purifying the MMWH-Red.

[0153] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solution to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample; (f) Isolating the medium molecular weight heparin from the dialysed heparin sample; (g) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (h) purifying the MMWH-Red.

[0154] The method of synthesis may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the medium molecular weight heparin solution in a dialysate to provide a dialysed medium molecular weight heparin sample; (f) Isolating the medium molecular weight heparin from the dialysed heparin sample; (g) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (h) purifying the MMWH-Red.

[0155] Both MMWH and MMWH-Red inhibit von Willebrand Factor, and so are appropriate active pharmaceutical ingredients (APIs) and drug products for the purposes of the invention.

[0156] Advantageously, MMWH-Red may display greater stability as an API and drug product compared to MMWH. Furthermore, MMWH-Red may reduce the potential for side reaction with excipients comprised in the drug product and MMWH-Red may reduce interactions with proteins in vivo.

[0157] MMWH-Red may be characterised by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), Factor X analysis.

[0158] The MMWH-Red may comprise a chemical modification. The chemical modification may be selected from the group comprising N-acetylation, N-deacetylation, N-sulfation, O-sulfation, 2-0 desulfation, and complete desulfation.

[0159] For the avoidance of doubt, embodiments related to each aspect of the invention apply mutatis mutandis to the other aspects of the invention. Further aspects and embodiments of the present invention will be evident from the discussion herein.

[0160] Every document cited herein, including any cross-referenced or related patent or application, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited.

[0161] It will be appreciated that various modifications may be made to the embodiments shown without departing from the spirit and scope of the invention as defined by the accompanying claims.EXAMPLES

[0162] The invention will now be demonstrated by reference to the following non-limiting examples.

[0163] Unless otherwise mentioned, room temperature and pressure are 20 °C (293.15 K, 68 °F) and 1 atm (14.696 psi, 101.325 kPa), respectively.Experimental MethodsSize Exclusion Chromatography (SEC)

[0164] SEC is performed on a GEC Superdex75 (10 / 100) column on an Agilent 1200 HPLC system with variable wavelength UV monitor or GEC AKTA system with variable wavelength UV monitor. The mobile phase is 0.15M NaCI in water, which is passed through the column at 0.4 mL / min. The optical density is measured at 232 nm. SEC is performed with reference to an 11 kD standard.Freeze drying

[0165] Freeze drying (also known as lyophilisation or cryodesiccation) is a drying process carried out at low temperature. Freeze drying generally involves reducing temperature and pressure to below the substance's triple point and removing the frozen solvent (e.g. water ice) by sublimation. For aqueous compositions, such as those disclosed herein, freeze drying may be carried out at temperatures of from about -20 °C to -80 °C, preferably about -40 °C, and pressures of from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).Method of MMW Heparin Manufacture

[0166] 3 grams of unfractionated heparin was dissolved in 50 mL of milli Q water at room temperature. 1.19 grams of solid NalO4 was then added under stirring and the reaction was allowed to proceed at room temperature under stirring for 3 hours.

[0167] Then 0.9 grams of solid NaCI was added to the reaction mixture to obtain a NaCI concentration of 1.8% m / v. The solution was cooled to 15 °C. 14 mL methanol is added to the reaction mixture. The solution was cooled to 5 °C and 116 mL of methanol is added to obtain a final methanol concentration of 73% v / v while maintaining the temperature between 5 and 10 °C.

[0168] After completion of the methanol addition, the precipitate was allowed to settle 2 hours at room temperature. The supernatant is removed by decanting and the precipitate is washed with methanol several times to obtain a water content less than 5%. The precipitate was then isolated by filtration, washed with methanol and dried in vacuo at room temperature overnight.

[0169] The above method is representative of the method of MMWH synthesis. It will be appreciated that conditions may vary as outlined in Table I.

[0170] Table I below shows the molecular weight analysis of unfractionated heparin (UF heparin) and UF heparin treated according to the reaction conditions specified in the table (n / a - not applicable; ND - not determined; RT - room temperature; none - none added).

[0171] Samples ID24-006-4, ID24-006-6 and ID24-006-7, for example, share similar reaction conditions except that the time of reaction was varied. These examples demonstrate that Mw of MMWH can be controlled by varying the reaction conditions. Thus, the invention disclosed herein provides a tuneable method of MMWH preparation, allowing the targeting of specific MMWH properties, in particular Mw.Table I:Method of MMWH-Red ManufactureOxidation of Heparin

[0172] UFH heparin is oxidised as described above under Method ofMMW Heparin Manufacture.Reduction of the Oxidized Heparin

[0173] To a 10 mg / mL solution of the oxidised heparin in deionised water (110 mL) is added 20 mg of sodium borohydride. The reaction mixture is stirred for 3 hours at 25 °C. The reduced medium molecular weight heparin is purified by exhaustive dialysis using phosphate buffer (pH = 7.0).

