Method for synthesizing low molecular weight heparin from low molecular weight heparin precursor chemoenzyme

Abstract

Low molecular weight heparin (LMWH) suitable for equivalent use with USP enoxaparin sodium is prepared from starting materials isolated from engineered E. coli K5 capsular polysaccharides (e.g., E. coli K5 heparin precursors). The Escherichia coli CPS is treated with an acid to remove 3-deoxy-D-mann-oct-2-ketonic acid (Kdo) residues and is further hydrolyzed by alkali treatment to form a low molecular weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH) having a molecular weight and a degree of N-acetylation comparable to that of enoxaparin. The LMW-NSNAH is modified and converted into the LMWH through a series of enzymes of C < 5 >-epimerase, 2-O-sulfotransferase, 6-O-sulfotransferase and 3-O-sulfotransferase. The composition including LMWH is prepared without the use of porcine heparin, so that the raw materials can be obtained more easily, the production process can be better controlled, and the concern of contamination, adulteration or animal viruses or impurities is reduced. In addition, the LMWH product is proved to be structurally and functionally comparable to the traditional drug LMWH.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 276,212, filed on November 5, 2021, and U.S. Provisional Application No. 63 / 310,410, filed on February 15, 2022, which are incorporated by reference as if fully disclosed herein. Statement Regarding Federal Funding of Research and Development

[0002] This invention was made with U.S. Government support through the National Science Foundation under Grant No. DMR-1933525. The U.S. Government may have certain rights in this invention. Background Art

[0003] Heparin products are widely used clinical anticoagulants in modern medical practice. Low molecular weight heparin (LMWH) is currently produced by controlled chemical or enzymatic depolymerization of unfractionated heparin (UFH) extracted from animal tissue. LMWH has replaced UFH in many clinical applications and currently accounts for over 60% of the heparin market. In the past, significant efforts have been made to produce bioengineered UFH through chemical and enzymatic processes to address concerns about animal-derived UFH.

[0004] Heparin is typically produced from animal tissues rich in heparin proteoglycans, primarily from porcine intestine. Heparin is a linear, highly sulfated polysaccharide covalently attached to the core protein serglycan, serglycan, and is stored in intracellular granules in mast cells. It consists of repeating disaccharide units composed of either β-D-glucuronic acid (GlcA) or α-L-iduronic acid (IdoA) linked 1,4-glycosidically to D-glucosamine (GlcN). Unlike DNA and protein synthesis, heparin biosynthesis is not template-driven, resulting in polysaccharides that are heterogeneous in length and substitution patterns. Heparin biosynthesis in certain animal cells begins in the endoplasmic reticulum and involves the formation of a tetrasaccharide linker (D-xylose (Xyl)-D-galactose (Gal)-Gal-GlcA) attached to a serine residue in its core protein. Next, driven by two polymerases, exosome glycosyltransferase (EXT) 1 and EXT 2, chain polymerization proceeds through the formation of repeating disaccharide building blocks of GlcA linked to N-acetyl-α-D-glucosamine (GlcNAc) 1,4-linked to form the heparin backbone—the heparin precursor.

[0005] Now refer to Figure 1 The heparin precursor is a repeating disaccharide unit [→4)GlcA(1→4)GlcNAc(1→] nThe backbone is subsequently modified by de-N-acetylation and N-sulfation, C5-epimerization, and a series of 3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent O-sulfation reactions, all of which occur in the Golgi compartment. These reactions are catalyzed by the following enzymes: N-deacetylase / N-sulfotransferase (NDST), which forms N-sulfo-α-D-glucosamine (GlcNS) residues; C5-epimerase (Epi), which converts GlcA residues to L-iduronic acid (IdoA); and 2-O-sulfotransferase, 6-O-sulfotransferase, and 3-O-sulfotransferase (ST), which transfer sulfo groups to the polysaccharide chain. The drug heparin is polydisperse and isomerized, with an average molecular weight of 18-20 kDa.

[0006] As mentioned above, LMWHs are currently produced by controlled chemical or enzymatic depolymerization of UFH. Compared with UFH, LMWHs have several advantages in therapeutic anticoagulation, including high subcutaneous bioavailability, more predictable pharmacokinetic characteristics, longer plasma half-life, and a lower incidence of heparin-induced thrombocytopenia (HIT). Commercially available LMWHs are polydisperse, fractionated heparins with an average molecular weight range of 3-8 kDa. For example, enoxaparin (~4,500 Da) is produced by benzylation and alkaline hydrolysis, dalteparin (~6,000 Da) is derived from controlled nitrous acid depolymerization, and tinzaparin (~6,500 Da) is prepared by controlled heparinase digestion. Of these three drugs, enoxaparin produced by Sanofi is the most LMWH holds a major share of the global market and has the most extensive clinical evidence of efficacy and safety across various applications, resulting in the widest range of therapeutic indications. Recently, the patent and supplementary protection certificate for the original drug enoxaparin expired. In 2010, the US Food and Drug Administration (FDA) approved a generic version of enoxaparin, reducing the drug's price and making LMWH available to a wider patient population. However, the quality and supply of LMWH depend on the quality of animal-derived heparin. There are growing concerns about the shortage of porcine heparin, and the heparin and LMWH supply chains are facing threats from impurities, contamination, and adulteration. Consequently, efforts are underway to develop and improve technologies and methods for synthesizing UFH and LMWH.

[0007] With the successful expression of recombinant heparin biosynthetic enzymes (including glycosyltransferases, C5-Epi, and 2-OST, 6-OST, and 3-OST), the chemoenzymatic synthesis of UFH has become possible. This chemoenzymatic method closely mimics the heparin biosynthetic pathway. The preparation of bioengineered UFH starts with Escherichia coli K5 capsular polysaccharide (CPS), a heparin precursor. The heparin precursor is chemically deacetylated and N-sulfonated to produce the N-sulfoheparin precursor, which is then modified with C5-Epi and 2-OST, 6-OST, and 3-OST. This bioengineered UFH exhibits chemical and biological equivalence to pharmaceutical porcine heparin.

[0008] Using N-acetylglucosaminyltransferase (KfiA) and heparin precursor synthase (pmHS2), an isostructural, monodisperse, fondaparinux-like super-LMWH was synthesized chemoenzymatically from a uridine-5'-diphosphate (UDP)-sugar donor and a disaccharide acceptor derived from the heparin precursor. Furthermore, a single targeting construct of the isostructural dodecasaccharide LMWH was synthesized and demonstrated as a viable candidate to replace LMWH in thrombosis prevention. These chemoenzymatic processes rely on the iterative synthesis of isostructural molecular species using expensive UDP sugar donors. Summary of the Invention

[0009] Various aspects of the present disclosure relate to methods for preparing low molecular weight heparin (LMWH). In some embodiments, the method comprises providing a certain amount of heparin precursor (heparosan); contacting the heparin precursor with one or more acids to form an acid-treated heparin precursor; converting the acid-treated heparin precursor into a low molecular weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH) by depolymerization and de-N-acetylation; and enzymatically converting the LMW-NSNAH into LMWH. In some embodiments, the heparin precursor is E. coli capsular polysaccharide. In some embodiments, the heparin precursor is synthesized by an engineered strain of E. coli K5.

