GB1602439A - Purified heparin - Google Patents

Description

(54) PURIFIED HEPARIN (71) We, CHAOY S.A., a French Company, of 48 Avenue Theophile-Gautier, 75782 Paris, Cedex 16, France, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: The invention relates to a purification process of heparin and of heparin salts, as well as to the products obtainable by such process and the properties of which are improved with regard to the previously known heparin salts.

Sodium salts of heparin (also termed as sodium heparinates") are conventionally used for the production of injectable solutions of heparin. Mixed calcium-sodium heparinates or calcium-magnesium heparinates and advantageously calcium heparinates have more recently been brought into use, in order to do away with a number of indesirable vascular reactions, particularly in the injection area, due to the sodium ions.

Mixed heparinates, i.e. calcium-sodium heparinates or calcium-magnesium heparinates, and sodium-free heparinates, particularly calcium heparinates are advantageously prepared from an initial heparinate, say of sodium, by the process defined in British patent 1 471 482. This process comprises contacting the initial simple salt of heparin in an aqueous medium with a salt of the desired metal to be substituted at least partially for the metal in the initial heparin salt, to form an intermediate mixed heparin salt containing the desired metal, and separating the so formed intermediate heparin salt from free metal ions contained in said medium. To the extent where a heparin salt further enriched with the metal of substitution is desired, the above said intermediate heparin salt can be recontacted in an aqueous medium with a salt of the desired metal. If desired, the conditions of operation of this process can be adjusted so as to obtain a simple heparinate from the substitution metal free of the metal contained in the initial heparin salt.

It has been found, occasionally, that on storage of solutions of heparin salts containing calcium ions, deposits or precipitates could form or a turbidity would appear in the solutions. Obviously this tendency to the formation of precipitates or turbidity, which happens to be all the greater as the said solutions are more concentrated in heparin, is of particular disadvantage in the case of injectable solutions of heparin intended for therapeutical use prepared in advance industrially, notably in the form of predetermined doses, such as in the form of ampullae or dispensable syringes, and which accordingly must remain perfectly clear even after several months of storage. The formation of small deposits or even but light turbidity does of course render the said solutions inappropriate for the therapeutical administration.

It has been found that these difficulties may be attributed to the presence in the solutions of heparin, of injectable quality, of mineral salts of various nature and in variable quantities (up to 2.5% by weight) depending upon the origin of the tested batch and of the processes used for their extraction from natural sources, even though such contents of mineral salts remain within limits tolerated according to today's regulations, for instance the standards of the French CODEX.

The greater part of the residual salts is constituted by chlorates and sulfates which may respectively amount up to 1 %. Other salts, particularly carbonates, sulfites, are present in smaller proportions.

The transformation of sodium salts of heparin into calcium containing salts of heparin, say according to the process recal led hereabove, should reduce the proportions of these salts, taking into account the low solubility of the calcium sulfates sulfites and carbonates. The latter should as a matter of fact be separable as solids from the clear solution of the calcium-containing salts of heparin.

It has however been observed that, most surprisingly, at the end of the transformation of sodium heparinate into calcium heparinate, particularly in accordance with the above mentioned process, the contents of the above mentioned residual mineral salt is but little modified compared to that of the initial sodium heparinate.

Even more surprising was the finding that salts of heparin, including those which contain a metal formed at least in part of calcium, are amenable in the form of liquid aqueous solutions, even though they may still contain minor, yet non negligible amounts of oxalic ions. This was all the more unexpected as oxalates of metals like calcium are, as it well known, highly waterinsoluble.

The reason for this behaviour is not yet well understood. Nevertheless, a change in the normal solubility conditions of calcium oxalates has been ascertained, particularly when calcium chloride is in excess. Thus, it has been found that commercially available injectable heparins or heparinates may have residual salt-contents varying from 1.0 to 2.5% in weight, and from about 90 to 300 ppm oxalate ions, and sometimes even more.

It has been found that the elimination of the mineral salts from the treated heparins results in the disappearing of turbidity and deposits which were liable to occur in the calcium heparinate solutions after the preparation of said solutions.

The calcium or mixed calcium heparinate solutions which have undergone a purification process according to the invention as hereinafter disclosed, remain clear, even after having been stored for several months.

No turbidity or deposit appears.

Heparins or heparin salts according to the invention are thus those, the content of which in residual mineral salts is sufficiently low for enabling the injectable solutions prepared starting from such heparins - and more especially calcium heparinate solutions - and under concentrations normally used for this type of administration. to remain perfectly clear, even after several months of storage.

