OA20550A - Method for obtaining low molecular weight heparins by tangential flow filtration. - Google Patents

Abstract

Method for obtaining low molecular weight heparins (LMWH) with a molecular weight distribution between 3.0 and 5.0 kDa comprising at least one concentration step by tangential flow filtration (TFF). The method is particularly useful for the preparation of bemiparin and enoxaparin without the use of fractional precipitation nor the use of alcoholic solutions. In particular, the preparation of LMWH is obtained by depolymerization of heparin and filtration (TFF ultrafiltration and/or diafiltration) of the depolymerized heparin without the use of fractional precipitation and without an alcoholic solution.

Description

METHOD FOR OBTAINING LOW MOLECULAR WEIGHT HEPARINS BY TANGENTIAL

FLOW FILTRATION

DESCRIPTION OF THE TECHNIQUE

[0001] The présent invention relates to a method for obtaining low molecular weight heparins with a spécifie molecular weight distribution, comprising at least one concentration step by tangential flow filtration. Apartfrom the tangential flow filtration concentration, the method may encompass other steps such as diafiltration or hydrogen peroxide treatment. Thus, the invention may be included in the field of pharmaceutical technology.

Background of the Invention

[0002] Heparin is a polysaccharide of the glycosaminoglycan family, formed by uronic acid (Liduronic acid or D-glucuronic acid) and D-glucosamine, linked alternafely. L-iduronic acid may be 2-O-sulfated and D-glucosamine may be ΛΖ-sulfated and/or 6-O-sulfated, and to a lesser extent N-acetylated or3-O-sulfated. Heparin is preferably used as a sodium sait, but can also be used as a sait of other alkali or alkaline earth metals and is mainly used as an antithrombotic and anticoagulant médicament.

[0003] Heparins can be classified according to their molecular weight into, unfractionated heparin (UFH), low molecular weight heparin (LMWH), and very low molecular weight heparin (VLMWH). LMWH and VLMWH are obtained from the depolymerization of the original UFH molécule.

[0004] In any case, for UFH as well as for the various LMWH or VLMWH obtained by the known depolymerization methods (enzymatic, nitrous acid, β-elimination, etc.), the current purification processes are performed by sélective précipitation of their oligosaccharide chains with alcohols (mainly methanoi and éthanol) and to a lesser extent with other solvents such as acetone. Taking into account that several volumes of alcohol are used with respect to the volume ofthe aqueous solution contained in the product of interest and that, moreover, several purifications are generally necessary, from an industrial point of view this purification strategy poses a problem in terms of the volume of alcoholic waste generated in relation to its management, storage and recycling.

[0005] Thus, from a waste management point of view, other purification alternatives where the use of alcohol is not necessary are more interesting. Dialyzing the aqueous solution containing the product of interest against a membrane of the appropriate pore size is an alternative, but it has the drawback of dialyzing against large amounts of water and it is also difficult to scale optimized laboratory scale processes fo an industrial scale.

[0006] In view of the prier art, there are numerous methods of obtaining enoxaparin sodium, albeit much more complex than that of the présent invention or with the presence of alcoholic solvents.

[0007] Thus, patent document CN103342761 discloses a method for preparing enoxaparin sodium, which includes two serial ultrafiltrations by membranes of 8 kDa and 2 kDa respectively, with the aim of eliminating dégradation products and impurities of low molecular weight and controlling the molecular weight and molecular weight distribution of the product in the presence of alcoholic solvents. Once the purified product is obtained, it is lyophilized to obtain enoxaparin sodium. In this case, lyophilization is used to remove solvents and moisture that may hâve remained housed in the structure of enoxaparin sodium obtained from the process of this patent.

[0008] Patent document CN102050888 also discloses a final purification process of enoxaparin sodium in the presence of alcoholic solvents with a 1 kDa membrane concentration step and subséquent lyophilization wherein the molecular weight and molecular weight distribution of the product is further controlled. As in the previous patent, lyophilization is used to remove solvents and moisture that may hâve remained housed in the structure of enoxaparin sodium obtained from the process of this patent

[0009] Other processes for préparation and/or purification of polysaccharides are disclosed in the priorart: ES2161615A1 by Laboratorios Farmacéuticos ROVI, S.A., US2009105194A1 by Flengsrud, US5767269 by Hirsh, WO2010/111710A1 by Solazyme, Inc., US2007/0154492A1 by Michon, Griffin et al. (“Isolation and characterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin” Carbohyd. Res. (1995), 276, 183-197), and US5110918 by Casu. These processes require the fractional précipitation of depolymerized heparin.

[0010] Given the closest prior art, it is noteworthy that ail previous documents suffer from the drawback of using alcohols at one stage or another of the process, which is currently undesirable due to a greater concern for the environment. In some documents,buffered media with salts are used in one of the tangential filtration steps. However, the control of molecular weights by the pore size employed and the parameters indicated by prior art documents is not spécifie and does not allow defining a spécifie structural profile of the enoxaparin sodium obtained. Accordingly, it would be désirable to provide a simplîfied and efficient procedure for obtaining LMWH, eliminating the use of alcohols, which would also allow control overthe profile of the product obtained in such a way that it can be implemented continuously.

Summary of the Invention

[0011] As an alternative, the present invention makes use of tangential flow filtration (TFF) using non-alcoholic diafiltration buffers, which represents a significant advantage with respect to the methods described in the prior art, as it minimizes the residual solvent contents in the product formed and therefore the purity profile with respect to sa me, Further, the method designed herein provides a method that can be performed continuously as it allows profiling of the obtained product without the need for structural adjustments. This means that compared to production methods that involve purification by fractional précipitâtes, the production time is minimized, with the resulting improvement in cost réduction and increased production capacity. [0012] In some embodiments of the process of the invention, the crude depolymerized heparin is the product of a heparin depolymerization process. In some embodiments, the process of the invention excludes the use fractional précipitation of depolymerized heparin, in particular excluding the use of fractional précipitation of depolymerized heparin produced by heparin depolymerization. In addition, the invention considers embodiments wherein the low molecular weight heparin is prepared in two main steps: a) depolymerization of heparin to form crude depolymerized heparin; and b) purification of crude depolymerized heparin by TFF (concentration and/or diafiltration using membranes as described below) and without the use of fractional précipitation,

[0013] In some embodiments, the depolymerized heparin is enoxaparin sodium or bemiparin sodium, preferably enoxaparin sodium.