[0010] In some embodiments, contacting the heparin precursor with one or more acids to form the acid-treated heparin precursor comprises removing 3-deoxy-D-mannose-2-ulose acid (Kdo) residues from the heparin precursor by acid hydrolysis. In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH comprises hydrolyzing the acid-treated heparin precursor by treating the acid-treated heparin precursor with one or more bases; treating the acid-treated heparin precursor with one or more other acids; contacting the acid-treated heparin precursor with one or more enzymes; or a combination thereof. In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH by depolymerization and de-N-acetylation further comprises re-acetylation of the acid-treated heparin precursor after de-N-acetylation, and N-sulfation of the acid-treated heparin precursor to obtain LMW-NSNAH.

[0011] In some embodiments, reacetylation of the acid-treated heparin precursor after de-N-acetylation comprises contacting the acid-treated heparin precursor with acetic anhydride. In some embodiments, N-sulfation of the acid-treated heparin precursor to obtain LMW-NSNAH comprises contacting the acid-treated heparin precursor with trimethylamine sulfur trioxide, pyridine sulfur trioxide, or a combination thereof. In some embodiments, reacetylation of the acid-treated heparin precursor after de-N-acetylation comprises contacting the acid-treated heparin precursor with about 53 μM / L acetic anhydride. In some embodiments, N-sulfation of the acid-treated heparin precursor to obtain LMW-NSNAH comprises contacting the acid-treated heparin precursor with about 76 mM / L trimethylamine sulfur trioxide.

[0012] In some embodiments, enzymatically converting the LMW-NSNAH to LMWH comprises contacting the LMW-NSNAH with C5-Epi and 2-OST to form a heparin precursor comprising N-sulfo, N-acetyl, 2-O-sulfo IdoA (NSNA2SH). In some embodiments, enzymatically converting the LMW-NSNAH to LMWH comprises contacting the NSNA2SH with 6-O-sulfotransferase-3, 6-O-sulfotransferase-1, or a combination thereof to form a heparin precursor comprising N-sulfo, N-acetyl, 2-O-sulfo, 6-O-sulfo IdoA (NSNA2S6SH). In some embodiments, enzymatically converting the LMW-NSNAH to LMWH comprises contacting the NSNA2S6SH with 3-O-sulfotransferase-1 to form LMWH.

[0013] In some embodiments, the LMW-NSNAH has a molecular weight between about 3,800 Daltons and about 4,500 Daltons. In some embodiments, the LMW-NSNAH comprises about 10% to about 15% N-acetyl groups. In some embodiments, the LMWH is an isomeric, polydisperse form of enoxaparin having an anti-factor Xa concentration between about 90 and about 125 IU / mg.

[0014] Various aspects of the present disclosure relate to an intermediate LMW-NSNAH produced by a method comprising: providing a quantity of a heparin precursor, wherein the heparin precursor is an E. coli capsular polysaccharide; contacting the heparin precursor with one or more acids to remove Kdo residues from the heparin precursor by hydrolysis, thereby forming an acid-treated heparin precursor; and converting the acid-treated heparin precursor by depolymerization and de-N-acetylation to form the LMW-NSNAH. In some embodiments, the LMW-NSNAH has a molecular weight and a ratio of N-sulfo groups to N-acetyl groups such that enzymatic treatment with a C5-epimerase and at least one sulfotransferase produces a final product having a molecular weight and chemical properties consistent with animal-derived enoxaparin. In some embodiments, the LMW-NSNAH has a molecular weight between about 3,800 and about 4,500 Daltons. In some embodiments, the LMW-NSNAH comprises about 10% to about 15% N-acetyl groups.

[0015] In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH comprises hydrolyzing the acid-treated heparin precursor by treating it with one or more bases; treating the acid-treated heparin precursor with one or more other acids; contacting the acid-treated heparin precursor with one or more enzymes; or a combination thereof. In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH by depolymerizing and de-N-acetylation further comprises adding the acid-treated heparin precursor to a reaction medium comprising methanol, anhydrous sodium carbonate, and about 53 μM / L acetic anhydride to form a re-acetylated heparin precursor, and adding the re-acetylated heparin precursor to a reaction medium comprising anhydrous sodium carbonate and about 76 mM / L trimethylamine sulfur trioxide to obtain LMW-NSNAH.

[0016] Various aspects of the present disclosure relate to compositions comprising LMWH, wherein the LMWH is prepared by enzymatic conversion of LMW-NSNAH prepared from E. coli capsular polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For the purpose of illustrating the invention, the accompanying drawings show embodiments of the disclosed subject matter. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown in the drawings. In which:

[0018] Figure 1 It is the chemical structure of the heparin precursor;

[0019] Figure 2 is a diagram of a method for preparing low molecular weight heparin (LMWH) according to some embodiments of the present disclosure;

[0020] Figure 3 The present invention shows the removal of 3-deoxy-D-manno-octyl-2-ulosonic acid (Kdo) from low molecular weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH). 1 HNMR analysis chart;

[0021] Figure 4A is a graph showing molecular weight analysis of chemically biosynthesized LMW-NSNAH by gel permeation chromatography (GPC);

[0022] Figure 4B is a graph showing molecular weight analysis of LMWH for chemical biocatalysis by GPC;

[0023] Figure 5A A diagram showing the conversion of NS2S by 2-O-sulfotransferase and C5-epimerase reactions during the enzymatic synthesis of chemically biosynthetic LMWHs according to some embodiments of the present disclosure;

[0024] Figure 5B A diagram showing the conversion of Tris by a 6-O-sulfotransferase reaction during the enzymatic synthesis of a chemically biosynthetic LMWH according to some embodiments of the present disclosure;

[0025] Figure 5C is a diagram of 3S conversion by a 3-O-sulfotransferase reaction during enzymatic synthesis of chemically biosynthesized LMWH according to some embodiments of the present disclosure, the 3S conversion being confirmed by anti-Xa activity;

[0026] Figure 6 is a table of chemically biosynthesized LMWH and its intermediate disaccharide structures determined by treatment with heparin lyases I, II, and III according to some embodiments of the present disclosure;

[0027] Figure 7A is a graph showing disaccharide profiling of chemically biosynthesized LMWH by strong anion exchange high performance liquid chromatography (SAX-HPLC) according to some embodiments of the present disclosure;

[0028] Figure 7Bis a graph showing tetrasaccharide profile analysis of chemically biosynthesized LMWH by SAX-HPLC according to some embodiments of the present disclosure;

[0029] Figure 8A is a diagram showing the disaccharide composition analysis of LWMH chemically biosynthesized according to some embodiments of the present disclosure;

[0030] Figure 8B is a diagram showing the tetrasaccharide composition analysis of LWMH chemically biosynthesized according to some embodiments of the present disclosure;

[0031] Figure 9 The chemical structures of five 3-O-sulfated tetrasaccharide structures of LWMH synthesized by chemical biosynthesis according to some embodiments of the present disclosure are shown;

[0032] Figure 10A is a graph showing that according to some embodiments of the present disclosure, enoxaparin and chemically biosynthesized LMWH 1 HNMR spectrum;