The appearance even of a light turbidity is always related to concentration conditions of the pharmaceutical solutions. In practice, the concentrations of the heparin solutions used for injections vary according to the type of injection (either subcutaneous, intramuscular, intravenous). The risks of turbidity or deposit increasing with the concentration according to the invention, said solutions must remain clear.

Heparin salts.according to the invention, containing residual oxalate ions liable to precipitate under the concentration conditions ordinarily used for the preparation of injectable solutions and more particularly calcium and calcium-sodium heparinates, to remain clear, have a content in residual mineral salts below 0.5% and preferably below 0.3% and below 30, preferably 20 ppm or less oxalate ions.

It must be understood that the expression "mineral salts" is not restricted to salts of mineral acids, such as sulfuric or hydrochloric acid, but also includes the salts of acids like oxalic or carbonic acid, or even of organic acids of low molecular weight, such as acetic acid.

It has been found that such heparin salts can be used for the production of injectable solutions which can be stored or shelved over prolonged periods of time, even when the metal of the heparin salt is, at least in part, one which like calcium, forms oxalates which are not water-soluble.

A process for obtaining purified heparins according to the invention, starting from commercially available products, brings into play a selective precipitation utilizing the difference of solubility between heparin salts and residual mineral salts.

The process according to the invention for obtaining such heparin or heparin salts having very reduced contents of oxalate, starting from a heparin preparation to be purified or, more generally, a mixture of heparin and other salts, particularly mineral salts in admixture therewith, comprises adding to an aqueous solution containing the heparin-mineral salts mixture an amount of a non-ionic precipitating agent such as alcohol, for instance ethanol, under conditions to cause a selective precipitation of the heparin or heparin salts while the mineral salt(s) remain in solution, recovering the heparin or heparin salt and repeating this selective separation on a new aqueous solution of the heparin so recovered until the concentration of oxalate in the resulting product is less than 30 ppm, preferably even less than 20 ppm.

According to a preferred embodiment of the process of the invention, the heparin used is in the form of a salt of a metal, the oxalates of which are themselves watersoluble. Another metal, particularly like calcium, of which the oxalates are waterinsoluble, can then be substituted, at least partially, for the metal of the purified heparin finally obtained, substantially freed from its oxalates.

The concentration of the heparin solution used for such treatment may largely vary. It is preferable, for practical reasons, that the solution should not be too diluted as the volume of ethanol used is proportional to that of the treated solution. For an equal quantity of heparin, the more diluted the solution, the larger the amount of ethanol required. To the contrary, the heparin solutions must not exceed a certain degree of concentration as their viscosity increases rapidly, and the heparin precipitation, starting from these solutions, could possibly carry down a large amount of the initial impurities which are intended to be separated.

Taking these conditions into account, the process is advantageously carried out on solutions the concentration of which is of the order of that of the solutions usually used for injections, that is containing from about 40 to about 250 g/l of sodium heparinate (corresponding to about 5000 to 30000 IU/ml).

The pH of the solution influences the result of the purification. An acid solution would rather retain the mineral salts in the solution, particularly oxalates, and consequently improve the separation.

Besides, heparin may be affected by strong acids. A solution having a pH above 3.5 and preferably comprised between 5 and 7 is advantageously used.

A sufficient amount of ethanol is added to the heparin solution thus prepared, so that practically the whole heparin precipitates while the mineral salts remain in the aqueous/alcoholic solution. For one volume of heparin solution, 0.5 to 1.5 volume of ethanol is preferably used.

Preferably, a practically pure neutral alcohol (99 to 100 GL) is resorted to.

The heparin precipitate will be separated from the supernatant and then the precipitate is kneaded and washed in order to eliminate all remaining traces of solution.

The washing is preferably done with absolute alcohol. Heparin is then filtrated and dried.

Should these first separations be incomplete, this treatment of the heparin precipitate may possibly be renewed till one obtains heparin salts meeting the above indicated requirements.

Most of the initial heparin is recovered in the course of the above described precipitation; the rest remains in the hydroalcoholic solution. The latter may be treated with another alcohol quantity in order to obtain a new heparin precipitation.

The purification process of the invention has been found to be applicable in all instances for the removal of the free oxalates contained in commercial heparins, whichever be its source.

However, while this process certainly enables the removal of any measurable free oxalates until providing heparins which contain less than 30 and even than 20 ppm, when applied to a heparin salt of a metal, like sodium, of, which the corresponding oxalate is water-soluble, it has occurred in few instances that upon converting at least partially such heparin salt into one of a metal, like calcium, of which the oxalates are insoluble, the resulting product was finally found to contain greater amounts of free oxalates.