[0174] As shown in Figure 2, monitoring of the reduction reaction by ^-NMR shows that the aldehyde signal present in MMWH at ~9.25 ppm disappears during the reduction reaction to prepare MMWH-Red, the hydroxyl form, thus indicating preparation of MMWH-Red.

[0175] The MMWH-Red prepared according to the above method has a Factor Xa activity of <10 lU / mg. MMWH (intermediate in this process) has a Factor Xa activity of <5 lU / mg. Unfractionated heparin has a Factor Xa activity of 169.24 lU / mg. Reduced medium molecular weight heparin displayed a very low activity against Factor Xa as compared to unfractionated heparin and low molecular weight heparin. Thus, unlike UF Heparin or LMW Heparin, MMWH-Red does not affect Factor Xa-mediated coagulation.

[0176] MMWH-Red was also found to display inhibition in Ristocetin-induced platelet aggregation(RIPA) assay.

[0177] Protocol according to "Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC / ISTH", Journal of Thrombosis and Haemostasis, 2013, 11: 1183-1189.

[0178] Blood samples were taken from non-smokers not on any anti-platelet therapy (for example, aspirin). An anti-platelet therapy may also be known as a platelet agglutination inhibitor or a platelet aggregation inhibitor.

[0179] Blood was drawn, with no venostasis, from a donor into 109 mM sodium citrate solution (VACUETTE, 3.5 mL #454327, lot#A230836U). The first 3 to 4 mL of blood drawn was discarded.

[0180] Blood samples were allowed to 'rest' at room temperature for 15 min before centrifugation. Platelet rich plasma (PRP) was prepared by centrifuging blood samples at 200 g for 10 min at 21 °C, without using brake. Platelet poor plasma (PPP) was prepared by centrifuging blood samples, from which PRP was removed, at 1500 g for 15 min at 21 °C.

[0181] An assessment of PRP quality was made by carrying out a platelet count of the PRP. Platelet count in PRP was 421 G / L. The platelet count of PRP samples was not (and should not be) adjusted to a standardised value with autologous PPP.

[0182] After centrifugation, PRP samples were allowed to sit at room temperature for 15 min before Light Transmission Aggregometry (LTA) studies were carried out. PRP was used to set 0% light transmission in the aggregometer. Autologous PPP was used to set 100% light transmission in the aggregometer. LTA studies were carried out at 37 °C. Before adding agonist, baseline tracings for LTA were observed for oscillations and stability for at least 1 minute. The volume of agonist added for LTA was consistent, and never more than 10% of the total sample volume.

[0183] 5.2 mM H2O solutions of MMWH or MMWH-red were prepared.Ristocetin working solution:- 20 pL of Ristocetin stock (24 mg / mL ristocetin in saline).Sample working solution:-10 pL of 5.2mM MMWH or MMWH-red solution was combined with 163 pL saline to provide a 300 pM working solution of each of the samples.RIPA protocol:360 piL PRP was diluted with Agrastat (5 pg / mL; 1 / 10, antagonist of GPIIIbllla). 20 pL of sample working solution or 20 pL saline (control) was added (to give a final concentration of sample of 15 pM). The solution was stirred for 2 seconds and then incubated for 10 minutes at 37 °C without stirring. The solution was then incubated for 1 minute at 37 °C with agitation (baseline tracking). 20 pL of ristocetin working solution was added and the platelet agglutination measured.

[0184] Each experiment was carried out in duplicate. The amplitude, slope and Area under the curve (AUC) calculated by software.Results:

[0185] Both MMWH and MMWH-Red were found to lower the amplitude, slope and area under the curve measured to a greater extent than control (see Figure 1). Thus, MMWH and MMWH-Red prepared according to the methods described herein were found to display inhibition in Ristocetin- induced platelet aggregation (RIPA). This indicates that the von Willebrand Factor interaction of the heparin polysaccharide remains intact.Factor Xa activity

[0186] Factor Xa is the activated form of the coagulation factor X. Factor X is a serine endopeptidase enzyme, which plays a key role at several stages of the coagulation system.

