IE912487A1 - Process for preparing heparin calcium - Google Patents

Abstract

The preparation of the calcium salt of heparin or of products derived from heparin, such as the low molecular weight fractions obtained by extraction or by partial depolymerisation is described. The process according to the invention is characterised in that the calcium salt of heparin or of its derivatives is prepared by employing ion exchange membranes which, when subjected to an electrical field, allow a cation exchange to be carried out on the sodium heparin in a single stage.

Description

- ΙΑThe present invention relates to the preparation of heparin salts or of products derived from heparin such as the fractions of low molecular weight obtained by extraction or by partial depolymerisation. The process which constitutes the subject of the invention makes it possible, more particularly, to prepare the calcium salt of heparin or of its derivatives.

Heparin, known for its anti-coagulating action on blood, is extracted in the form of the sodium salt (heparin sodium) from various animal organs such as bovine or pig lungs or intestines. The therapeutic use of heparin sodium is carried out by intravenous administration. Patent FR-2,225,149 (73 13581) discloses heparin calcium which makes it possible to carry out a heparin15 based therapy by subcutaneous administration, which is a lot easier.

The calcium salt manufacturing process most often used at the industrial level consists in carrying out the sodium-calcium exchange by dissolving the heparin sodium in a calcium salt solution. Although calcium shows a greater affinity for heparin than sodium, a single exchange, even in the presence of excess calcium, is insufficient for completely eliminating the sodium which, according to the different pharmacopoeias, should only be present at less than 0.1% in the final product. It is therefore necessary to alcohol-precipitate the sodium and calcium heparinate mixture obtained during the first exchange and then to redissolve the precipitate obtained in a new calcium-rich solution. This procedure has to be repeated several times before the sodium content is sufficiently low. The precipitation in alcoholic medium being a slow procedure (one to two days), this process has the disadvantage of being long, alcohol-consuming and labour intensive. Moreover, the precipitation yields not being 100%, part of the heparin, recovered in the form of subfractions in the supernatants, has to be subjected to other purification procedures.

Other methods have been proposed for carrying out the exchange of cations in heparins - 2 - Static dialysis of heparin sodium with calcium salts, which is a process too slow to be applied industrially. The use of a hemodialyser-type apparatus is described in Patent US-4,409,103.

- The use of cation exchange resins has the disadvantage of requiring the use of heparin in the form of heparinic acid which is extremely unstable because it undergoes rapid autohydrolysis (Patent US-4,168,377).

- Diafiltration using a calcium salt solution is 10 disclosed in Patent US-4,409,103. It uses ultrafiltration membranes (ndn-ion exchange membranes). The salts are carried by the water flux which crosses the membranes under the action of a pressure gradient. Heparin is retained through a filtration effect, its size being larger than that of the pores of the membrane. The disadvantage of this process is that, in order to be sufficiently rapid, the membranes used should have high water permeabilities and therefore large-diameter pores which cause heparin losses through the membranes.

In a study on the solubility of various heparin and chondroitin sulphate salts, these products were electrodialysed using parchment membranes (E. Jorpes, Biochem. J. 1935, 29, 1817-1830). The method of assay not being sufficiently accurate, the author did not detect loss of activity before and after electrodialysis.

Nevertheless, he found 1% of the missing initial activity in the anode compartment. Furthermore, the method described by E. Jorpes requires the use of heparin in the acid, unstable form.

The Japanese Patent Application published under the number JP-01-149801 (Chemical Abstracts 112. 22607 m) discloses a method for preparing modified cellulose ether, particularly carboxymethylcellulose salts, according to which the sodium salt is subjected to an electro35 dialysis through an ion exchange membrane or an ultrafiltration membrane to give an acid form and the acid thus obtained is treated with the cation hydroxide or with a salt of the cation in order to obtain the desired salt. This method also requires the use of the acid form - 3 and, when applied to heparin sodium, involves a loss due to the degradation of heparin in the acid form.

It is also known that it is possible to separate from a solution various metal ions and in particular sodium, calcium or iron (ferric) ions by forming complexes with a chelating agent such as EDTA and then subjecting the complexes thus obtained to electrodialysis (Patent Abstracts of Japan Vol. 13 No. 286 (c-613) /3634/, 29.06.89; JP-1080408).