Though atthis stage no scientific explanation can be offered for interpreting these phenomena, it is assumed that part of the oxalates contained in the commercial heparins of some sources behaves as if it were absorbed or fixed on the heparin molecules, the latter then behaving, apparently, as an anion-exchanger. Therefore, and as a result of these phenomena, the final heparin salts may prove again to no longer be suitable for the production of injectable solutions of the heparin which can be stored over prolonged periods of time.

It has however been further found that this difficulty can be overcome when resorting to the further improved process of this invention which comprises contacting said non-ionic heparin-precipitating-agent in the above described purification process steps with an initial aqueous solution of the heparin to be purified (or of the heparinmineral salt mixture) which contains watersoluble mineral salts other than oxalates in a concentration sufficient to favour a separation of the oxalates, including the apparently initially fixed or adsorbed oxalates which are then freed and remain in the aqueous solution upon the attendant precipitation of the heparin salts.

Thus some of the other salts present in the heparin solution to be purified may even, upon proper adjustment of their own concentration in the solution, whenever appropriate, participate to the more complete extraction of the oxalate ions.

Thus in preferred embodiments of the process according to the invention it will be usually required to preliminary adjust the concentration of said water-soluble mineral salts in the initial aqueous solution of heparin, prior to contacting the latter with the abovesaid non-ionic heparin precipitating agent.

As a matter of fact, it has been found that a sufficient concentration, particularly of salts comprising divalent anions and preferably too, monovalent anions, have a behaviour as if they were causing the displacement from the oxalates possibly adsorbed on or fixed to the heparin.

If need be, it will then be appropriate to repeat the precipitation steps upon recontacting a solution of the heparin so recovered and of water-soluble mineral salts other than oxalates in an adjusted concentration as hereabove defined until the concentration of total oxalates in the final heparin is less than 30 ppm, preferably even less than 20 ppm.

Preferably, the metal (or metals) of the salts having divalent anions, other than oxalates, which are contained in or possibly added to the initial heparin solution, is (or are) selected among those containing metals the oxalates of which are water-soluble.

Carbonates, preferably sodium carbonate, have been found most effective in the process according to the invention.

Preferably too the heparin in the abovesaid solution is in the form of a heparin salt of the same metal as that of the mineral salts. Any possibility of exchange of the metal contained in the latter mineral salt for the metal of the heparin salt is then avoided whereby the metal contents of the purified heparin salts is kept under close control.

Advantageously the concentration of the said mineral salts in the solution to be contacted with the non ionic precipitating agents such as alcohol, is adjusted to a valve from 0.3 to 2.5, for instance of about 0.5 % in weight with respect to heparin.

Advantageously too the starting solution also contains salts having monovalent anions of at least one metal, the oxalate of which is water-soluble. Sodium chloride is representative of such salts. In a preferred embodiment of the process according to the invention, the concentration of said monovalent salt is or is adjusted to a value ranging from 1 to 7%. for instance of about 2.5% in weight/volume of solution.

It has been found advantageous. though not necessary. that the pH of the solution contacted with the non-ionic precipitating agent be comprised between 7 and 10, for instance of the order of 8.5. This is actually the pH which establishes spontaneously when the salt having divalent anions used is sodium carbonate.

These salts having divalent and/or monovalent anions can then be easily eliminated in the final stage of the process, for instance in a final contacting step of the heparin solution with the non-ionic agent. Advantageously the pH is then adjusted for instance with hydrochloric acid. to a slightly acid value, sufficient for destroying the carbonates, particularly at a pH ranging from 3 to 7. The chloride ions remain in the aqueous solution, when the final heparin salt is precipitated.

Heparin compositions. particularly heparin salts are thus obtained which are substantially oxalate-free. which are - either directly suitable for the prepara tisf pharmaceutical compositions, particularly injectable or perfusable solutions having long storage-life, even when metallic cations of said heparin salts are formed at least in part of metals, the oxalates of which are highly insoluble, like calcium, - or suitable as starting heparin salts from which the metallic cations contained therein can be substituted at least in part advantageously, though not necessarily, according to the process of British patent 1.471.482 already mentioned hereabove, to provide other substantially oxalate-free heparin salts, which are then formed into pharmaceutical compositions having long storage life.