[0014] In some embodiments of the process of the invention, the crude enoxaparin sodium (or bemiparin sodium) is the product of a heparin depolymerization process. In some embodiments, the process of the invention excludes the use fractional précipitation of enoxaparin sodium (or bemiparin sodium), in particular excluding the use of fractional précipitation of enoxaparin sodium (or bemiparin sodium) produced by heparin depolymerization. In addition, the invention considers embodiments wherein the enoxaparin sodium (or bemiparin sodium) is prepared in two main steps: a) depolymerization of heparin toform crude enoxaparin sodium (or bemiparin sodium) without fractional précipitation; and b) purification of crude enoxaparin sodium (or bemiparin sodium) by TFF (concentration and/or diafiltration using membranes as described below) and without the use of fractional précipitation.

[0015] In some embodiments, the molecular weight (Mw) of the enoxaparin falls within the following ranges:

Sample	Mw, Da	M1 <2000 Da, %	M2 2000-8000 Da, %	M3 >8000 Da, %
Raw sodium	enoxaparin	3000- 5000	<25	60-80	<20
Enoxaparin produced process)	sodium (new	3800- 5000	12.0-20.0	68.0-82.0	<18.0

[0016] In some embodiments, the molecular weight (Mw) of the bemiparin faHs within the following ranges:

Sample	Mw, Da	M1 <2000 Da, %	M2 2000-6000 Da, %	M3 >6000 Da, %
Raw bemiparin sodium	2500- 5000	<40	50-75	<25
Bemiparin sodium produced (new process)	3000- 4200	<35.0	50.0-75.0	<15.0

[0017] To control the parameters that aliow the implémentation of the method employed in the présent invention, certain filtration membranes with a spécifie pore size are selected that allow their use on products in a wide range of molecular weights. Available membranes generally range from 1 kDa to 1000 kDa (or < 1 kDa) nominal cut-off. The nominal molecular weight cutoff (NMWCO) is defined as the minimum molecular weight of a soluté that is 90% retained by the membrane, and is determined evaluating the membrane rétention of components with different molecular weight (Figure 6).

[0018] In LMWH such as bemiparin sodium or enoxaparin sodium, with average molecular weights of 3600 and 4400 Da, respectively, the range of available membranes is therefore limited to those with nominal cut-off approximately <1 kDa, as higher pore sîzes would resuit in loss of the lowest molecular weight oligosaccharide chains.

[0019] The inventors of the présent invention hâve developed a method that allows obtaining LMWH, and in particular enoxaparin sodium and bemiparin sodium, by concentration by TFF without the need to use alcohols or other organic solvents, nor médiums buffered with salts, obtaining a product with an improved purity than those described in prior documents, with suitable quality attributes, according to the parameters described in the monograph for this product of the European Pharmacopoeia (latest édition) and the United States Pharmacopoeia 4 (latest édition). In addition, the product has an average molecular weight profile and an oligosaccharide chain distribution suitablefor its possible pharmacological applications.

[0020] In a first aspect, the présent invention relates to a method for obtaining iow molecular weight heparins (LMWH) with an average molecular weight distribution of between approximately 3.0 and approximately 5.0 kDa, comprising the following steps:

a) providing a depolymerized heparin solution with an oligosaccharide chain distribution range of between approximately 0.6 and approximately 10 kDa and a heparin concentration of up to approximately 4% w/v;

b) performing a concentration step by aqueous phase tangential flow filtration (TFF) using an approximately 1 kDa nominal cut-off membrane to achieve a heparin concentration of up to approximately 25% w/v.

[0021] Preferably, the solution of step a) is an aqueous solution.

[0022] The heparin concentration in step a) is preferably from between approximately 3% and approximately 4% w/v, more preferably from between approximately 3.5% and approximately 4% w/v, and even more preferably approximately 4% w/v.

[0023] Preferably, the membrane used for the concentration by tangential flow filtration has a nominal cut-off of approximately 0.7 to approximately 1 kDa; and more preferably of approximately 0.9 to approximately 1 kDa. In a spécifie embodiment, it has a nominal cut-off of approximately 1 kDa.

[0024] Tangential flow filtration (TFF), as well as the remaining steps of the method of the invention (clarification, depth filtration, diafiltration, treatment with H2O2), can be performed in aqueous phase without alcohol or any other organic solvents.

[0025] Step b) can include performing at least one concentration step by aqueous phase tangential flow filtration (TFF) using an approximately <1 kDa nominal cut-off membrane, e.g. 1,2 or 3, until achieving a maximum heparin concentration of up to approximately 25% w/v.

[0026] In step b) the heparin concentration obtained can be at least 5% w/v, preferably at least 8% w/v. Preferably, in step b) a heparin concentration of at least 10% w/v is obtained, preferably between approximately 10% and approximately 25% w/v; more preferably between approximately 10% and approximately 22% w/v; even more preferably between approximately 10% approximately 20%. In a spécifie embodiment, in step b) a heparin concentration is obtained of between approximately 10% and approximately 22% w/v.

[0027] In one embodiment, in step b) a single concentration step by TFF is performed. In a spécifie embodiment, in step b) a single concentration step by TFF is performed until obtaining a heparin concentration of at least 10% w/v, preferably between approximately 10% and approximately 22% w/v, more preferably between approximately 12% and approximately 22% w/v.

[0028] According to another embodiment of the invention, in step b) a single concentration step by TFF is performed until obtaining a heparin concentration of at least 5% w/v, preferably at least 10% w/v, more preferably between approximately 5% and approximately 15% w/v or between approximately 10% and approximately 22% w/v.