[0033] Figure 10B is a graph showing that according to some embodiments of the present disclosure, enoxaparin and chemical biocatalysis of LMWH 13 CNMR analysis chart;

[0034] 11A-11B are graphs showing surface plasmon resonance (SPR) sensorgrams of antithrombin III (AT) binding to heparin (surface competition between enoxaparin and chemical biocatalyzed LMWH), respectively, according to some embodiments of the present disclosure;

[0035] FIG11C is a graph showing the IC values ​​calculated for enoxaparin and chemical biocatalyzed LMWH using AT inhibition data, according to some embodiments of the present disclosure. 50 ;

[0036] 11D-11E are graphs showing SPR sensorgrams of platelet factor IV (PF4) binding to heparin (surface competition between enoxaparin and chemical biocatalyzed LMWH), respectively, according to some embodiments of the present disclosure; and

[0037] FIG11F is a graph showing the IC values ​​of enoxaparin and chemical biocatalyzed LMWH calculated using PF4 inhibition data, according to some embodiments of the present disclosure. 50 . DETAILED DESCRIPTION

[0038] Now refer to Figure 2, some embodiments of the present disclosure relate to a method 200 for preparing low molecular weight heparin (LMWH) (also referred to herein as "chemical biosynthetic" or "chemical biocatalyzed" LMWH). In some embodiments, at 202, a certain amount of heparin precursor is provided. In some embodiments, the heparin precursor is synthesized by a bacterial source (i.e., one or more bacteria). In some embodiments, the heparin precursor is isolated from the one or several bacteria and used in the steps of method 200, i.e., method 200 is performed extracellularly. In some embodiments, the heparin precursor is secreted by the bacterial source and subsequently isolated therefrom for use in method 200. In some embodiments, after lysing the bacterial source, the heparin precursor is collected for use in method 200 by any suitable process to release the heparin precursor. In some embodiments, at least some steps of method 200 are performed intracellularly, i.e., within the bacterial source itself. In some embodiments, the heparin precursor is provided 202 to a reaction vessel, wherein at least one subsequent step in method 200 is performed.

[0039] In some embodiments, the bacterial source is any suitable wild-type or engineered bacterium. In some embodiments, the bacterial source comprises an Escherichia coli (E. coli) strain. In some embodiments, the bacterial source comprises E. coli K5. In some embodiments, the bacterial source comprises an engineered strain of E. coli K5. In some embodiments, the engineered strain of E. coli K5 has had the fructosyltransferase removed.

[0040] In some embodiments, the heparin precursor is Escherichia coli capsular polysaccharide (CPS). Without wishing to be bound by theory, the heparin precursor used in method 200 isolated from the bacterial source (e.g., an engineered strain of Escherichia coli K5 from which fructosyltransferase has been removed) is acidic CPS. As described above, the heparin precursor is a linear chain of repeating structures (1→4)-β-GlcA)(1→4)-α-GlcNAc(1→). In some embodiments, the average molecular weight of the heparin precursor used in method 200 is between about 35 kDa and about 65 kDa. In some embodiments, the average molecular weight of the heparin precursor is between about 45 kDa and about 55 kDa. In some embodiments, the average molecular weight of the heparin precursor is between about 48 kDa and about 52 kDa. In an exemplary embodiment, the average molecular weight of the heparin precursor CPS provided in step 202 can be 49 kDa, which is much larger than the molecular weight of commercially available UFH and LMWH.

[0041] In some embodiments, at 204, 3-deoxy-D-mannose-2-oxaloyl (Kdo) residues are removed from the provided heparin precursor. In some embodiments, the Kdo residues are removed from the heparin precursor by hydrolysis. In some embodiments, the Kdo residues are removed from the heparin precursor by acid hydrolysis. In some embodiments, the heparin precursor is contacted with one or more acids to remove the Kdo residues and form an acid-treated heparin precursor by acid hydrolysis. In some embodiments, the one or more acids include any acid or combination of acids suitable for removing the Kdo residues without degrading the heparin precursor to the extent that it can no longer be enzymatically converted to heparin, which will be discussed in more detail below. In some embodiments, the one or more acids include hydrochloric acid (HCl).

[0042] In some embodiments, at 206, the heparin precursor from which the Kdo residue has been removed (also referred to herein as "de-Kdo heparin precursor") is converted by depolymerization and de-N-acetylation to form a low molecular weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH). In exemplary embodiments, the one or more acids used in step 204 also serve to reduce the molecular weight of the heparin precursor, de-N-acetylate the heparin precursor, or a combination thereof. In some embodiments of step 206, it is the acid-treated heparin precursor that is converted by depolymerization and de-N-acetylation to form the LMW-NSNAH. In some embodiments, the de-Kdo heparin precursor is de-N-acetylated and depolymerized to obtain a low molecular weight form of the heparin precursor, such as LMW-NSNAH, having an average molecular weight between about 3,000 and about 10,000 Daltons. In some embodiments, the des-Kdo heparin precursor is de-N-acetylated and depolymerized to obtain LMW-NSNAH having an average molecular weight ranging from about 4,000 to about 7,000 Daltons. In some embodiments, the des-Kdo heparin precursor is de-N-acetylated and depolymerized to obtain LMW-NSNAH having an average molecular weight ranging from about 3,800 to about 4,500 Daltons. In some embodiments, the des-Kdo heparin precursor is de-N-acetylated and depolymerized to obtain a de-N-acetylation rate of about 85% to about 90%.

[0043] In some embodiments, the des-Kdo heparin precursor (e.g., the acid-treated heparin precursor) is converted 206 by hydrolysis by depolymerization and de-N-acetylation. In some embodiments, the hydrolysis is the result of treating the des-Kdo heparin precursor with one or more bases, one or more other acids, one or more enzymes, or a combination thereof. In some embodiments, the one or more bases comprise a base composition, such as one or more alkali metals. In some embodiments, the one or more bases comprise hydroxides. In some embodiments, the one or more bases comprise sodium hydroxide (NaOH). In some embodiments, the concentration of the one or more bases is between about 1N and about 3N. In some embodiments, the concentration of the one or more bases is about 2N. In some embodiments, the one or more enzymes comprise endo-β-glucuronidase.

[0044] As described above, in some embodiments, the des-Kdo heparin precursor is depolymerized to achieve an average molecular weight of between about 3,000 and about 10,000 Daltons. In some embodiments, the des-Kdo heparin precursor is depolymerized to achieve an average molecular weight of between about 4,000 and about 7,000 Daltons. In some embodiments, the des-Kdo heparin precursor is depolymerized to achieve an average molecular weight of between about 3,800 and about 4,500 Daltons. In some embodiments, the des-Kdo heparin precursor is de-N-acetylated to achieve about 10% to about 15% N-acetyl groups on the des-Kdo heparin precursor. In some embodiments, reaction temperatures (55°C, 60°C, 65°C, and 70°C) and times (24 hours, 48 ​​hours, 72 hours, and 96 hours) can be used for the conversion step 206. In an exemplary embodiment, after 48 hours of reaction at 65°C, the average molecular weight of the des-Kdo heparin precursor is reduced to 3.9 kDa as determined by GPC.