The invention thus concerns more particularly among the oxalate-free heparin preparations, the metallic salts of heparin, i.e.

either simple salts of heparin, such as the sodium, potassium, calcium or magnesium salts of heparin, or mixed salts of heparin containing at least two of the above - said metallic cations in any relative proportions, all of these heparin salts being substantially free of oxalates, in that they contain less than 30, preferably less than 20 ppm of total oxalates. Among the mixed salts one may cite the preferred series of those which contain sodium and calcium.

The invention also concerns more particularly the oxalate-free heparin preparations having an activity of at least 120 International Units (IU)/mg, free of pyrogens, as well as the highly concentrated solutions of heparin useful for their application in therapy for the control of blood-coagulation, particularly oxalate-free solutions suitable for sub-cutaneous injection containing from 5000 to 35 000 IU/ml of heparin, preferably from 20 000 to 30 000, such as 25 000 IU/ ml, or oxalate-free solutions suitable for intravenous injection, containing from 1 000 to 10 000, for instance 5 000 IU/ml of heparin, etc.

The purified heparin according to the invention may be used, either directly to prepare, e.g. injectable solutions under usual dosages and concentration conditions or, possibly, to prepare derived salts such as calcium heparinates or mixed calciummagnesium, calcium-sodium salts, for instance according to the process described in British patent No. 1 471 482.

First example of a preferred purification procedure of heparin In a preferred alcoholic fractionating purification process of injectable sodium heparinate, sodium heparin of bovine or porcine origin, of injectable quality is used.

This heparinate is dissolved in demineralised water (having a resistivity from 300 000 to 800 000 Q/cm, preferably 500 000 62/cam.

The concentration of the heparinate solution is settled between 5 000 and 30 000 IU/ml, preferably 25 000 IU/ml. 0.3 % metacresol is added in order to prevent from any contamination. The pH of the solution is maintained between 5 and 7, preferably 6.5, by adding either a reagent grade 5 N NaOH solution, or a reagent grade 5 N hydrochloric acid solution. The solution conductivity remains between 7 000 and 15 000 11 Mhos-cm.

0.7 volume of neutral ethyl alcohol (99 to 100" GL) is added to the heparinate solution thus prepared. One leaves the precipitate to settle and possibly adds a small amount of sodium chloride if necessary.

The precipitate is separated from the supernatant. It is once more solubilized in demineralised water, having the above described characteristics, so as to obtain a concentration roundabout 12 500 IU/ml.

The pH is controlled to 5.5 if need be, having recourse to a NaOH 5 N or Hcl 5 N solution. The solution is filtered on a Millipore (Registered Trade Mark) 0.3 F filter.

1.2 volume of neutral ethylic alcohol (99 100" GL) for 1 volume of solution is added under stirring. One leaves the precipitate to settle, possibly with addition of a small amount of sodium chloride.

The so obtained heparin precipitate is dehydrated by crushing in absolute ethyl alcohol, then filtered under vacuum upon industrial Bchner, washed with absolute alcohol and dried under 1 torr vacuum at a temperature of 35/40"C.

When further precipitations are needed, they are carried out in the same way as for the second precipitation step; i.e. heparin concentration of 12 500 IU/ml, pH 5.5. and 1.2 volume of absolute alcohol per 1 volume solution.

Second example of a general preferred procedure First alocholic fractionation A sodium salt of heparin of injectable quality, of bovine or porcine origin is dissolved in demineralized water having a resistivity ranging from 300 000 to 800 000 ohms, and preferably of 500 000 ohms, to provide a solution having a heparin concentration of from 5 000 to 30 000 IU/ml and preferably 25 000 IU/ml. Metacresol is added in an amount to provide a 0.3% concentration. The pH of the solution is adjusted to a value from 7 to 10, preferably 8.5 by addition of a sodium carbonate solution (anhydrous sodium carbonate in a proportion of 0.5% relative to the initial heparin weight.

The conductivity of the solution ranges then from 7 000 to 15 000 per, Mhos/cm.

Sodium chloride crystals are then added to the solution in a proportion of 2.5% weight/ volume. 0.7 volume of neutral ethylic alcohol titrating 99 to 100" G.L. (Gay-Lussac degrees) is then added, under stirring, to the volume of the heparin solution. The heparin precipitate is then collected, after 6 hours of standing of the medium.

2nd. alcoholic fractionation The heparin precipitate thus obtained is redissolved in demineralized water having the above mentioned characteristics so as to obtain a concentration close to 12 500 IU/ ml. The pH approximates then 8.5. Crystals of sodium chloride are then added in a 2.5 % weight/volume proportion relative to the solution. One volume of neutral ethyl alcohol titrating from 99 to 1000 G.L. is added under stirring.