[0029] In an additional embodiment, the process of the invention comprises:

a) providing a depolymerized heparin solution with an oligosaccharide chain distribution range of between 0.6 and 10 kDa and a heparin concentration of between approximately 3% and approximately 4% w/v, preferably 4% w/v;

b) performing a single concentration step by aqueous phase tangential flow filtration (TFF) using a kDa nominal cut-off membrane to achieve a heparin concentration of up to 25% w/v, preferably between approximately 12% and approximately 22% w/v;

[0030] In another embodiment, step b) comprises two concentration steps by TFF.

[0031] In a preferred embodiment, in step (b) a first concentration is performed by TFF to achieve a heparin concentration of between approximately 4% and approximately 10% w/v, preferably between approximately 5% and approximately 10% w/v, and a second concentration step by TFF to achieve a heparin concentration of between approximately 10% and approximately 25% w/v. In a more preferred embodiment, the second concentration step achieves a heparin concentration of between approximately 12% and approximately 25% w/v, and more preferably of between approximately 12% and approximately 22% w/v.

[0032] In another preferred embodiment a concentration by TFF is performed in a single step from approximately 4% w/v to approximately 12-22% w/v (or approximately 10-25% w/v).

[0033] The process of the invention can include one or several additional steps, such as clarification, depth filtration, diafiltration with water, treatment with hydrogen peroxide or lyophilization.

[0034] In another preferred embodiment, at least one step of clarifying the heparin solution of step (a) is performed.

[0035] In another preferred embodiment, at least one depth filtration step is performed that may be prior to or subséquent to any of the TFF concentration steps. For example, before or after the TFF concentration step (if only one such step is performed) or before or afterthe first TFF concentration step if step b) comprises more than one such step.

[0036] In another preferred embodiment, at least one diafiltration step is performed with water that may be prior to or subséquent to any of the TFF concentration steps. For example, before or after the TFF concentration step, or before or after the first TFF concentration step if step b) comprises more than one such step.

[0037] In a preferred embodiment a treatment step with H2O2 is performed that can be prior to the TFF concentration step or prior to any of the concentration steps if step b) comprises more than one TFF concentration steps. For example, if step b) comprises two TFF concentration steps, a treatment step with H2O2 can be performed prior to the first concentration step or prior to the second concentration step.

[0038] In another preferred embodiment, at least one diafiltration step with water is performed that can be prior to the TFF concentration step or to the first TFF concentration step (if step b) comprises more than one TFF concentration step) or prior to the aforementioned treatment step with H2O2.

[0039] In another preferred embodiment, a step of lyophilizing the concentrate obtained in step (b) is performed.

[0040] In the présent invention, LMWH” is understood to mean heparins with an average molecular weight of less than approximately 8000 Da. Bearing in mind that one of the objectives of the method of the invention is to remove impurities associated with the manufacturing process, which are generally of low molecular weight (< 500 Da), the membranes available for use are limited since ideally they présent a cut-off lower than the average molecular weight of the LMWH so as to allow the removal of low molecular weight impurities without loss of oligosaccharide chains. Preferably, the nominal cut-off of the membranes employed is si kDa, although this may vary depending on the molecular weights to be obtained.

[0041] As one skilled in the art is aware, among the various steps of concentration by TFF preferably the maintenance steps that may be required for cleaning and/or regenerating the membranes are carried out using water, NaOH or any other product according to the spécifications thereof.

[0042] The spécification describes one or more embodiments incorporating features of this invention. The scope of the présent invention is not limited only to the embodiments described in it. The invention includes ail combinations and subcombinations of the various aspects and embodiments disclosed in this document. These and other aspects of the invention will be made évident with référencé to the following detailed description, claims and accompanying drawings.

Brief Description of the Drawings

[0043] The accompanying drawings, which are appended herein and fomn part of the spécification, illustrate one or more embodiments of the present invention and, together with the description, also contribute to the explanation of the principles of the present invention and to allow a person skilled in the art to reproduce and use the invention. The following drawings are provided for illustration purposes only and in no way limit the fuil scope of the present invention.

[0044] Figure 1: Comparative schematic for normal flow filtration and tangential flow filtration. [0045] Figure 2: General schematic of the tangential filtration process.

[0046] Figure 3: Comparison of concentration and diafiltration processes.

[0047] Figures 4A and 4B: Schematics of the purification process according to the invention. [0048] Figures 5A-5D: Comprise various graphs representing the variation in both average molecular weight (FIG. 5A) and distribution of molecular weights (FIG, 5B: <2000 kDa; FIG. 5C: >8000kDa; FIG. 5D: 2000-8000 kDa) is linear in the second concentration from 10% to 20% of nominal concentration ofthe product in the retentate.

[0049] Figure 6: Nominal cut-off: définition.

Description ofthe Invention

[0050] In the present invention tangential flow filtration” or “TFF” is understood as the filtration technique in which the solution to be fîltered passes tangentially over the surface of the filter, such that the pressure différence that is generated allows components that are smaller than the pore size to pass through same (permeate). Larger components are retained over the filter surface and returned to the feed tank (retentate).

[0051] In the present invention “clarification” is understood as the filtration performed to eliminate particles in suspension present in the solution, such as filtration performed by filters from 1-60 microns, preferably from 1-25 microns.

[0052] In the present invention, depth filtration is understood to mean filtration in which a multi-step labyrinth filter medium is used, which helps retain the particles. The larger particles will be retained on the surface and the finer ones follow their path towards the inside of the filter medium being trapped in the innerlayers, so thatthe turbidity ofthe dissolution is reduced. In a spécifie embodiment, this is a filtration performed with filters of 1-5 microns, preferably of 2-4 microns. The filtration can be performed with water or buffered solution.

[0053] In the present invention, concentration is understood as the tangential filtration step in which the retained product increases its concentration in the solution by removing permeate (see Figure 3).

[0054] In the présent invention, diafiltration” is understood as the tangential filtration step in which, while the permeate is removed, the solution is fed with the same buffer solution of water flow rate, so that the concentration of the retentate in the solution is not modified (see Figure 3). In this case, a membrane such as that used in concentration by TFF can be used; that is, a membrane with a nominal cut-off of approximately £1 kDa , preferably approximately 0.7 to approximately 1 kDa, more preferably approximately 0.9 to approximately 1kDa, and even more preferably approximately 1kDa.