[0045] In some embodiments, converting 206 the des-Kdo heparin precursor includes reacetylation 206A of the des-Kdo heparin precursor after de-N-acetylation and / or depolymerization. In some embodiments, the depolymerized heparin precursor is at least partially reacetylated. In some embodiments, reacetylation 206A of the des-Kdo heparin precursor includes contacting the des-Kdo heparin precursor with acetic anhydride. In some embodiments, reacetylation 206A includes adding methanol, anhydrous sodium carbonate, and acetic anhydride. In some embodiments, the amount of acetic anhydride added is sufficient to achieve approximately 10% to approximately 15% N-acetyl groups on the des-Kdo heparin precursor. In some embodiments, the concentration of acetic anhydride is between approximately 40 μM / L and approximately 60 μM / L. In some embodiments, the concentration of acetic anhydride is between approximately 45 μM / L and approximately 55 μM / L. In some embodiments, the concentration of acetic anhydride is between approximately 50 μM / L and approximately 55 μM / L. In some embodiments, the concentration of acetic anhydride is about 53 μM / L. In some embodiments, the des-Kdo heparin precursor is contacted with acetic anhydride multiple times. In some embodiments, the des-Kdo heparin precursor is contacted with acetic anhydride at least 4 times at predetermined intervals. In some embodiments, the intervals are regular. In some embodiments, the intervals are irregular. In some embodiments, the intervals are between about 10 minutes and about 30 minutes. In some embodiments, the intervals are about 20 minutes.

[0046] In some embodiments, converting 206 the des-Kdo heparin precursor further comprises N-sulfating 206B the des-Kdo heparin precursor. In some embodiments, N-sulfating 206B the des-Kdo heparin precursor yields LMW-NSNAH. In some embodiments, N-sulfating 206B the des-Kdo heparin precursor comprises contacting the des-Kdo heparin precursor (e.g., acid-treated heparin precursor) with trimethylamine sulfur trioxide, pyridine sulfur trioxide, or a combination thereof. In some embodiments, the heparin precursor is N-sulfated 206B by adding equal parts of anhydrous sodium carbonate and trimethylamine sulfur trioxide. In some embodiments, the concentration of the trioxide reactant (e.g., trimethylamine sulfur trioxide, pyridine sulfur trioxide, etc.) is between about 60 mM / L and about 90 mM / L. In some embodiments, the concentration of the trioxide reactant is between about 70 mM / L and about 80 mM / L. In some embodiments, the concentration of the trioxide reactant is about 76 mM / L.

[0047] In some embodiments, the molecular weight of the LMW-NSNAH is between about 3,000 and about 10,000 Daltons. In some embodiments, the molecular weight of the LMW-NSNAH is between about 4,000 and about 7,000 Daltons. In some embodiments, the molecular weight of the LMW-NSNAH is between about 3,800 and about 4,500 Daltons. In some embodiments, the LMW-NSNAH comprises about 10% to about 15% N-acetyl groups.

[0048] At 208, the LMW-NSNAH is enzymatically converted to LMWH. In some embodiments, the enzymatic conversion 208 is performed by one or more sequential enzymatic treatments, each treatment comprising one or more enzymes. In some embodiments, the enzymatic conversion 208 of the LMW-NSNAH to LMWH comprises contacting the LMW-NSNAH with a C5-epimerase (C5-Epi) and a 2-O-sulfotransferase (2-OST) to form a heparin precursor (NSNA2SH) comprising N-sulfo, N-acetyl, 2-O-sulfo IdoA. In some embodiments, the enzymatic conversion 208 of the LMW-NSNAH to LMWH comprises contacting the NSNA2SH with one or more sulfotransferases. In some embodiments, enzymatically converting 208 the LMW-NSNAH into LMWH comprises contacting the NSNA2SH with 6-O-sulfotransferase-3 (6-OST-3), 6-O-sulfotransferase-1 (6-OST-1), or a combination thereof to form a heparin precursor comprising N-sulfo, N-acetyl, 2-O-sulfo, 6-O-sulfo IdoA (NSNA2S6SH). In some embodiments, enzymatically converting 208 the LMW-NSNAH into LMWH comprises contacting the NSNA2S6SH with 3-O-sulfotransferase-1 (3-OST) to form the LMWH. In some embodiments, the LMWH is an isomeric, polydisperse form of enoxaparin with an anti-factor Xa level between about 90 and about 125 IU / mg. In some embodiments, an amount of 1,6-anhydromannose comprising a chain is introduced into the LMWH.

[0049] Some embodiments of the present disclosure relate to intermediate LMW-NSNAH. As described above, the intermediate LMW-NSNAH is the result of one or more processing steps on a heparin precursor starting material synthesized from a bacterial source. Obtaining heparin precursor starting materials from such bacteria and subsequently converting them into heparin products offers numerous advantages over animal-derived heparin products, such as raw material availability and purity. The methods of the present disclosure ensure that bacterial-derived heparin precursors are converted into intermediate LMW-NSNAH, which is then converted into LMWH that is functionally equivalent to the animal-derived heparin.

[0050] In some embodiments of the method for preparing LMW-NSNAH, a certain amount of heparin precursor is provided. As described above, in some embodiments, the heparin precursor is E. coli CPS. In some embodiments, the heparin precursor is contacted with one or more acids to remove Kdo residues from the heparin precursor by hydrolysis, thereby forming an acid-treated heparin precursor. In some embodiments, the acid-treated heparin precursor does not contain Kdo after acid hydrolysis and can be separated from the rest of the reaction medium using a separation membrane of appropriate size and configuration (e.g., a separation membrane with a molecular weight cutoff of 1 kDa).

[0051] The main difference between the heparin precursor intermediates in animals and the heparin precursors used in the CPS methods described in the present disclosure lies in the receptors for their biosynthesis. In animals, the heparin precursors assemble on receptors corresponding to the tetrasaccharide linker region (Xyl-Gal-Gal-GlcA) attached to the serine residues of the core protein serglycin. However, the biosynthesis of the heparin precursor CPS begins with a glycolipid receptor, which is composed of multiple linked Kdo residues.

[0052] Now refer to Figure 3 As discussed above in step 204 of the disclosed embodiment, the glycolipid termini (including the Kdo residue) are removed prior to the other LMWH synthesis steps (e.g., steps 206-208) because the glycolipid termini are not found in porcine-derived LMWH products. The reaction conditions of step 204 act to remove the Kdo, but may also hydrolyze the N-acetyl group and reduce the molecular weight of the heparin precursor. 1 H NMR and GPC analysis determined that, in an exemplary embodiment, treatment of a heparin precursor from E. coli K5 (fructosyltransferase removed) with hydrochloric acid at pH 1 at 90°C for 1 hour releases Kdo without modifying the heparin precursor chain. 1 The Kdo signal observed at 1.5–2.5 ppm in H NMR disappeared in the retentate, indicating that Kdo had been successfully removed.