[0187] Heparin (unfractionated heparin) and its derivatives, e.g. low molecular weight heparin, bind to a plasma cofactor, antithrombin (AT) to inactivate several coagulation factors Ila, Xa, Xia and XI la. This inactivation of Factor Xa by heparins is termed "indirect" since it relies on the presence of AT and not on a direct interaction with Factor Xa.

[0188] As shown in the table below, MMWH prepared by incubating unfractionated heparin and an oxidising agent at room temperature exhibits low activity against factor Xa. MMWH prepared by incubating unfractionated heparin an oxidising agent at low temperature has low activity against factor Xa.HPLC analysis

[0189] Unfractionated heparin has a molecular weight around 16000 Daltons. The oxidized material should have a reduced molecular weight of around 11,000 Daltons. One way to confirm the reaction has been completed would be by a retention time shift towards lower molecular weight using size exclusion chromatography (SEC).

[0190] HPLC conditions:

[0191] Sample Bl: 27.16mg Sodium heparin in 10 mL of 0.05M phosphate buffer, pH 7

[0192] 6 mL of sample Bl was combined with 35 mg sodium periodate. 3 mL of this mixture was stored overnight at 3°C (sample B2) and 3 mL of this mixture was stored at room temperature overnight.

[0193] Sample Bl, sample B2 and sample B3 were analysed using SEC. FIG. 3 shows the chromatogram for sample Bl, FIG. 4 shows the chromatogram for sample B2 and FIG. 5 shows the chromatogram for sample B3. The x-axis is retention time and the y-axis is light absorbance.

[0194] Sample Bl had a retention time of 27.106 minutes. Sample B2 had a retention time of 27.673 minutes. Sample B3 had a retention time of 28.290 minutes.EMBODIMENTS OF INVENTION1. A method of synthesis of medium molecular weight heparin, the method comprisin the steps of: a. Dissolving unfractionated heparin in an aqueous solution to provide a first solution; b. Adding an oxidising agent to the first solution to provide a second solution; and c. Incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.2. The method according to embodiment 1, wherein the method further comprises step (d) which comprises: i. Quenching the oxidising agent to provide a quenched solution; ii. precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture; or iii. quenching and precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture.3. The method according to embodiment 2 wherein step (d) comprises the addition of a quenching and precipitating agent to the medium molecular weight heparin solution to provide a medium molecular weight heparin precipitate in a precipitation mixture.4. The method according to embodiment 3 wherein the quenching and precipitating agent comprises an alcohol.5. The method according to embodiment 4 wherein the alcohol comprises methanol.6. The method according to any one of embodiments 3-5 wherein the quenching and precipitating agent is added to give a final concentration the quenching and precipitating agent in the precipitation mixture of between about 50% v / v to about 90% v / v.7. The method according to any one of embodiments 2-6 wherein a salt is added to the medium molecular weight heparin solution prior to step d.8. The method according to embodiment 7 wherein the salt is added to a final salt concentration in the medium molecular weight heparin solution of from about 0.5% m / v to about 3% m / v.9. The method according to embodiment 7 or embodiment 8 wherein the salt is a sodium salt.10. The method according to embodiment 9 wherein the sodium salt is sodium chloride.11. The method according to any one of embodiments 2-10 wherein the medium molecular weight heparin is isolated by filtration.12. The method according to any preceding embodiment, wherein the aqueous solution is water.13. The method according to any preceding embodiment, wherein the incubating step c. is carried out for from about 1 hour to about 48 hours.14. The method according to any preceding embodiment, wherein the incubation temperature in step c. is about 18 °C.15. The method according to any preceding embodiment, wherein the temperature of the aqueous solution in step a. is from about 15 °C to about 30 °C.16. The method according to any preceding embodiment, wherein the oxidising agent is a periodate, preferably sodium periodate or potassium periodate.17. The method according to any preceding embodiment, wherein the medium molecular weight heparin has an average molecular weight of from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol), preferably wherein the medium molecular weight heparin has an average molecular weight of about 11000 Da (g / mol).18. The method according to any preceding embodiment, wherein the medium molecular weight heparin comprises at least three units of a ldoA2S-GlcNS6S disaccharide.19. The method according to any preceding embodiment wherein the method does not include an alkaline elimination step.20. The method according to any preceding embodiment wherein the method does not include the addition of an alkali metal salt.The method according to embodiment 20 wherein the alkali metal salt comprises NaOH, KOH or LiOH. The method according to embodiment 20 wherein the alkali salt is NaOH, KOH or LiOH. The method according to any preceding embodiment, wherein the method further comprises incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin. The method according to embodiment 23 wherein the reducing agent is a mild reducing agent, preferably wherein the mild reducing agent is sodium borohydride. A kit suitable for preparing medium molecular weight heparin, wherein the kit comprises: a. unfractionated heparin; b. an aqueous solution; c. an oxidising agent; and d. optionally, an inactivating agent.