Electrodialysis has also been used for the continuous preparation of solutions containing various inorganic salts of low molecular weight, in particular sodium, ammonium or magnesium sulphite from calcium sulphite (Patent US-4,009,088).

It has now been found that by using an alternating sequence of anion-permeable membranes (APM) and cation-permeable membranes (CPM) in a conventional electrodialysis apparatus, these membranes being assembled so as to allow the circulation of liquids between two adjacent membranes, and by circulating a solution of the sodium salt of heparin or of heparin fragments or fractions and a solution of the calcium salt used for the ion exchange, the corresponding calcium salt is obtained directly without requiring the use of heparin in the acid form.

It has also been found that the reaction is practically quantitative and that less than 0.1% of the initial heparin (or of its fractions or fragments) is detected in the brine solution, such as is defined below, and in the electrode-rinsing solution.

The present invention therefore relates to a process for preparing the calcium salt of heparin or of its fragments or fractions, characterised in that a solution of the sodium salt of heparin or of its frac35 tions or fragments and a solution of a water-soluble salt of calcium is circulated through an electrodialysis apparatus comprising anion-permeable membranes and cation-permeable membranes in an alternating sequence allowing the circulation of the liquids. - 4 By way of starting products, heparin sodium or one of its fractions obtained by fractionation or by depolymerisation, particularly nadroparin also known under the name CY 216 and disclosed in Patent US-4,686,288, in the form of a sodium salt is preferably used. In this latter case, the process of the present invention is particularly advantageous because the molecular mass of nadroparin, of about 2,000 to about 8,000 daltons with a peak which is around 4,500 daltons, is substantially lower than that of heparin; the Na-Ca exchange during an ultrafiltration or dialysis process would involve high losses.

There may be used as starting products heparin sodium or heparin fragments, such as nadroparin sodium, which are purified or which have not been subjected to final purification and optionally contain small amounts of ethanol, sodium sulphate or sodium chloride. Comparative tests have shown that such products when present in small amounts in the starting products, do not affect the quality (purity) of heparin calcium or heparin fragments in the form of calcium salts obtained by the process of the invention.

All these starting products are hereafter called heparins whereas the final products are called heparin calcium.

The water-soluble salt of calcium preferably used is the chloride.

The process of the present invention comprises in particular the use of anion-permeable membranes (APM) and cation-permeable membranes (CPM) in a conventional electrodialysis apparatus. These ion exchange membranes are assembled in an alternating sequence which allows the circulation of liquids between two adjacent membranes. The process comprises circulating through the sequence of membranes a solution containing the sodium salt of heparin or one of its fractions or fragments and a watersoluble salt containing the calcium cation which is to be exchanged with sodium (for example CaCl2).

The membranes preferably have a permeability such - 5 that they allow the passage only of molecules having a molecular weight lower than about 500 daltons.

By way of non-restrictive examples, ASAHI GLASSRAMV/CSV membranes or Neo-SeptaRCMl and AMI membranes (Tokayama Soda, supplier) may be mentioned.

The process according to the invention is illustrated in the appended drawings. In these drawings: - Figure 1 schematically shows the functioning of an electrodialyser in the process according to the invention.

- Figure 2 schematically shows the migration of ions in the electrodialyser in the case of the preparation of calcium heparinate (heparin calcium).

Into an electrodialyser E (Fig. 1), on the one 15 hand, is introduced and therefore circulates therein a solution 1, hereinafter called product solution, containing heparin sodium (Hn, nNa+) and a water-soluble salt of calcium, for example calcium chloride and, on the other hand, there circulates a solution 2, hereinafter called brine, which initially contains only a small amount of salt (for example CaCl2) to ensure conductivity and which subsequently becomes enriched in salts (for example NaCl and CaCl2) and which is replaced during the electrodialysis by the addition of the water-soluble salt of calcium (for example CaCl2) to the product solution 1.