[0053] In some embodiments, the acid-treated heparin precursor is then converted to form the LMW-NSNAH by depolymerization and de-N-acetylation. In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH comprises hydrolyzing the acid-treated heparin precursor by treating the acid-treated heparin precursor with one or more bases, treating the acid-treated heparin precursor with one or more other acids, contacting the acid-treated heparin precursor with one or more enzymes, or a combination thereof. In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH comprises adding the acid-treated heparin precursor to a reaction medium comprising methanol, anhydrous sodium carbonate, and about 53 μM / L acetic anhydride to form a re-acetylated heparin precursor.

[0054] Now refer to Figures 4A-4B , for example, by alkaline hydrolysis, a heparin precursor consistent with the disclosed embodiments is chemically de-N-acetylated to partially (or completely) remove the N-acetyl groups of the GlcNAc residues and depolymerize the polysaccharide chains by β-elimination. In an exemplary embodiment, due to the reaction conditions in the de-N-acetylation in step 206 above, no acetyl groups are found based on NMR analysis (100% de-N-acetylation). Then, in step 206A, after the alkaline treatment and before N-sulfation (e.g., in step 206B), a certain amount of acetic anhydride is added to re-acetylate the heparin precursor. In some embodiments, converting the acid-treated heparin precursor to LMW-NSNAH includes adding the re-acetylated heparin precursor to a reaction medium comprising anhydrous sodium carbonate and about 76 mM / L trimethylamine sulfur trioxide to N-sulfate the re-acetylated heparin precursor and obtain LMW-NSNAH.

[0055] Specific reference Figure 4A In this exemplary embodiment, 146 mg of low molecular weight N-sulfo, N-acetylheparin precursor with a molecular weight of 4,200 Da was obtained from 1 g of acid-treated heparin precursor. The peaks at 5.31 ppm (corresponding to GlcNAc residues) and 5.55 ppm (corresponding to GlcNS residues) were analyzed. 1H NMR analysis reveals an N-acetyl / N-sulfo group ratio in the range of 10%-15%, resulting in a product that meets the United States Pharmacopeia (USP) standard for enoxaparin. In certain embodiments, the molecular weight and N-sulfo to N-acetyl ratio of the LMW-NSNAH allow for enzymatic treatment with C5-Epi and a sulfotransferase (e.g., 2-OST, 6-OST-1, 6-OST-3, 3-OST, etc., or a combination thereof) to yield a final product with a molecular weight and chemical properties consistent with animal-derived enoxaparin. As described above, in some embodiments, the molecular weight of the LMW-NSNAH is between about 3,000 and about 10,000 Daltons. In some embodiments, the molecular weight of the LMW-NSNAH is between about 4,000 and about 7,000 Daltons. In some embodiments, the molecular weight of the LMW-NSNAH is between about 3,800 and about 4,500 Daltons. In some embodiments, the LMW-NSNAH comprises from about 10% to about 15% N-acetyl groups.

[0056] Now refer to Figures 5A-5C Some embodiments of the present disclosure relate to compositions comprising LMWH. In some embodiments, the LMWH is prepared by enzymatic conversion of LMW-NSNAH prepared from bacterial (e.g., E. coli) CPS. In some embodiments, the LMWH in the composition complies with USP enoxaparin specifications.

[0057] The chemoenzymatic synthesis of LMWH (compliant with USP enoxaparin specifications) was demonstrated by subjecting LMW-NSNAH to a series of enzymatic modifications consistent with the disclosed embodiments. The conversion of NSNAH to NSNA2SH is catalyzed by C5-Epi, which converts a GlcA residue to an IdoA residue in a reversible reaction. The IdoA residue is then locked into place by 2-OST via 2-O-sulfation, resulting in an IdoA2S residue. NSNA2SH is converted to NSNA2S6SH using 6-OST-1 and 6-OST-3. NSNA2S6SH is converted to LMWH using 3-OST. The C5-Epi / 2-OST and 6-OST-X reactions are monitored by disaccharide composition analysis. The 3-OST reaction is monitored by an anti-Xa activity assay. Disaccharide composition analysis is used to determine the sulfation state, with a target range of 68-74% for NS2S based on commercially available enoxaparin.

[0058] Specific reference Figure 5AThe conversion of NSNAH to NSNA2SH was determined at time points of 4 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours. Without wishing to be bound by theory, the synthesis of LMWHs consistent with the disclosed embodiments is much slower than the synthesis of chemically biosynthesized UFH due to the reduced activity of these enzymes on shorter chain substrates. At the 96 hour time point, the maximum conversion of NS2S achieved was 69.3%, which meets the USP enoxaparin specification. Reference is now made to Figure 5B , the conversion of NS2S to NS2S6S is complete within 24 hours. The chain length of UFH is sufficient to bind AT and thrombin, resulting in a ternary complex that inactivates thrombin and thereby prevents blood clot formation. In contrast, LMWH is composed of smaller chains than UFH, and most of these chains are long enough to bind AT, inactivating factor Xa. Therefore, the synthesis of LMWH consistent with the embodiments of the present disclosure is monitored by anti-Xa activity. Now referring to Figure 5C On a dry basis, the anti-factor Xa potency of enoxaparin was not less than 90 IU / mg and not more than 125 IU / mg. This activity was achieved after 120 hours of treatment with 3-OST, and the anticoagulant activity was not enhanced by further enzymatic reactions.

[0059] Reference again Figure 4B The molecular weight of LMWHs consistent with the disclosed embodiments was determined by GPC using USP enoxaparin sodium molecular weight calibrant. The USP standard for the weight average molecular weight of enoxaparin sodium is 4,500 Da, with a range between 3,800 and 5,000 Da. Since sulfation increases the molecular weight of the final product, a target molecular weight of 3,800 to 4,500 Da was set for the LMW-NSNAH intermediate. As expected, starting from 4,200 Da for the low molecular weight NSNAH, the molecular weight of the final LMWH product has increased to 4,350 Da.

[0060] The anticoagulant activity of the NSNA2S6SH intermediate and final LMWH product was measured using the method described in the current USP enoxaparin monograph. The target anticoagulant potency for enoxaparin sodium, calculated on a dry basis, is no less than 90 and no more than 125 International Units (IU) / mg for anti-Factor Xa, and no less than 20.0 and no more than 35.0 IU / mg for anti-Factor IIa. The ratio of anti-Xa activity to anti-IIa activity is between 3.3 and 5.3.

[0061] 20 μL of the reaction solution was taken out regularly at different time points and analyzed for anti-Xa activity and concentration by HPLC-GPC. Figure 5C ) increases over the first 48 hours and then decreases until the activity reaches 105 IU / mg.