[0055] In one embodiment, the heparin (after depolymerization) is a sodium sait of heparin, e.g. enoxaparin sodium or bemiparin sodium.

[0056] Crude enoxaparin sodium can be obtained by alkali (e.g. NaOH) depolymerization of the benzyl ester of heparin obtained from pig intestinal mucosa.

[0057] In one spécifie embodiment, the product obtained after depolymerization of enoxaparin sodium corresponds to a solution which, in addition to containing raw enoxaparin sodium, contains impurities corresponding to the saponification in alkaline medium of the benzyl ester of heparin, in addition to salts corresponding to the pH adjustments made during the breaking process. According to one embodiment of the invention, the TFF process is carried out on this raw enoxaparin sodium solution, so that the concentration of this solution is carried out with the aim, on the one hand, of eliminating low molecular weight impurities, and on the other, of reaching the appropriate concentration to carry out the bleaching treatment with hydrogen peroxide. Alternatively, bleaching treatment with H2O2 could be performed prior to the step of concentration by TFF. These steps can be performed without the use of fractional précipitation of the crude product (crude depolymerized heparin).

[0058] Alternatively, bleaching treatment with H₂O₂ could be performed prior to the step of concentration by TFF.

[0059] Additionally, a diafiltration process (optîonal) can be carried out for a thorough removal of low molecular weight impurities, before or after the concentration step. At the end of this step, optionally a second concentration is performed in order to remove the generated saline impurities and adjust the content in low molecular weight oligosaccharide chains, for which the average molecular weight of the solution is monitored; once the optimal value is reached, it is lyophilized to obtain enoxaparin sodium of the appropriate purity.

[0060] Therefore, in one spécifie embodiment the process of the invention comprises:

a) providing a depolymerized heparin solution with an oligosaccharide chain distribution range of between 0.6 and 10 kDa and an enoxaparin sodium concentration of approximately 4% w/v;

b) carrying out a concentration step by TFF in aqueous phase using a membrane with nominal cut-off <1 kDa until obtaining a heparin concentration of up to approximately 9

25% w/v, of up to approximately10% w/v, or preferably between approximately 5% and approximately 10% w/v;

c) optionally, performing a dîafiltration step with water (e.g. Non-buffered water) before or after step b),

d) performing a treatment step with H₂O₂ before or after step b),

e) optionally, performing a single concentration step by aqueous phase TFF using a <1 kDa nominal cut-off membrane to achieve a heparin concentration of up to 25% w/v, preferably between approximately 12% and approximately 25% w/v; and

f) performing a lyophilization step on the product obtained.

[0061] In a spécifie embodiment, the process of the invention comprises;

a) providing a depolymerized enoxaparin sodium solution with an oligosaccharide chain distribution range of between approximately 0.6 and approximately 10 kDa and an enoxaparin sodium concentration of up to 4% w/v;

b) carrying out a concentration step by TFF in aqueous phase using a membrane with nominal cut-off ^1 kDa until obtaining a heparin concentration of up to approximately 25% w/v, of up to approximately 10% w/v, or preferably between approximately 5% and approximately 10% w/v;

c) performing a treatment step with H₂O₂ on the product obtained in step b),

d) performing a single concentration step by aqueous phase TFF using a <1 kDa nominal cut-off membrane to achieve a heparin concentration of up to 25% w/v, preferably between approximately 12% and approximately 25% w/v; and

e) performing a lyophilization step on the product obtained.

Preferably, the process of this invention includes a clarification and/or depth filtration step before step b).

[0062] In a spécifie embodiment, the process of the invention comprises:

a) providing a depolymerized enoxaparin sodium solution with an oligosaccharide chain distribution range of between approximately 0.6 and approximately 10 kDa and an enoxaparin sodium concentration of up to 4% w/v;

b) carrying out a concentration stage by TFF in aqueous phase using a membrane with nominal cut-off si kDa until obtaining a heparin concentration of up to approximately 25% w/v, of up to approximately 10% w/v, or preferably between approximately 5% and approximately 10% w/v;

c) performing a dîafiltration step with water on the product obtained in step b),

d) performing a treatment step with H₂O₂ on the product obtained in step c),

e) optionally, performing a depth filtration step on the product obtained in step d),

f) performing a single concentration step by aqueous phase TFF using a £1 kDa nominal cut-off membrane to achieve a heparin concentration of up to 25% w/v, preferably between approximately 12% and approximately 25% w/v; and

g) performing a lyophilization step on the product obtained.

Preferably, the process of this invention includes a clarification and/or depth filtration step before step b).

[0063] In a spécifie embodiment, the process of the invention comprises:

a) a) providing a depolymerized enoxaparin sodium solution with an oligosaccharide chain distribution range of between 0.6 and 10 kDa and an enoxaparin sodium concentration of up to 4% w/v;

b) performing a single concentration step by aqueous phase TFF using a <1 kDa nominal cut-off membrane to achieve a heparin concentration of up to 25% w/v, preferably between approximately 5% and approximately 10% w/v;

c) performing a depth filtration step on the product obtained in step b),

d) performing a treatment step with H₂O₂ on the product obtained in step c),

e) performing a single concentration step by aqueous phase TFF using a <1 kDa nominal cut-off membrane to achieve a heparin concentration of up to 25% w/v, preferably between approximately 12% and approximately 25% w/v; and

f) performing a lyophilization step on the product obtained.

Preferably, the process of this invention includes a clarification and/or depth filtration step before step b).

[0064] In a spécifie embodiment, the process of the invention comprises:

a) providing a depolymerized enoxaparin sodium solution with an oligosaccharide chain distribution range of between 0.6 and 10 kDa and an enoxaparin sodium concentration of up to 4% w/v;

b) performing a diafiltration step with water on the solution of step a),

c) performing a treatment step with H₂O₂ on the product obtained in step c),

d) performing a single concentration step by aqueous phase TFF using a S1 kDa nominal cut-off membrane to achieve a heparin concentration of up to approximately 25% w/v, preferably between approximately 5% and approximately 20% w/v; and

e) performing a lyophilization step on the product obtained.