The electrodialyser E comprises a combination of cells, each consisting of an APM and a CPM. The number of membranes M used is 30 to 60 and preferably 40 to 50 and they are permeable to molecules smaller than about 500 daltons. The electrodes are rinsed in a known manner, for example with a solution 3 of HCl (0.5%) or of KC1 (1%). The product solution 1 circulates in one of two compartments, whereas the brine 2 circulates in the other compartments (see Figure 2).

Under the effect of a continuous electric field perpendicular to the membranes (provided continuously by a source V): - the Na+ ions go through the CPMs towards the cathode and are confined in the adjacent brine - 6 compartment because they encounter an APM therein; - the ions which are to be exchanged with Na+, in this case Ca2+, also go through the CPMs towards the cathode and are confined in the adjacent brine compart5 ment because their migration is blocked by the encounter with an APM; - the anions of the calcium salt added to displace sodium go through the APMs towards the anode and are confined in the brine compartment because they encounter a CPM therein; - the heparinate ions (Hn) migrate towards the APMs but cannot go through them because their molecular mass is too high. The electrodialysis membranes are in fact only permeable to species of molecular weight lower than about 500. The heparinate ions therefore remain in the product solution 1.

As the excess sodium cations and calcium cations are transferred into the brine in the form of the salt of the anion used to displace sodium, preferably in the chloride form, the desired cation, calcium in this case, is added to the product solution so as to completely displace the sodium.

It then suffices to recover the product solution by draining the product circuit and to add ethanol (or another heparin-precipitating agent) to this solution in order to precipitate the calcium salt of heparin or of one of its fractions.

The potential which is applied to the electrodes may range from 2 to 38 volts/cm, preferably from 5 to 30 volts/cm. The process is carried out at a temperature of to 80‘C, preferably at room temperature.

The process of the present invention is very simple to implement, it is very rapid and it produces very pure calcium salts, with a sodium content lower than 0.1% and with a very good yield in biological activity.

Furthermore, relative to a simple electrodialysis which is a desalting process, the process of the present invention produces a direct exchange of cations on an anionic macromolecule. Moreover, the macro-anions are - 7 known to foul the surface of the electrodialysis membranes; surprisingly, after a simple rinsing with water between two tests, no reduction in the performances of the membranes was observed as might have been expected at the beginning.

Relative to the known processes, the process of the present invention has considerable improvements, in particular: - relative to ultrafiltration or dialysis, the 10 process of the invention combines speed and low losses, which are difficult to reconcile by the two abovementioned methods; - relative to the successive precipitations, the duration of the treatment, the number of manipulations and the consumption of precipitating agent are considerably reduced.

The following examples illustrate the invention.

The material used in these examples is as follows: - SRTI electrodialyser model Pl - ASAHI GLASS AMV/CSV membranes (20 cells of 69 cm2) (a cell being made up of one APM and one CPM). The electrodialyser is therefore made up of a total of 41 membranes.

The operating conditions are as follows: - potential applied to the electrodes: 25 volts for Example 1 and 30 volts for Example 2; - temperature: room temperature.

EXAMPLE 1: Manufacture of heparin calcium PREPARATION A IN THE PRESENCE OF IMPURITIES 115 g of heparin sodium (or 20.6 χ 106 units according to the European Pharmacopoeia, 1990 ed., page 333, sodium heparinate monograph - assay: V.2.2.6.) are dissolved in 900 g of water. 15 g of ethanol, 1.2 g of Na2SO4 and 170 mg of NaCl are added. This solution is subjected to electrodialysis and the ions are progressively transferred into a brine solution with the following initial composition: CaCl2 : 5 g/H2O : 1 kg. The electrodes of the apparatus are rinsed with 1.2 litres of - 8 1% KCl which recirculates continuously. 100 g of a 38.5% solution of CaCl2 are added to the product at 0, 30 and 90 minutes. The salt-enriched brine is replaced with 1 kg of 0.5% CaCl2 at 30 and 90 minutes. After 150 minutes, the product solution is recovered by draining. Heparin is then precipitated with ethanol, ground, washed with alcohol and then dried in an oven under vacuum. 120 g of a powdered product with the following characteristics are recovered: - biological activity - sodium content - calcium content - biological activity : 20.6 χ 106 units (yield 100%) 0.03% 9.2% Only 0.6% of the initial heparin was detected in the brine and electrode-rinsing solutions by the tolu15 idine blue assay (J.P. DUCLOS, HEPARINE, Masson ed., p. 285). This effectively demonstrates the nearly total impermeability of the membranes to heparin.