[0062] Referring to Table 1 below, LMWH consistent with embodiments of the present disclosure has an anti-Xa activity of 105 IU / mg and an anti-IIa activity of 24 IU / mg, with an anti-Xa / anti-IIa ratio of 4.4, consistent with USP enoxaparin. Table 1: Anticoagulant activity and IC of LMWH in triplicate preparations 50 Value Summary

[0063] Now refer to Figure 6 , disaccharide composition analysis of LMWH and its intermediates consistent with the disclosed embodiments was performed using treatment with heparin lyases I, II, and III. These treatments yielded eight different disaccharide products based on the level and position of sulfation. These disaccharides were then analyzed by strong anion exchange high performance liquid chromatography (SAX-HPLC) to monitor the biosynthesis of the intermediates and the final products (see Figures 7A-7B The disaccharide compositions of a heparin control, an enoxaparin control, and LMWHs consistent with embodiments of the present disclosure are shown in Table 2 below. Table 2: Analysis of the disaccharide and tetrasaccharide composition of LMWH in triplicate formulations

[0064] Now refer to Figures 8A-8B Heparin isolated from the bacterial source was treated with chemical N-sulfation, 2-OST / C5-Epi, 6-OST, and 3-OST consistent with the above-described embodiments to yield a disaccharide composition similar to enoxaparin. The TriS content of the chemically biocatalyzed LMWH was 62.6%, compared to 66.3% for enoxaparin. The NS6S content of the chemically biocatalyzed LMWH was 17.3%, higher than the 10.3% for enoxaparin, while the N2S2 content of the LMWH was 3.5%, lower than the 7.0% for enoxaparin. This indicates that 2-OST conversion is lower than 6-OST conversion. Notably, the 3-O-sulfated glucosamine residue is resistant to cleavage by heparinase. Therefore, in addition to the anticoagulant activity analysis, tetrasaccharide analysis was performed by treatment with heparinase I, II, and III, followed by SAX-HPLC analysis of the resulting resistant tetrasaccharides.

[0065] Now refer to Figure 9, five tetrasaccharides including 3-OST have been characterized: (1) ΔUA-GlcNAc6S-GlcUA-GlcNS3S (wherein ΔUA is deoxy-α-L-threo-hex-4-enopyranosyl uronic acid); (2) ΔUA-GlcNAc6S-GlcUA-GlcNS3S6S; (3) ΔUA-GlcNS6S-GlcUA-GlcNS3S; (4) ΔUA2S-GlcNAc6S-GlcUA-GlcNS3S6S; (5) ΔUA2S-GlcNS6S-GlcUA-GlcNS3S6S. The results show that the chemical biocatalyzed LMWH has a similar distribution of tetrasaccharides including 3-OST compared to enoxaparin (see Table 2 again). The LMWH produced by the embodiments of the present disclosure is highly similar to enoxaparin in terms of disaccharide and tetrasaccharide composition analysis.

[0066] Now refer to Figures 10A-10B , one-dimensional 1 H and 13 C NMR spectroscopy to characterize the structure of enoxaparin and LMWH produced by embodiments of the present disclosure. 1 All H peaks can be clearly attributed. The spectra of the two LMWHs appear very similar, but there are some differences. The GlcNS3S peak overlaps with H1 of ΔUA2S at 5.44 to 5.42 ppm. The IdoA2S peak is attributed to 5.17 to 5.09 ppm. Compared with enoxaparin, the chemical biocatalyzed LMWH has a lower H4ΔUA intensity at 5.90 ppm. The signal peaks at 5.48 ppm, 5.43 ppm, 5.33 ppm, 5.13 ppm, 5.07 ppm and 4.51 ppm correspond to anomeric hydrogen. The LMWH also has two peaks within 5.09 to 5.00 ppm, which, without wishing to be bound by theory, may be IdoA2S or impurities. Compared with enoxaparin or imitation enoxaparin, the chemical biocatalyzed LMWH produced by the embodiments of the present disclosure has a very small amount of 1,6-anhydromannose.

[0067] Referring now to Figures 11A-11C, the anticoagulant activity of heparin is primarily mediated by its binding to and modulation of AT. Therefore, the interaction between heparin and AT is a step in the anticoagulation process. Competitive surface plasmon resonance (SPR) was used to measure the competitive AT binding of USP heparin immobilized on the chip surface with LMWH produced by embodiments of the present disclosure. The IC value for a 50% reduction in response units (RU) was 0. 50 The IC values ​​can be calculated from the graph for a range of LMWH solution concentrations (up to 50 μg / mL). 50The values ​​were 11.0 and 12.0 μg / mL, respectively. Thus, the AT binding activity of the LMWH produced by the disclosed embodiments was slightly lower than that of enoxaparin, but still within an acceptable range.

[0068] 11D-11F, of particular concern is heparin-induced thrombocytopenia (HIT), which is caused by the interaction between heparin and platelet factor IV (PF4), leading to adverse immune disorders. The HIT potential of LMWH produced by the disclosed embodiments was analyzed. PF4 binding was assessed by solution competition SPR using a rapid method and IC was calculated. 50 IC of LMWH produced by the disclosed embodiments 50 The measured value was 2.8 μg / mL, while enoxaparin was 2.7 μg / mL. These results are comparable to those of LMWH samples, which ranged from 2.4 to 2.9 μg / mL. LMWH binds to PF4 with much less affinity than UFH, making HIT less likely to be caused by LMWH. Example

[0069] Materials. Escherichia coli K5 heparin precursor CPS was prepared by fermentation. 2-OST, 6-OST, 3-OST, and C5-Epi enzymes were prepared. Enoxaparin LMWH standards and enoxaparin sodium molecular weight calibrators A (1400 Da, 2250 Da, 4550 Da, and 9250 Da) and B (1800 Da, 3350 Da, and 6650 Da) were purchased from United States Pharmacopeia (USP, Rockville, MD). Human antithrombin III (AT) and platelet factor 4 (PF4) were purchased from Hyphen BioMed (Neuville-sur-Oise, France). Recombinant Flavobacterium heparin lyases I, II, and III (EC numbers 4.2.2.7, 4.2.2.X, and 4.2.2.8, respectively) were expressed in Escherichia coli and purified. Unsaturated heparin disaccharide standards were purchased from Iduron (Manchester, UK). Biophen heparin anti-Xa (2 stages) and anti-IIa (2 stages) kits were purchased from Aniara (West Chester, OH, USA).

[0070] Removal of the glycolipid termini of the K5 heparin precursor. The heparin precursor CPS, prepared from Escherichia coli K5 (fructosyltransferase-depleted), was treated with hydrochloric acid to remove the glycolipid receptor 3-deoxy-D-manno-octano-2-ulosonic acid (Kdo). The heparin precursor was dissolved in a hydrochloric acid solution, the pH adjusted to 1, and then incubated at 90°C for 1 hour. The solution was readjusted to pH 7 with sodium hydroxide and desalted by dialysis. Results were confirmed by NMR analysis.

[0071] A low-molecular-weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH) was prepared by chemical cleavage. The Kdo-free heparin precursor was de-N-acetylated and depolymerized by a controlled alkaline reaction. A sample (20 g / L) was dissolved in 50 mL of 2N NaOH and incubated in a shake flask at 65°C for 48 hours, cooled to room temperature, and the pH was adjusted to 7.0 with HCl. Controlled reacetylation was performed by adding methanol (3.5 mL), anhydrous sodium carbonate (130 mM / L), and acetic anhydride (53 μM / L, four times with 20-minute intervals). The amount of acetic anhydride added was sufficient to achieve 10-15% N-acetyl groups, as determined by NMR. Next, N-sulfation was performed by adding equal parts of anhydrous sodium carbonate (130 mM / L) and trimethylamine sulfur trioxide (76 mM / L), followed by mixing at 47°C for 48 hours. Sulfation levels were monitored by measuring unsubstituted amines using an o-phthalaldehyde (OPA) assay. The ratio of sulfonyl groups to acetyl groups was determined by NMR. Low molecular weight LMW-NSNAH was precipitated with 85% methanol overnight at 4°C. Remaining salts were removed by washing four times with 85% methanol and centrifuging at 1800×g.