Preferably, the process of this invention includes a clarification and/or depth filtration step before step b).

Examples of the Invention

[0065] The foilowing spécifie examples provided below serve to illustrate the nature of the présent invention. These examples are included for illustrative purposes only and are not to be construed as limitations to the invention claimed herein. Crude enoxaparin sodium and bemiparin sodium were prepared by depoîymerization of heparin without performing fractional précipitation.

[0066] The process is described below for obtaining crude enoxaparin sodium, the starting product used in described examples 1, 2, 3,4 and 5. Dissolve 10 g of heparin sodium in purified water and under stirring add benzéthonium chloride solution, forming benzéthonium heparinate. Wash the formed product several fîmes with waterto remove excess chlorides and finally the dry the product by lyophilization. Dissolve the benzéthonium heparinate in methylene chloride and adjust the température. Add benzyl chloride and allow to react. The product obtained is heparin benzyl ester. Dissolve the heparin benzyl ester in water and add sodium hydroxide. At the end of the reaction, neutralize the solution; the product obtained is crude enoxaparin sodium. After obtaining the crude enoxaparin sodium, the examples described below were performed.

Example 1

[0067] The method of the invention was performed with the following main steps:

a) first concentration by TFF to obtain a product with a heparin concentration of 4% to 10% w/v;

b) diafiltration and treatment with H2O2;

c) second concentration by TFF to obtain a final product with a heparin concentration of 10% to 20% w/v.

[0068] Initially, crude enoxaparin sodium with a product concentration of 40 g/L and an oligosaccharide chain distribution between 0.6 and 10 kDa was used as the starting product. This initial product was prefiltered with a 3.0 μm Clarigard® filter. The product of the heparin depoîymerization process is crude enoxaparin sodium.

[0069] Next, the first concentration step was carried out by TFF, intended to increase the concentration of enoxaparin to a value between 4% and 10%, as well as to reduce the concentration of contaminants (mainly salts with a molecular weight < 0.5 kDa and other small Products resulting from previous manufacturing processes). To do this, a Millipore® regenerated cellulose membrane with a nominal cut-off of £1 kDa was used. The concentration step was started with about 2005 g of product with a transmembrane pressure (TMP) of 3.25 12 bar prior to passing the permeate flow to a separate container, the system was kept in total recirculation for about 15 minutes.

[0070] During concentration, permeate flow ranged from 9 initial to 4.8 final LMH (L/m²/h) (53% of initial flow), for an average flow of 6.2 LMH. 2 x permeate samples were collected, one in VCF (volume concentration factor) = 1.26X (P1 ) and the other of the permeate volume at the end of concentration (P2). A sample of the volume of material retained at the end of concentration (R1 ), as well as a sample of the initiai feed, was also taken after préfiltration by the 3.0 μm filter (B 1 ). The main data of the concentration study are presented in Table 1.

Table 1: LMWH Concentration Study (4 —> 10%)
Initial volume (L)	Extractable volume (L)	VCF (X)	Mean permeate flow (LMWH)	T fC)	Time (min)	APressure (bar)	Flow rate Tangential (LMM)
2.005	1.2	2.49	6.2	23.6 ->25.7	106	0.5 -> 0.7	5.3

[0071] Subsequently, a diafiltration step was carried out using purified water, intended to clarify the resulting product. During diafiltration, permeate flow continuously decreased from an initial 8.4 to a final 2.4 LMH (approx. 29%), for an average flow of 5.03 LMH. 3 x permeate samples were collected, each at the end of each diavolume (D1, D2 and D3 respectively) and one from the volume of material retained at the end of diafiltration (D5). The conditions under which the diafiltration study was carried out can be seen in Table 2:

Table 2: LMWH diafiltration study
Initial volume (L)	Extractable volume (L)	Diafiltration volumes (N)	Mean permeate flow (LMWH)	T rc)	Time (min)	APressure (bar)	Flow rate Tangential (LMM)
0.798	2.3	3	5.03	26.5 ->27.8	390	0.3 -> 0.8	5.3

[0072] The approximately 0.8 L of product obtained after diafiltration was subsequently subjected to Chemical reaction with H2O2.

[0073] Before continuing with the second concentration step by TFF, an additional (optionai) clarification step was carried out, again using a 3.0 pm Clarigard® filter, to remove any particles that may hâve decanted at the bottom of the vessei.

[0074] Next, a second concentration step was carried out by TFF, this time aimed at achieving a concentration of enoxaparin of between 10% and 20% w/v, also using a Millipore® regenerated cellulose membrane of Si kDa nominal cut-off.

[0075] During concentration, permeate flow ranged from 3 initial to 0.75 final LMH (25%), for an average flow of 1.5 LMH. 2 x permeate samples were collected, one in VCF = 1.34X (P3) and the other in the permeate volume at the end of concentration (P4). A sample was also taken of the volume of material retained after concentration (R2), after depolarizing the membrane by leaving the system running at a low TMP (1.2 bar) for 10 minutes.

[0076] Table 3 below shows the molecular weights of the samples taken during the process described above, analyzed according to the method established by the European Pharmacopoeia (EP).

Table 3: Molecular weights of samples obtained at different stages of the process
Sample	Mw, Da	<2000 Da, %	2000-8000 Da, %	>8000 Da, %
Raw enoxaparin sodium	3737	23.8	71.1	8.1
B1 (Clarigard® clarification)	3723	24.0	71.1	5.0
P1 (1 st permeate sample, 1 st concentration)	<600	—	—	—
P2 (2nd permeate sample, 1 st concentration)	<600	—	—
RI (retentate afterfirst concentration)	3645	25.2	69.9	4.8
D1 (permeate after first diavolume)	1196	96.5	3.5	0.0
D2 (permeate after second diavolume)	1491	84.5	15.5	0.0
D3 (permeate after third diavolume)	1646	78.1	21.9	7.0
D5 (retentate after completion of diafiltration)	4158	14.6	78.4	7.0
P3 (1 st permeate sample, 2nd concentration)	1866	67.3	32.7	0.0
P4 (2nd permeate sample, 2nd concentration)	1991	60.3	39.7	0.0

Table 3: Molecular weights of samples obtained at different stages of the process
Sample	Mw, Da	<2000 Da, %	2000-8000 Da, %	>8000 Da, %
R2 (retentate after second concentration)	4444	8.1	84.0	7.9

[0077] As shown in the percentages of the MW fractions of the target product (less than 2000 Da, 2000 to 8000 Da and more than 8000 Da) in the table above, it appears that the first concentration step does not negatively affect the product profile (there is no loss of any fraction in the permeate as seen in samples P1 and P2).