PREPARATION B USING PURE HEPARIN SODIUM 115 g of heparin sodium (or 20.6 χ 106 units according to the European Pharmacopoeia, 1990 ed., page 333, sodium heparinate monograph - assay: V.2.2.6.) are dissolved in 900 g of water. This solution is subjected to electrodialysis and the ions are progressively transferred into a brine solution with the follow25 ing initial composition: CaCl2 : 5 g/H2O : 1 kg. The electrodes of the apparatus are rinsed with 1.2 litres of 1% KCl which recirculates continuously. 100 g of a 38.5% solution of CaCl2 are added to the product at 0, 30 and 90 minutes. The salt-enriched brine is replaced with 1 kg of 0.5% CaCl2 at 30 and 90 minutes. After 150 minutes, the product solution is recovered by draining. Heparin is then precipitated with ethanol, ground, washed with alcohol and then dried in an oven under vacuum. 120 g of a powdered product with the following characteristics are recovered: .6 x 10® units (yield 100%) 0.03% 9.2% - biological activity - sodium content - calcium content - 9 EXAMPLE 2 : Manufacture of the calcium salt of nadroparin 220 g of nadroparin sodium (or 62.0 χ 106 anti-Xa units according to Path. Biol. 1988, 36, 335-337), obtained as described in Example 1 of Patent US-4,686,288, are dissolved in 1860 g of water. This solution is divided into two equal parts, and each part is subjected to electrodialysis (the apparatus can only treat one litre per batch), the ions are progressively transferred into a brine solution with the following initial composition: CaCl2 : 5 g/H2O : 1 kg. The electrodes of the apparatus are rinsed continuously with 1.2 litres of 0.5% HCl which recirculate continuously. 100 g of a 38.3% solution of CaCl2 are added to the product at 0, 26 and 47 minutes for electrodialysis of the first batch and at 0, 37 and 77 minutes for electrodialysis of the second batch. The brine is replaced with 1 litre of 0.5% CaCl2 on each addition of CaCl2 to the solution. The pH of the solution is maintained between 6 and 7 by additions of Ca(OH)2. After 83 minutes for the first electrodialysis and 127 minutes for the second, the product is recovered by draining and rinsing of the product circuit with distilled water. Nadroparin calcium is then precipitated with ethanol, ground, washed with ethanol and dried in an oven under vacuum. 220 g of the powder with the following characteristics are recovered: - biological activity : 62.7 χ 106 anti-Xa units (yield of approximately 100%) - sodium content : 0.08% - calcium content : 9.8% The amount of nadroparin which is lost through the membranes is estimated by the ethanol precipitation procedure to be less than 0.01%.

Claims (9)

CLAIMS Process for the preparation of the calcium salt

1. of heparin or of its fragments or fractions, characterised in that a solution of the sodium salt of heparin or of its fractions or fragments and a solution of a water-soluble salt of calcium is circulated through an electrodialysis apparatus comprising anion-permeable membranes and cation-permeable membranes in an alternating sequence allowing the circulation of the liquids.

2. Process according to Claim 1, characterised in that electrodialysis is carried out at a potential of 2 to 38 volts/cm.

3. Process according to Claim 1, characterised in that electrodialysis is carried out at a potential of 5 to 30 volts/cm.

4. Process according to any one of Claims 1 to 3, characterised in that the membranes used allow the passage only of molecules having a molecular weight lower than about 500 daltons.

5. Process according to any one of Claims 1 to 4, characterised in that the chloride is used as calcium salt.

6. Process according to any one of Claims 1 to 5, characterised in that electrodialysis is carried out at a temperature of 0 to 80*C.

7. Process according to Claim 6, characterised in that electrodialysis is carried out at room temperature.

8. A process according to claim 1 for the preparation of a calcium salt of heparin or of its fragments or fractions, substantially as hereinbefore described and exemplified.

9. A calcium salt of heparin or of its fragments or fractions whenever prepared by a process claimed in a preceding claim.