[0072] Chemically biosynthesized LMWH was prepared by enzyme modification. A 50 mg sample of LMW-NSNAH was treated with C5-Epi and 2-OST to yield a low molecular weight N-sulfo, N-acetyl, 2-sulfoheparin precursor (LMW-NSNA2SH). Detailed reaction conditions were as follows: substrate concentration of 1 mg / mL, PAPS concentration of 5 mM, and concentration of each immobilized enzyme (C5-Epi / 2-OST) of 1 mg / mL in a 50% slurry. The reaction was incubated at 37°C for 120 hours in 50 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 7.2) containing 0.05% NaN3 and 125 mM NaCl. After completion of the reaction, the mixture was filtered to remove the enzyme resin and dialyzed against distilled water using a 1 kDa molecular weight cutoff membrane to remove salts and other small molecule impurities. Disaccharide composition analysis was used to monitor and confirm the sulfation reaction. Next, controlled reactions of 6-OST and 3-OST to produce LMWHs were performed using immobilized enzymes, i.e., chemically biocatalyzed LMWHs. Reaction conditions were similar to those used in the C5-Epi / 2-OST reaction. Reaction status was monitored using disaccharide composition analysis and anti-Xa activity assays, respectively.

[0073] Molecular weight was determined by gel permeation chromatography (GPC). Molecular weight was determined by GPC high performance liquid chromatography (HPLC) using enoxaparin sodium molecular weight calibrant. A guard column BioSuite 7.5×75 mm was used to protect two analytical columns connected in series: Waters BioSuiteTM 125, 5 μm HR SEC 7.8×300 mm column (Waters Corporation, Milford, MA). The mobile phase was 0.5 M lithium nitrate, the flow rate was set at 0.6 mL / min, the injection volume was 20 μL, and the concentration was 5 mg / mL.

[0074] Anticoagulant activity. The anticoagulant activity of the products was determined using the BIOPHEN Heparin Anti-Xa (2-stage) and Anti-IIa (2-stage) kits according to the manufacturer's protocol. Briefly, AT (Anti-Xa Reagent 1 (r1)), Factor Xa (r2), and Factor Xa-specific chromogenic substrate (r3) were used for anti-Xa activity, while AT (Anti-IIa Reagent 1 (R1)), human thrombin (R2), and Factor IIa-specific chromogenic substrate (R3) were used for anti-II activity. Each reagent was reconstituted with 1 mL of distilled water and shaken until completely dissolved. After diluting r1 / R1 and r2 / R2 1 / 5 with the appropriate buffer (Tris-EDTA-NaCl-PEG, pH 8.4), r3 / R3 was diluted with distilled water to reduce the reagents immediately before use. Reference standards and diluted samples were prepared to the appropriate concentrations. The sample (40 μL) was added to a 96-well plate and incubated at 37°C for 5 minutes. 40 μL of r1 / R1 was added, mixed thoroughly, and incubated for 2 minutes. Then, 40 μL of r2 / R2 was added and incubated for 2 minutes. Finally, 40 μL of r3 / R3 was added and incubated for an additional 2 minutes. The reaction was stopped by adding 80 μL of 50 mM acetic acid. The absorbance was then measured at 405 nm. Anti-Xa and anti-IIa activities were calculated using a standard curve of different enoxaparin concentrations.

[0075] Analysis of disaccharide and tetrasaccharide composition. TM Disaccharide and tetrasaccharide compositions were determined by strong anion exchange (SAX)-HPLC using a UV detector on an LC-2030 system (Shimadzu, Kyoto, Japan). Samples (100 g) were thoroughly digested at 37°C for 2 h using a mixture of heparin lyases I, II, and III (10 mU each) in digestion buffer (50 mM ammonium acetate, including 2 mM calcium chloride, pH 7.0). The reaction was terminated by boiling for 10 min, and the denatured enzyme was removed by centrifugation at 10,000 × g for 10 min. The supernatant, concentrated to 1 μg / μL, was then eluted with Shimadzu TMThe analysis was performed using an HPLC system coupled to an LC-20AD pump, CBM-20A controller, SIL-20AHT autosampler, and SPD-20AVUV detector. A Spherisorb SAX column (4.0×250 mm, 5.0 μm, Waters) was equilibrated with mobile phase A (1.8 mM sodium dihydrogen phosphate, pH=3) and then gradient eluted with mobile phase B (1.8 mM sodium dihydrogen phosphate and 2 M sodium perchlorate, pH=3). Disaccharide analysis used a mobile phase B gradient that increased from 5% to 50% over 30 minutes, held for 5 minutes, then changed to 5% and held for 15 minutes. Tetrasaccharide analysis used a gradient of 15-32.5% mobile phase B from 0-40 minutes, 42.5% mobile phase B at 50 minutes, 50% at 54 minutes, and held for 1 minute at a flow rate of 0.45 mL / min.

[0076] Nuclear magnetic resonance (NMR) spectroscopy analysis. NMR spectroscopy was performed by Z-axis gradient 1 H / 2 H / 13 C / 15 The N2 cryoprobe was used to obtain the data on a Bruker 800 MHz (18.8 T) standard aperture NMR spectrometer. The sample was dissolved in 0.4 mL of 99.96% D2O and lyophilized, and then repeated twice. The results were carried out at 298 K. 1 H / 13 C 1D NMR.

[0077] Surface plasmon resonance (SPR) analysis. SPR measurements were performed on BIAcore TM 3000 instrument (GE, Uppsala, Sweden) using BIAcore 3000 control and BIAevaluation TM Software (version 4.0.1) was used. Biotinylated heparin, prepared by conjugating the reducing end of heparin to amine-PEG3-biotin (Pierce, Rockford, IL), was immobilized on a streptavidin-coated chip according to the manufacturer's protocol. IC 50, competition studies of surface heparin and LMWH (produced by embodiments of the present disclosure) binding to proteins were performed using SPR. AT (250 nM) or PF4 (125 nM) was mixed with HBS-EP buffer solutions (0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% surfactant P20, pH 7.4) of different concentrations of LMWH and injected onto the chip at a flow rate of 30 μL / min. Dissociation and regeneration were performed using sequential injections of 10 mM glycine-HCl (pH 2.5) and 2 M NaCl to obtain a fully regenerated surface after each run. For each set of competition experiments, a control experiment was performed to ensure that the surface was fully regenerated, so the results obtained from each run were comparable.

[0078] The methods and systems of the present disclosure facilitate the production of LMWH suitable for use equivalent to enoxaparin sodium, the most widely used low molecular weight heparin product. The chemically biocatalyzed LMWH described in the present disclosure is intended to be used as a comparable version of traditional pharmaceutical LMWH.