The medium is left standing for 12 hours.

The new heparin precipitate is collected.

The "second alcoholic fractionation" is repeated three times in the same conditions as above, thus totalizing 4 fractionation steps. The precipitate originating from the 4th alcoholic fractionation is redissolved in demineralized water having the previously mentioned characteristics, so as to obtain a concentration approximating 12 500 IU/ml.

The pH is adjusted to 3 with hydrochloric acid, under vigorous stirring. Stirring is then maintained for another 15 minutes. The pH is then adjusted to 5.5 with 5 N sodium hydroxide. Sodium chloride crystals are added thereto in an amount of 2.5% weight/ volume and the solution thus obtained is filtrated on a MILLIPORE 0.3 F membrane. 1 volume/volume of neutral ethyl alcohol titrating 99 to 100" G.L. is added to the filtrate under stirring.

The medium is left standing for 12 hours.

The obtained heparin precipitate is dehydrated by crushing in absolute ethyl alcohol and filtrated under vacuum. It is finally washed in absolute alcohol and dried under a 1 Torr vacuum at a temperature of 35/40"C. An injectable sodium salt of heparin is thus obtained in which the proportion of total mineral salts is less than 0.5% and the total oxalate contents is below or equals at most 20 ppm.

Example I In this example, commercially available sodium heparinate titrating 160 lU/mg is used.

The total mineral salts content is 2.5 % and the oxalate content is of the order of 220 ppm (i.e. 0.022 %).

10000 g of this heparinate are dissolved in a 300 litre steel-inox reactor by adding 50 1 of demineralised water of a 500 000 Q/cm resistivity, previously filtrated on a Millipore membrane 0.22 Il.

The dissolution is achieved after one hour agitation. The pH is measured and adjusted at 6.5 by addition of NaOH 5 N or Hcl 5 N.

The volume is brought up to 64 1 by means of the same demineralised water. The conductivity measured is 15 000 FMhos/cm.

The temperature is kept between 15 and 30"C.

192 ml of newly distilled metacresol are added and 0.7 volume of a neutral ethyl alcohol (99/100 GL) is added for 1 volume of the initial solution, that is 44.8 litres. The solution is left until a precipitate appears; if need be, a small amount of sodium chloride is added. The supernatant and the sodium heparinate precipitate are separately recovered; the mineral salt contents of the latter is below 0.5 %, yet, about 200 ppm oxalate ions are still to be found therein.

The sodium heparinate is again solubilized in 50 litres of the same demineralised water of a 500 000 Q/cm conductivity. If need be, the pH is adjusted to 5.5 by addition of 5 N NaOH or 5 N Hcl. The measured conductivity is 15 000 F Mhoscm. The volume is brought up to 128 litres with the same demineralised water, 384 g of newly distilled metacresol are added and the whole preparation is left for 24 hours at room temperature. The heparin solution is filtrated on a 0.3 ij CWSS Millipore filter.

The filtrate is recovered. 30 litres of neutral ethyl alcohol (99/100 GL) i.e. 1.2 volume, are slowly added under stirring to 25 1. of the filtrate. The product is then left until a precipitate appears. If need be, a small amount of sodium chloride is added.

The supernatant and the sodium heparinate precipitate are separately recovered.

The latter is then dehydrated with absolute alcohol, crushed and filtrated under industrial vacuum on a Bchner funnel and finally dried and lyophilized. 9.400 gr of sodium heparinate are thus obtained, that is a yield of 94 %. This heparinate titrates 160 IU/ mg. Its content in mineral salts is 0.15 %, including 30 ppm oxalate ions.

In order to recover the heparinate which is still retained in the aqueous alcoholic supernatants, the treatment is prolonged as follows: There is added, under stirring, one volume of neutral ethyl alcohol (99/100 GL) to one volume of supernatant of the first alcoholic precipitation. After 24 hours settling, the clear to slightly opalescent supernatant is transferred and directed towards residual alcohols.

The precipitate is separated and recovered in absolute ethyl alcohol, dehydrated, crushed and dried.

Neutral ethyl alcohol (90/100 GL) is added under stirring to the supernatant of the second preparation, in a proportion of 0.5 volume to one volume of supernatant, and then left to settle for 24 hours.

Sodium heparinate is recovered in conditions similar to those described in the case of the first supernatant.

The sodium heparinate thus obtained is used in the preparation of the calcium salt.

This preparation is effected according to the method described in the British patent N0 1 471 482.