Claims

1. CLAIMSWhat is claimed is:

1. A method of synthesis of medium molecular weight heparin, the method comprising the steps of: a. Dissolving unfractionated heparin in an aqueous solvent to provide a first solution; b. Adding an oxidising agent to the first solution to provide a second solution; and c. Incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution.

2. The method according to claim 1, wherein the method further comprises purifying the medium molecular weight heparin from the medium molecular weight heparin solution.

3. The method according to claim 2, wherein purifying the medium molecular weight heparin comprises precipitating and filtering the medium molecular weight heparin, dialysis, ion exchange, lyophilization, size exclusion chromatography, ultrafiltration, or a combination thereof.

4. The method according to claim 1, wherein the method further comprises step (d) which comprises: i. Quenching the oxidising agent to provide a quenched solution; ii. precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture; or iii. quenching and precipitating the medium molecular weight heparin to provide a medium molecular weight heparin precipitate in a precipitation mixture.

5. The method according to claim 4 wherein step (d) comprises the addition of a quenching and precipitating agent to the medium molecular weight heparin solution to provide a medium molecular weight heparin precipitate in a precipitation mixture.

6. The method according to claim 5 wherein the quenching and precipitating agent comprises an alcohol.

7. The method according to claim 6 wherein the alcohol comprises methanol.

8. The method according to any one of claims 5-7 wherein the quenching and precipitating agent is added to give a final concentration the quenching and precipitating agent in the precipitation mixture of between about 50% v / v to about 90% v / v.

9. The method according to any one of claims 4-8 wherein a salt is added to the medium molecular weight heparin solution prior to step d.

10. The method according to claim 9 wherein the salt is added to a final salt concentration in the medium molecular weight heparin solution of from about 0.5% m / v to about 3% m / v.

11. The method according to claim 9 or claim 10 wherein the salt is a sodium salt.

12. The method according to claim 11 wherein the sodium salt is sodium chloride.

13. The method according to any one of claims 4-12 wherein the medium molecular weight heparin is isolated by filtration.

14. The method according to any preceding claim, wherein the aqueous solvent is water.

15. The method according to any preceding claim, wherein the incubating step c. is carried out for from about 1 hour to about 48 hours.

16. The method according to any preceding claim, wherein the incubation temperature in step c. is about 18 °C.

17. The method according to any preceding claim, wherein the temperature of the aqueous solvent in step a. is from about 15 °C to about 30 °C.

18. The method according to any preceding claim, wherein the oxidising agent is a periodate, preferably sodium periodate or potassium periodate.

19. The method according to any preceding claim, wherein the medium molecular weight heparin has an average molecular weight of from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol), preferably wherein the medium molecular weight heparin has an average molecular weight of about 11000 Da (g / mol).

20. The method according to any preceding claim, wherein the medium molecular weight heparin comprises at least three units of a ldoA2S-GlcNS6S disaccharide.

21. The method according to any preceding claim wherein the method does not include an alkaline elimination step.

22. The method according to any preceding claim wherein the method does not include the addition of an alkali metal salt.

23. The method according to claim 22 wherein the alkali metal salt comprises NaOH, KOH orLiOH.

24. The method according to claim 22 wherein the alkali salt is NaOH, KOH or LiOH.

25. The method according to any preceding claim wherein the method does not include an acid hydrolysis step.

26. The method according to any previous claim wherein the method does not include the addition of an acid following step c.

27. The method according to any previous claim comprising preparing a composition for biological testing comprising the medium molecular weight heparin prepared in step c.

28. The method according to any preceding claim, wherein the method further comprises incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

29. The method according to claim 28 wherein the reducing agent is a mild reducing agent, preferably wherein the mild reducing agent is sodium borohydride.

30. A kit suitable for preparing medium molecular weight heparin, wherein the kit comprises: a. unfractionated heparin; b. an aqueous solvent; c. an oxidising agent; and d. optionally, an inactivating agent.

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