[0079] Enoxaparin is typically obtained by alkaline depolymerization of heparin benzyl ester, which is isolated exclusively from porcine intestinal mucosa. However, the preparation and use of porcine-derived enoxaparin present significant drawbacks, namely the variability of the animal-derived heparin starting material, its limited availability and poor control, and its impurities.

[0080] The LMWH and compositions comprising the LMWH described in the present disclosure are prepared without the use of porcine-derived heparin and are therefore prepared without the depolymerization step of porcine-derived UFH. In contrast, the method of the present disclosure utilizes a bacterial source (e.g., engineered E. coli K5) to generate a heparin precursor used as the backbone precursor of the LMWH product. The depolymerization method (e.g., by an alkaline composition) can obtain a suitable chain length backbone, which can then be modified by C5-Epi, 2-O-sulfotransferase, 6-O-sulfotransferase, and 3-O-sulfotransferase. These methods are relatively low in cost but can produce enoxaparin in a highly pure, isomeric, polydisperse form. In addition, this chemoenzymatically synthesized LMWH has several advantages over LMWH prepared from animal-derived UFH, including easier access to raw materials, better control of the production process, and reduced concerns about contamination, adulteration, or animal viruses or impurities.

[0081] While the present invention has been described and illustrated with respect to exemplary embodiments thereof, it will be understood by those skilled in the art that the foregoing and other modifications, omissions, and additions may be made thereto without departing from the spirit and scope of the invention.

Claims

1. A method for preparing low molecular weight heparin (LMWH), comprising: Providing a certain amount of heparin precursor; contacting the heparin precursor with one or more acids to form an acid-treated heparin precursor; converting the acid-treated heparin precursor by depolymerizing and de-N-acetylation to form a low molecular weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH); and The LMW-NSNAH is enzymatically converted into LMWH, The heparin precursor is Escherichia coli capsular polysaccharide.

2. The method according to claim 1, wherein the heparin precursor is synthesized by an engineered strain of Escherichia coli K5.

3. The method of claim 1 , wherein contacting the heparin precursor with one or more acids to form an acid-treated heparin precursor comprises: The 3-deoxy-D-manno-octano-2-ulosonic acid (Kdo) residue is removed from the heparin precursor by acid hydrolysis.

4. The method of claim 1 , wherein converting the acid-treated heparin precursor into LMW-NSNAH comprises hydrolyzing the acid-treated heparin precursor by: treating the acid-treated heparin precursor with one or more bases; treating the acid-treated heparin precursor with one or more additional acids; contacting the acid-treated heparin precursor with one or more enzymes; or A combination of them.

5. The method of claim 1 , wherein converting the acid-treated heparin precursor into LMW-NSNAH by depolymerizing and de-N-acetylation further comprises: After de-N-acetylation of the acid-treated heparin precursor, it is re-acetylated; and The acid-treated heparin precursor is N-sulfated to obtain LMW-NSNAH.

6. The method according to claim 5, wherein: Reacetylation of the acid-treated heparin precursor after de-N-acetylation comprises: contacting the acid-treated heparin precursor with acetic anhydride; and N-sulfating the acid-treated heparin precursor to obtain LMW-NSNAH comprises: The acid-treated heparin precursor is contacted with trimethylamine sulfur trioxide, pyridine sulfur trioxide, or a combination thereof.

7. The method of claim 5, wherein re-acetylation of the acid-treated heparin precursor after de-N-acetylation comprises: The acid-treated heparin precursor was contacted with approximately 53 μM / L acetic anhydride.

8. The method of claim 5, wherein N-sulfating the acid-treated heparin precursor to obtain LMW-NSNAH further comprises: The acid-treated heparin precursor is contacted with about 76 mM / L trimethylamine sulfur trioxide.

9. The method of claim 1 , wherein enzymatically converting the LMW-NSNAH to the LMWH further comprises: The LMW-NSNAH is contacted with C5-Epi and 2-OST to form a heparin precursor (NSNA2SH) comprising N-sulfo, N-acetyl, 2-O-sulfo IdoA.

10. The method of claim 9, wherein enzymatically converting the LMW-NSNAH into LMWH further comprises: The NSNA2SH is contacted with 6-O-sulfotransferase-3, 6-O-sulfotransferase-1, or a combination thereof to form a heparin precursor comprising N-sulfo, N-acetyl, 2-O-sulfo, 6-O-sulfo IdoA (NSNA2S6SH).

11. The method of claim 10, wherein enzymatically converting the LMW-NSNAH into LMWH further comprises: The NSNA2S6SH is contacted with 3-O-sulfotransferase-1 to form LMWH.

12. The method of claim 1, wherein the LMW-NSNAH has a molecular weight between about 3,800 and about 4,500 Daltons.

13. The method of claim 1, wherein the LMW-NSNAH comprises about 10% to about 15% N-acetyl groups.

14. The method of claim 1, wherein the LMWH is an isomeric, polydisperse form of enoxaparin with between about 90 and about 125 IU / mg of anti-Factor Xa.

15. An intermediate low molecular weight N-sulfo, N-acetylheparin precursor (LMW-NSNAH), said LMW-NSNAH being produced by a method comprising the steps of: providing an amount of a heparin precursor, wherein the heparin precursor is an Escherichia coli capsular polysaccharide; contacting the heparin precursor with one or more acids to remove 3-deoxy-D-mannose-2-ulosonic acid (Kdo) residues from the heparin precursor by hydrolysis, thereby forming an acid-treated heparin precursor; and Converting the acid-treated heparin precursor into LMW-NSNAH by depolymerization and de-N-acetylation; The LMW-NSNAH has a molecular weight and a ratio of N-sulfo groups to N-acetyl groups such that enzymatic treatment thereof with a C5-epimerase and at least one sulfotransferase produces a final product with a molecular weight and chemical properties consistent with animal-derived enoxaparin.

16. The intermediate LMW-NSNAH of claim 15, wherein the LMW-NSNAH has a molecular weight between about 3,800 and about 4,500 Daltons.

17. The intermediate LMW-NSNAH of claim 15, wherein the LMW-NSNAH comprises about 10% to about 15% N-acetyl groups.

18. The intermediate LMW-NSNAH according to claim 15, wherein converting the acid-treated heparin precursor to LMW-NSNAH comprises hydrolysis via the following pathway: treating the acid-treated heparin precursor with one or more bases; treating the acid-treated heparin precursor with one or more additional acids; contacting the acid-treated heparin precursor with one or more enzymes; or A combination of them.

19. The intermediate LMW-NSNAH according to claim 15, wherein converting the acid-treated heparin precursor into LMW-NSNAH by depolymerization and de-N-acetylation further comprises: adding the acid-treated heparin precursor to a reaction medium comprising methanol, anhydrous sodium carbonate and about 53 μM / L acetic anhydride to form a re-acetylated heparin precursor; and The reacetylated heparin precursor is added to a reaction medium comprising anhydrous sodium carbonate and about 76 mM / L trimethylamine sulfur trioxide to obtain LMW-NSNAH.

20. A composition comprising low molecular weight heparin (LMWH), wherein the LMWH is prepared by enzymatic conversion of low molecular weight N-sulfo, N-acetylheparin (LMW-NSNAH) prepared from Escherichia coli capsular polysaccharide.

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