To sum it up, the treatment comprises: adding calcium chloride to a sodium heparinate solution so as to obtain a calcium enriched heparinate, then eliminating the freed sodium ions and re-adding calcium ions so as to obtain a calcium-ions-enriched heparinate.

By said process, a calcium salt of heparin is obtained, titrating 160 IU/mg, 10.3 % calcium, 0.2 % sodium, and 20 ppm oxalate ions.

Example 2 A sodium heparinate prepared as in example 1, titrating 160 IU/mg and having a content of about 30 ppm oxalate is processed as described in the British patent N" 1 471 482 for the preparation of a calciumsodium heparinate. In this example, calcium chloride is added only once to a sodium heparinate solution; freed sodium ions are then eliminated.

There has been thus prepared a calcium heparinate titrating 160 IU/mg and having a calcium content of about 7 %. The content in oxalate ions of this heparinate salt is 18 ppm.

A solution of this calcium-sodium heparinate, titrating 25 000 IU/ml, stored in ampullae for several months exhibits no trace of precipitate.

Example 3 The test carried out in example 2 has been repeated, this time completely transforming the sodium heparinate into calcium heparinate, according to the process described in the British patent N" 1 471 litres by addition of demineralized water.

The concentration is 25 000 IU/ml. 192 ml of freshly distilled metacresol, then 50 g of sodium carbonate in the form of an aqueous solution, and finally 1 600 g of sodium chloride crystals are added thereto. The solution is again left under stirring for 15 minutes. The pH, which should be of 8.5 is verified and; if needed, adjusted to 8.5 with a sodium carbonate solution. 44 litres of neutral ethyl alcohol titrating from 99 to 100"G.L are added thereto, under stirring (0.7 volume of alcohol per one initial volume of the solution). The medium is left standing for 6 hours and the heparin precipitate is collected.

This heparin precipitate is then subjected to a second alcoholic fractionation after having been redissolved in 110 litres of demineralized water having the previously mentioned characteristics, to obtain a concentration approximating 12 500 IU/ml. The solution is left under stirring for 15 minutes and demineralized water is added up to a volume of 128 litres. 384 ml of freshly distilled metacresol are added. The pH is adjusted to 8.5 with a solution of sodium carbonate. 3 200 gr. of sodium chloride are added. The solution is then left under stirring for 15 minutes. Neutral ethyl alcohol titrating from 99 to 100" G.L. is added in a porportion of 1 volume per volume of heparin solution, under stirring. The medium obtained is left standing for 12 hours. The heparin precipitate formed is recovered, after the removal of the supernatant.

The alcoholic fractionation is repeated three times again in the same conditions as in the second fractionation, on the heparin precipitate which is recovered each time from the preceding fractionation step.

The final heparin precipitate is then redissolved in demineralized water so as to obtain a concentration approximating 12 500 IU/ml, that is a solution having a volume of 128 litres. The pH is adjusted to 3 under vigorous stirring with hydrochloric acid. The solution is left under stirring for 15 minutes. The pH is adjusted to 5.5 with 5 N.

sodium hydroxide. 384 ml of freshly distilled metacresol and then 3 200 g of sodium chloride crystals are added. The obtained solution is filtrated on a 0.3 R MILLIPORE membrane. All these operations are carried out at a temperature ranging from 15 to 30"C. 1 volume of neutral ethyl alcohol titrating from 99 to 100 G.L. is added to the filtrate under stirring. The solution is left standing for 12 hours.

The final heparin precipitate thus obtained is dehydrated by crushing in absolute ethylic alcohol, filtrated under vacuum and subjected to repeated washings with absolute alcohol and then dried under 1 Torr vacuum at a temperature of 35/40"C for 24 hours. The powder is crushed an again dried under a 1 Torr vacuum for 24 hours at a temperature of 40"C, so as to eliminate the ultimate traces of solvent.

9 1 00 gr of a sodium salt of heparin are obtained, whose contents in mineral salts are less than 0.5%, and in oxalate less than 20 ppm. The obtained yield on that fraction is 90.43 %. The recovered titration IU/mg is : 157.

The dosages of the oxalate ions in the purified heparins were effected after their extractions by the method of J.R. HEL BERT and M.A. MARINI, Biochem.J.

(1963) 2 (5) pp. 1101-6, however modified in that the oxalate ions present in said heparins were extracted there from in the presence of an excess sodium carbonate, prior to being adsorbed on the IRA 400 anionic resin, the use of which has been recommended by the authors. After their elution from the resin, the oxalate ions were dosed according to the fluorometric method of P.M. ZAREMBSKI, and A. HODG KINSON, Biochem.J. (1965) 96,717-721).