[0078] For samples taken during diafiitration, Dl, D2, D3 and D5 indicate that:

- basically there is no loss of the highest MW fraction in the permeate, along the diafiitration;

- there is some loss of the smallest and average MW fractions in the permeate, where the highest loss is always relative to the lowest fraction;

- the rate of loss of the smallest and medium fractions decreases, respectively, along the diafiitration;

- in general, there is some réduction and enrichment along the diafiitration of smaller fractions and medium-high fractions respectively (according to the £1 kDa membrane cut-off).

[0079] The numbers for the instantaneous permeate samples P3, P4 and sample R2 at the end of the second concentration step indicate that

- basically there is no loss of the highest MW fraction in the permeate, throughout this step;

- there is some loss of the smallest and medium MW fractions in the permeate, where the highest loss is always relative to the smallest fraction (as in diafiitration);

- the rate of loss of the medium fraction increases in this second concentration step (as in diafiitration).

Example 2

[0080] The method of the invention was performed with the following main steps:

a) First concentration by TFF to obtain a product with a heparin concentration from 4% to 10% w/v;

b) H2O2 treatment;

c) Second concentration by TFF to obtain a final product with a heparin concentration from 10% to 20% w/v.

[0081] Approximately 1936 g of starting product was transferred to the tank and the system was operated in total recirculation at a TMP = 3.2 bar for 10 minutes at a cross-flow of 5.1 LMM.

[0082] During concentration, permeateflow ranged from an initial 9.6 to a final 4.2 LMH (44%), for an average flow of 6 LMH. A sample of instantaneous permeate was colîected at VCF = 1.43X (P5), another permeate sample after completion of the concentration (P6) and finally a sample of the retentate (R3) at the end of the concentration, after leaving the filter in total recirculation at TMP = 0.6 bar for 10' (membrane depolarization). A sample of the starting solution (B2) was also ta ken before transfer to the tank.

[0083] Approximately 794 g of product was transferred to the tank after being treated with H2O2 and proceeded to the second concentration step.

[0084] During concentration, permeate flow ranged from 5.7 initial to 0.9 final LMH (16.0%), for an average flow of 2.5 LMH. 1 x instantaneous permeate sample was colîected at VCF = 1 33X (P7) and another of the permeate volume at the end of concentration (P8). A sample of the retentate volume at the end of concentration (R4) was also colîected, after depolarizing the membrane by leaving the system running at a low TMP (0.8 bar) for 10 minutes.

Table 4: Molecular weights in the process steps
Sample	Mw, Da	<2000 Da, %	2000-8000 Da, %	>8000 Da, %
Raw enoxaparin sodium	3646	24.9	70.6	4.5
B2 (Clarigard® clarification)	3742	23.5	71.5	4.9
P5 (1st permeate sample, 1 st concentration)	< 600	—	—	—
P6 (2nd permeate sample, 1st concentration)	<600	—	—	—
R3 (retentate after first concentration)	3784	23.1	71.5	5.4
P7 (1 st permeate sample, 2nd concentration)	1311	90.8	9.2	0.0
P8 (2nd permeate sample, 2nd concentration)	1476	84.4	15.6	0.0
R4 (retentate after second concentration)	4308	13.0	79.2	7.8

[0085] Table 4 shows the molecular weights of the samples taken during the process described above, analysed according to the method established by the Européen Pharmacopoeia (EP).

[0086] As the numbers in the table above show, it appears that the first concentration step does not négatively affect the product profile (without loss of any fraction in the permeate, samples P5 and P6, as in samples P1 and P2),

[0087] The numbers relating to permeate samples P7, P8 and the retentate volume sample R4 at the end of the second concentration step indicate that:

there is no loss of the highest fraction of MW in the permeate, throughout said step;

there is some loss of the smallest and medium MW fractions in the permeate, the highest loss is always relative to the smallest fraction (as in the first diafiltration test);

the rate of loss of the medium fraction increases in the second concentration (as in the diafiltration process).

[0088] Finally, the retentate solution is lyophilized to obtain dry enoxaparin sodium.

Example 3

[0089] The method of the invention was performed with the following main steps:

a) First concentration by TFF to obtain a product with a heparin concentration from 4%to10%w/v;

b) Depth filtration;

c) H2O2 treatment;

d) Second concentration by TFF to obtain a final product with a heparin concentration from 10% to 20% w/v.

[0090] Approximately 2000 g of starting product (turbidity > 1000 NTU) was transierred to the tank and the system was operated in full recirculation at a TMP = 3.2 bar for 15 minutes at a cross-flow of 5.2 LMM.

[0091] During concentration, permeate flow ranged from an initial 9.6 to a final 3.9 LM H (41% of initial flow), for an average flow of 5.8 LMH. A permeate sample was collected at VCF = 1.43X (PT) and another at the end of concentration (P2’) as well as the retentate (RT) at the end of concentration after leaving the filter in total recirculation at TMP = 0.7 bar for 15’ (membrane depolarization). A sample of the initial feed (BT) was also taken prior to transfer to the tank.

[0092] The R1 ' solution was passed through a Millistak +® HC Pro COSP depth filter, reducing turbidity to 0.57 NTU, to be subsequently treated with H2O2. Approximately 0.8 litres of product were transferred to the tank after being treated with H2O2 and the second concentration step was performed.

[0093] This second concentration was performed by sequentially aliquoting both the retentate and the permeate from the initial nominal concentration of 10% (samples R’3 and P’3, respectively) to the final nominal concentration of 20% (samples R’12 and ΡΊ2, respectively), passing through the intermedîate concentrations of 11, 12, 13, 14, 15, 16, 17, 18 and 19%.

[0094] Table 5 below shows the molecular weights of the samples taken during the process described above, analyzed according to the method established by the Européen Pharmacopoeia (EP).

Table 5: Molecular weights in the process steps
Sample	Mw, Da	<2000 Da, %	2000-8000 Da, %	>8000 Da, %
BT (Clarigard® clarification)	3464	29.6	65.95	4.50
PT (1 st permeate sample, 1st conc.)	<600	—	—	—
P2’ (2nd permeate sample, 1 st conc.)	<600	—	—	—
R1’(retentate after first concentration)	3416	31.90	63.37	4.70
P3’ (1st permeate sample 11%, 2nd conc.)	1054	100.00	0.00	0.00
P4’ (1st permeate sample 12%, 2nd conc.)	945	99.20	0.77	0.00
P5’ (1st permeate sample 13%, 2nd conc.)	999	98.40	1.59	0.00
P6’ (1st permeate sample 14%, 2nd conc.)	1060	96.90	3.10	0.00
P7’ (Ist permeate sample 15%, 2nd conc.)	1139	94.20	5.76	0.00
P8’ (1st permeate sample 16%, 2nd conc.)	1197	92.20	7.82	0.00
P9’ (1st permeate sample 17%, 2nd conc.)	1255	90.00	10.04	0.00
P10’ (1st permeate sample 18%, 2nd conc.)	1288	88,70	11.29	0.00

Table 5: Molecular weights in the process steps
Sample	Mw, Da	<2000 Da, %	2000-8000 Da, %	>8000 Da, %
P11’ (1st permeate sample 19%, 2nd conc.)	1378	85.40	14.63	0.00
P12’ (1st permeate sample 20%, 2nd conc.)	1330	87.20	12.84	0.00
R3’ (1st retentate sample 11%, 2nd conc.)	3744	24.6	69.9	5.6
R4‘ (1st retentate sample 12%, 2nd conc.)	3757	24.3	70.0	5.7
R5’ (1st retentate sample 13%, 2nd conc.)	3795	23.5	70.8	5.8
R6’ (1st retentate sample 14%, 2nd conc.)	3827	22.6	71.6	5.9
R7’ (1st retentate sample 15%, 2nd conc.)	3881	21.5	72.4	6.1
R8’ (1st retentate sample 16%, 2nd conc.)	3937	20.2	73.5	6.3
R9' (1st retentate sample 17%, 2nd conc.)	3994	18.9	74.7	6.4
R10’ (1st retentate sample 18%, 2nd conc.)	4005	18.4	75.1	6.4
R1T (1st retentate sample 19%, 2nd conc.)	4079	16.7	76.6	6.7
R12’ (1st retentate sample 20%, 2nd conc.)	4116	15.9	77.3	6.8

[0095] The variation both in average molecular weight and in the distribution of molecular weights is linear during the 2nd concentration from 10% to 20% nominal concentration of the product in the retentate, so that, by adjusting the final value of the concentration of the product in the solution of the retentate, it is possible to define a certain molecular weight profile and distribution of oligosaccharide chains for obtaining enoxaparin sodium. This fact can be seen in Figure 5. Finally, the retentate solution is lyophilized to obtain dry enoxaparin sodium.

Example 4

[0096] The product obtained in the previous example was analysed to détermine its anti-FXa and anti-FIla activity. The results obtained were as follows:

Anti-FXa activity, lU/mg (dried substance)	Anti-Flla activity, IU/ mg (dried substance)	Ratio aFXa/a Fila
112	29.5	3.8

[0097] This quality attribute adequately fulfils the ranges defïned by both European Pharmacopoeia and US Pharmacopoeia for enoxaparin sodium:

- Anti-FXa activity: 90- 125 lU/mg (dried substance)

- Anti-Flla activity: 20.0 - 35.0 lU/mg (dried substance)

- Ratio aFXa/aFlla: 3.3 - 5.3

Example 5

[0098] The method of the invention was performed with the following main steps:

a) Diafiltration;

b) H2O2 treatment; and

c) Concentration by TFF to obtain a final product with a heparin concentration from 4% to 15% w/v.

[0099] Approximately 3010 g of starting product was transferred to the tank and the system was operated in total recirculation at a TMP = 4.9 bar for 15 minutes at a cross-flow of 2.0 LMM.

[00100] During diafiltration, permeate flow ranged from an initial 17.7 to a final 13.1 LMH, for an average flow of 12.6 LMH and for 6 diavolumes. After each dïafiltered volume samples were collected of both the permeate (DP1 to DP6) and the retentate (DR1 to DR6). The permeate was treated with H2O2 and approximately 2720 g of treated product were transferred to the tank.

[00101] During concentration, permeate flow ranged from 12.7 initial to 1.28 final LMH (89.9% réduction), for an average flow of 5.1 LMH. Concentration was performed from 4 to 15%, collecting both a permeate sample (CP1 to CP6) and a retentate sample (CR1 to CR6) from 10%. 1 x instantaneous permeate sample was collected at VCF = 1.33X (P7) and another of the permeate volume at the end of concentration (P8). A sample of the retentate volume at the end of concentration (R4) was also collected, after depolarizing the membrane by leaving the system running at a low TMP (0.8 bar) for 10 minutes.

[00102] Table 6 below shows the moiecular weights of the samples taken during the process described above, analyzed according to the method established by the European Pharmacopoeia (EP).