WHAT WE CLAIM IS: 1. A process for purifying heparin, or a heparin salt to reduce the mineral salt (including oxalate) content thereof which comprises adding to an aqueous solution of said heparin in a soluble form an amount of a non-ionic precipitating agent, under conditions to cause a selective precipitation of the heparin or heparin salt while the mineral salt(s) remain in solution, recovering the heparin or heparin salt and repeating the selective separation on a new aqueous solution of the heparin or heparin salt so recovered until the concentration of oxalate in the resulting product is less than 30 ppm.

2. A process according to Claim 1 wherein the selective separation is repeated until the concentration of oxalate in the final product is less than 20 ppm.

3. A process according to Claim 1 or Claim 2 wherein the non-ionic precipitating agent is an alcohol.

4. A process according to any preceding claim wherein the starting solution comprises a heparin salt of a metal, the oxalates of which metal are water-soluble.

5. A process according to any preceding claim wherein the starting solution comprises a sodium heparinate, and contains from 40 to 250 g/l of sodium heparinate.

6. A process according to Claim 5 wherein the starting solution contains from 5 000 to 30 000 IU/ml of sodium heparinate.

7. A process according to any preceding claim wherein the pH of the solution is adjusted to a value above 3.5 8. A process according to Claim 4 wherein the pH of the solution is adjusted to from 5 to 7.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (27)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   litres by addition of demineralized water.

   The concentration is 25 000 IU/ml. 192 ml of freshly distilled metacresol, then 50 g of sodium carbonate in the form of an aqueous solution, and finally 1 600 g of sodium chloride crystals are added thereto. The solution is again left under stirring for 15 minutes. The pH, which should be of 8.5 is verified and; if needed, adjusted to 8.5 with a sodium carbonate solution. 44 litres of neutral ethyl alcohol titrating from 99 to 100"G.L are added thereto, under stirring (0.7 volume of alcohol per one initial volume of the solution). The medium is left standing for 6 hours and the heparin precipitate is collected.

   This heparin precipitate is then subjected to a second alcoholic fractionation after having been redissolved in 110 litres of demineralized water having the previously mentioned characteristics, to obtain a concentration approximating 12 500 IU/ml. The solution is left under stirring for 15 minutes and demineralized water is added up to a volume of 128 litres. 384 ml of freshly distilled metacresol are added. The pH is adjusted to 8.5 with a solution of sodium carbonate. 3 200 gr. of sodium chloride are added. The solution is then left under stirring for 15 minutes. Neutral ethyl alcohol titrating from 99 to 100" G.L. is added in a porportion of 1 volume per volume of heparin solution, under stirring. The medium obtained is left standing for 12 hours. The heparin precipitate formed is recovered, after the removal of the supernatant.

   The alcoholic fractionation is repeated three times again in the same conditions as in the second fractionation, on the heparin precipitate which is recovered each time from the preceding fractionation step.

   The final heparin precipitate is then redissolved in demineralized water so as to obtain a concentration approximating 12 500 IU/ml, that is a solution having a volume of 128 litres. The pH is adjusted to 3 under vigorous stirring with hydrochloric acid. The solution is left under stirring for 15 minutes. The pH is adjusted to 5.5 with 5 N.

   sodium hydroxide. 384 ml of freshly distilled metacresol and then 3 200 g of sodium chloride crystals are added. The obtained solution is filtrated on a 0.3 R MILLIPORE membrane. All these operations are carried out at a temperature ranging from 15 to 30"C. 1 volume of neutral ethyl alcohol titrating from 99 to 100 G.L. is added to the filtrate under stirring. The solution is left standing for 12 hours.

   The final heparin precipitate thus obtained is dehydrated by crushing in absolute ethylic alcohol, filtrated under vacuum and subjected to repeated washings with absolute alcohol and then dried under 1 Torr vacuum at a temperature of 35/40"C for 24 hours. The powder is crushed an again dried under a 1 Torr vacuum for 24 hours at a temperature of 40"C, so as to eliminate the ultimate traces of solvent.

   9 1 00 gr of a sodium salt of heparin are obtained, whose contents in mineral salts are less than 0.5%, and in oxalate less than 20 ppm. The obtained yield on that fraction is 90.43 %. The recovered titration IU/mg is : 157.

   The dosages of the oxalate ions in the purified heparins were effected after their extractions by the method of J.R. HEL BERT and M.A. MARINI, Biochem.J.