Table 6: Moiecular weights in the process steps
Sample	MW, Da	<2000 Da, %	2000-8000 Da, %	>8000 Da, %
DR1 (retentate 1 diavolume)	3703	27.40	66.42	6.20
DR2 (retentate 2 diavolumes)	3770	26.10	67.51	6.40
DR3 (retentate 3 diavolumes)	3807	25.00	68.51	6.50
DR4 (retentate 4 diavolumes)	3841	24.10	69.36	6.50
DR5 (retentate 5 diavolumes)	3856	23.50	69.93	6.50
DR6 (retentate 6 diavolumes)	3884	22.90	70.40	6.70
CR1 (retentate concentration 10%)	3889	22.20	71.24	6.60
CR2 (retentate concentration 11%)	3912	21.60	71.77	6.60
CR3 (retentate concentration 12%)	3954	20.70	72.54	6.80
CR4 (retentate concentration 13%)	3967	20.10	73.08	6.80
CR5 (retentate concentration 14%)	3995	19.30	73.78	6.90
CR6 (retentate concentration 15%)	4029	18.50	74.54	7.00

[00103] The variation both in average moiecular weight and in the distribution of moiecular weights is linear from 10% to 15% nominal concentration of the product in the retentate, so that, by adjusting the final value of the concentration of the product in the solution of the retentate, it is possible to define a certain moiecular weight profile and distribution of oligosaccharide chains for obtaining enoxaparin sodium. The retentate solution is lyophilized to obtain dry enoxaparin sodium.

Example 6

[00104] The product obtained in the previous example was analysed to détermine its anti-FXa and anti-Flla activity. The results obtained were as follows:

Anti-FXa activity, lU/mg (dried substance)	Anti-Flla activity, 111/ mg (dried substance)	Ratio aFXa/a Fila
104	26.8	3.9

[00105] în view of the preceding description and the examples, a person skilled in the art would arrive at the invention as claimed without needing to resort to undue expérimentation. The above will be better understood with reference to the preceding examples, which describe certain processes for the préparation of embodiments of the présent invention. Ail the references made to these examples are for illustration purposes only. The examples must not be considered limiting, and are only illustrations of some of the many possible embodiments considered by the présent invention.

[00106] As used in the présent document, the term ''approximately means ± 10%, ± 5% or ± 1% of the specified value, preferably ± 10%. Moreover, ail the ranges specified in the présent document include the limits of the range and ail the whole and fractional values, particularly according to the définition of the term approximately.

Claims (22)

1. Method for obtaining low molecular weight heparins (LMWH) with an average molecular weight distribution of between approximateiy 3.0 and approximately 5.0 kDa comprising the following steps:

a) providing a crude depolymerized heparin solution with an oligosaccharide chain distribution range of between approximately 0.6 and approximately 10 kDa and a heparin concentration of up to approximately 4% w/v;

b) performing at least one concentration step by aqueous phase tangential flow filtration (TFF) using a nominal cut-off 1 kDa membrane to achieve a heparin concentration of up to approximately 25% w/v, thereby obtaining said LMWH.

2. Method according to claim 1, wherein step (b) is performed a first concentration step by TFF to achieve a heparin concentration of up to approximately 10% w/v and a second concentration step by TFF to achieve a heparin concentration of between approximately 10% and approximately 25% w/v.

3. Method according to claim 2, wherein the second concentration step is performed to a heparin concentration of between approximately 12% and approximately 22% w/v.

4. Method according to any one of claims 1 to 3, wherein a step of clarifying the heparin solution of step (a) is performed.

5. Method according to any one of claims 1 to 4, wherein at least one depth filtration step is performed.

6. Method according to claim 5, wherein the depth filtration step is performed prior to the TFF concentration step if only one is performed, or prior to the first TFF concentration step if more than one TFF concentration step is performed.

7. Method according to claim 6, wherein the depth filtration step is performed subsequently to the TFF concentration step if only one is performed, or prior to the first TFF concentration step if more than one TFF concentration step is performed.

8. Method according to any one of claims 1 to 7, wherein at least one step of diafiitration with water is performed.

9. Method for obtaining heparins according to claim 8, wherein the diafiltration step is performed prior to the TFF concentration step if only one is performed, or prior to the first TFF concentration step if more than one TFF concentration step is performed.

10. Method according to any one of claims 1 to 9, comprising at least one step of treating with H2O2.

11. Method according to claim 10, wherein the H2O2 treatment step is performed subsequently to the TFF concentration step if only one is performed, or prior to the first TFF concentration step if more than one TFF concentration step is performed.

12. Method according to claim 10, wherein the H2O2 treatment step is performed prior to the TFF concentration step if only one is performed, or prior to the first TFF concentration step if more than one TFF concentration step is performed.

13. Method according to any one of claims 1 to 12, wherein a step of lyophilizing the obtained concentrate is performed.

14. Method according to any one of claims 1 to 13, wherein the crude depolymerized heparin has not been obtained by fractional précipitation.

15. Method according to any one of claims 1 to 14, wherein the entire method excludes fractional précipitation of the heparin.

16. Method according to any one of claims 1 to 15, wherein the molecular weight (Mw) of the LMWH obtained is in the following ranges

Mw, Da M1 <2000 Da, % M2 2000-8000 Da, % M3 >8000 Da, %

LMWH 3800- 5000 12.0-20.0 68.0-82.0 <18.0.

17. Method according to any one of claims 1 to 16, wherein the molecular weight (Mw) of the crude depolymerized heparin is in the following ranges

Mw, Da M1 <2000 Da, % M2 2000-8000 Da, % M3 >8000 Da, % crude depolymerized heparin 3000- 5000 125 60-80 <20.

18. Method according to any one of daims 1 to 15, wherein the molecular weight (Mw) of the LMWH obtained is in the following ranges

Mw, Da M1 <2000 Da, % M2 2000-6000 Da, % M3 >6000 Da, %

LMWH 3000- 4200 <35.0 50.0-75.0 <15.0.

19. Method according to any one of daims 1 to 15 or 18, wherein the molecular weight (Mw) of the crude depolymerized heparin is in the following ranges

Mw, Da M1 <2000 Da, % M2 2000-6000 Da, % M3 >6000 Da, % crude depolymerized heparin 2500- 5000 <40 50-75 <25.

20. Method according to any one of daims 1 to 19, wherein the concentration by tangential flow filtration (TFF) employs a 0.7 to 1 kDa membrane.

21. Method according to claim 20, wherein a 0.9 to 1 kDa, preferably 1kDa, is employed.

22. Method according to any one of daims 1 to 21, wherein the heparin is enoxaparin sodium.