   (1963) 2 (5) pp. 1101-6, however modified in that the oxalate ions present in said heparins were extracted there from in the presence of an excess sodium carbonate, prior to being adsorbed on the IRA 400 anionic resin, the use of which has been recommended by the authors. After their elution from the resin, the oxalate ions were dosed according to the fluorometric method of P.M. ZAREMBSKI, and A. HODG KINSON, Biochem.J. (1965) 96,717-721).

   WHAT WE CLAIM IS: 1. A process for purifying heparin, or a heparin salt to reduce the mineral salt (including oxalate) content thereof which comprises adding to an aqueous solution of said heparin in a soluble form an amount of a non-ionic precipitating agent, under conditions to cause a selective precipitation of the heparin or heparin salt while the mineral salt(s) remain in solution, recovering the heparin or heparin salt and repeating the selective separation on a new aqueous solution of the heparin or heparin salt so recovered until the concentration of oxalate in the resulting product is less than 30 ppm.
2. 2. A process according to Claim 1 wherein the selective separation is repeated until the concentration of oxalate in the final product is less than 20 ppm.
3. 3. A process according to Claim 1 or Claim 2 wherein the non-ionic precipitating agent is an alcohol.
4. 4. A process according to any preceding claim wherein the starting solution comprises a heparin salt of a metal, the oxalates of which metal are water-soluble.
5. 5. A process according to any preceding claim wherein the starting solution comprises a sodium heparinate, and contains from 40 to 250 g/l of sodium heparinate.
6. 6. A process according to Claim 5 wherein the starting solution contains from 5 000 to 30 000 IU/ml of sodium heparinate.
7. 7. A process according to any preceding claim wherein the pH of the solution is adjusted to a value above 3.5
8. 8. A process according to Claim 4 wherein the pH of the solution is adjusted to from 5 to 7.
9. 9. A process according to any preceding

   claim wherein the precipitation is done using from 0.5 to 1.5 volume of ethanol per volume of said aqueous solution.
10. 10. A process according to any preceding claim wherein the concentration of mineral salts other than oxalates in the initial aqueous solution prior to contacting it with the non-ionic agent is sufficient, or is adjusted to a value sufficient, to favor the separation of the oxalates, including the apparently fixed or adsorbed oxalates, which are then freed and remain in the aqueous solution upon the subsequent precipitation of the heparin and which comprises recovering the precipitated heparin.
11. 11. A process according to Claim 10 wherein said aqueous solution contains at least one salt comprising a divalent anion and a metal, the oxalate of which metal is water-soluble.
12. 12. A process according to Claim 11 wherein the said salt is a carbonate.
13. 13. A process according to Claim 12 wherein said salt is sodium carbonate.
14. 14. A process according to any one of Claims 11 to 13 wherein the concentration of said salt ranges is from 0.3 to 2.5 per cent by weight with respect to the heparin or heparin salt.
15. 15. A process according to Claim 14 wherein the concentration of said salt is about 0.5 per cent by weight with respect to the heparin or heparin salt.
16. 16. A process according to any one of Claims 10 to 15 wherein said solution also comprises salts having monovalent anions of at least one metal, the oxalate(s) of which metal(s) is (are) water-soluble.
17. 17. A process according to Claim 16 wherein the said salt having monovalent anions is a chloride.
18. 18. A process according to Claim 17 wherein said salt is sodium chloride.
19. 19. A process according to any one of Claims 16 to 18 wherein the concentration of said salt having a monovalent anion in said aqueous solution ranges from 1 to 7% w/v.
20. 20. A process according to Claim 19 wherein the concentration of said salt is about 2.5% w/v.
21. 21. A process according to any one of Claims 10 to 20 wherein the heparin is in the form of a salt of a metal which is the same as that of said mineral salts.
22. 22. A process according to any one of Claims 10 to 21 which comprises further substituting at least in part a metal the oxalates of which are not water-soluble for the metal in the heparin obtained from the finally recovered heparin precipitate.
23. 23. A process according to Claim 22 wherein said substituted metal is calcium.
24. 24. Heparin salt of at least one metal, including at least in part calcium, and which contains less than 30 ppm of total oxalates.
25. 25. Heparin salt according to Claim 24 containing less than 20 ppm of total oxalates.
26. 26. Heparin salt according to Claim 24 or Claim 25 which contains less than 0.5 per cent of mineral salts.
27. 27. Heparin salt according to Claim 26 containing less than 0.3 per cent of mineral salts.