GB2176200A

GB2176200A - Process for the preparation of heparin salts

Info

Publication number: GB2176200A
Application number: GB08613593A
Authority: GB
Inventors: Gyorgy Kerey; Janos Illes; Bela Stefko; Erzesbet Neszmelyi; Pal Gere; Istvan Zambo
Original assignee: Richter Gedeon Vegyeszeti Gyar RT
Current assignee: Richter Gedeon Vegyeszeti Gyar Nyrt
Priority date: 1985-06-04
Filing date: 1986-06-04
Publication date: 1986-12-17
Anticipated expiration: 2006-06-04
Also published as: IT8620676A0; GB2176200B; IT1190124B; GB8613593D0; FR2582657B1; HU193769B; BE904867A; AT397250B; FR2582657A1; IT8620676A1; ATA144386A

Abstract

Heparin salts of pharmacopoeial purity, preferably calcium heparinate are prepared by treatment of an aqueous solution of a heparinate of pharmacopoeial quality or a pyrogen-free aqueous heparinate solution optionally containing heavy metal salts and inactive heparinoid fractions with a quaternary ammonium halide, separating the precipitated heparin-quaternary amine complex formed and washing it with pyrogen-free, deionized water, and dissolving the purified heparin-quaternary amine complex i) in an aqueous solution of a salt containing the cation of the heparinate to be prepared, or ii) in a water-immiscible organic solvent, and adding to the solution obtained an aqueous solution of a salt containing the cation of the heparinate to be prepared, or iii) in an organic solvent immiscible or restrictedly miscible with water, and adding to the solution obtained an aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the two phases to separate and separating them from each other, and if desired, adding to the organic phase obtained in step iii) an aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the phases to separate, separating them from each other and combining the aqueous phases. <IMAGE>

Description

SPECIFICATION Process for the preparation of heparin salts The invention relates to a new process for the preparation of heparin salts of pharmaceutical quality.

Heparin was discovered in 1913 and since that time has been widely employed in clinical therapy and prophylaxis. Until quite recently heparin has almost exclusively been used in the form of its sodium salt and the pharmacopoeial standards are measured as relative to the quality of sodium heparinate. The experiences gained during the use of sodium heparinate for several decades and the results of certain clinical trials performed in the past few years show, however, that in certain fields other heparin salts have more advantageous properties. The duration of activity of calcium heparinate is for example longer than that of sodium heparinate. Accordingly, the therapeutic dose can be maintained by less frequent administration of heparin patients.Moreover, the haemorrhaging observed in patients during the administration of sodium heparinate may be avoided by using heparin in the form of its calcium salt [Kakkar, V.V.: Clinical usage of heparin. Present and future trends, edit. M. Verstraete, S.J. Machin, Munsgaard, Copenhagen 1980, p. 158; Kakkar V.V: Heparin chemistry and chemical usage, Academic Press 1976].

Therefore there are more and more manufacturers offering various heparinates containing cations other than sodium (e.g. calcium, magnesium, lithium, potassium, ammonium heparinates) for clinical and diagnostic purposes.

According to the known processes the sodium cation of heparin can be replaced by another cation either by contacting an aqueous solution of sodium heparinate directly with a water-soluble salt of the selected cation ("reversible" process) or by isolating heparin as a free acid by means of an acidic ion exchange resin and subsequently converting this into the desired salt ("irreversible" process).

In the "reversible" process it is practically impossible to replace sodium cation by the desired other cation; hence the product should contain less than 1 % by mass of sodium cation. Even if the concentration of the new cation is increased, i.e. its salt is used in a large excess, the equilibrium of the reversible process can be shifted in the direction of the formation of the desired new heparin salt only to limited extent. A further shift of the equilibrium can be achieved by isolating the "mixed" heparin salt (i.e. the heparin salt containing sodium cation in addition to the desired new cation) by precipitation with a solvent and repeating the whole procedure several times. This multi-step procedure is obviously time consuming, labour intensive and has a high solvent demand, and the yield is substantially reduced by the multiple treatment.

According to the "irreversible" process the cation exchange is carried out using a cation exchange resin. In the first step of this process heparin is formed as a free acid, which is liable to decompose and has about a 20 to 30% lower anticoagulant activity.

An improved variant of the "reversible" process is disclosed in German Patent No. 2,417,859, in which the equilibrium of the reversible process is shifted in the desired direction not only by isolating the mixed salt formed but also by eliminating the cation of the starting sodium heparinate by dialysis. According to this process, calcium heparinate is for example prepared starting from sodium heparinate of pharmacopoeial purity by adding calcium chloride to an aqueous solution of sodium heparinate, stirring the mixture for 16 hours and then dialysing the solution in a small volume (300 ml.) against ion- and pyrogen-free water for 6 hours. From the dialysed solution diluted to about twice its original volume, the "mixed" heparin salt (sodium and calcium heparinate) is precipitated from an organic solvent.The isolated product is dissolved in distilled water and the whole procedure is repeated. The isolated calcium heparinate contains less than 0,1% by mass of sodium ion and its specific activity is about the same as that of the starting product.

The process is indeed advantageous since, by combining the precipitation with dialysis, cation exchange can be accomplished in two steps instead of the earlier multi-step processes. Dialysis has, however, several drawbacks: the reaction time is increased, there is a higher risk of pyrogenity and infection, dialysis carried out in small volumes is cumbersome and accordingly the losses are increased. The process is not suitable for the purification of heparin or for increasing its specific activity, since the starting material is already of pharmacopoeial purity.

Thus, in the known processes the cation of sodium heparinate is replaced by another cation either by using a cation exchange resin, i.e. by an "irreversible" process or by successive "reversible" processes, where the equilibrium state achieved in the individual steps is fixed by precipitating the "mixed" salt of heparin and a further shift of the equilibrium state to the desired extent is attempted starting from this fixed equilibrium.

The purpose of the present invention is thus to provide a process by which - the exchange of the original cation of heparin is made irreversible by preparing a water-insoluble intermediate (without the formation of heparic acid which is liable to decomposition), from which cation exchange can be accomplished quantitatively in a single step, - the cation exchange can be carried out not only from heparin of pharmacopoeial quality but from any aqueous heparin solution obtained following the elimination of pyrogenic substances during heparin production, and - which is suitable, in addition to cation exchange, for a further purification of heparin and for increas ing its specific activity.

We have surprisingiyfound that the quaternary ammonium cations of quaternary ammonium halides conventionally used for other purposes during the manufacture of heparin, are under certain conditions also suitable for the exchange of cations of heparinates. Due to the water-insoluble character of the heparin-quaternary amine complex the process is irreversible and therefore cation exchange may take place quantitatively.

According to one aspect, the present invention provides a process for the preparation of heparin salts of pharmacopoeial purity, preferably calcium heparinate, which comprises adding an aqueous solution to a quaternary ammonium halide capable of forming a heparin-quaternary amine complex to an aqueous solution of a heparinate of pharmacopoeial quality orto a pyrogenfree aqueous heparinate solution optionally containing heavy metal salts and inactive heparinoid fractions; and separating the precipitated heparin-quaternary amine complex and washing it with pyrogen-free, deionized water and dissolving the purified heparin-quaternary amine complex either i) in an aqueous solution of a salt containing the cation of the heparinate to be prepared; or ii) in a water-immiscible organic solvent, and adding to the solution obtained an aqueous solution of a salt containing the cation of the heparinate to be prepared; or iii) in an organic solvent immiscible or partially miscible with water, and adding to the solution obtained an aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the two phases to separate and separating them from each other, followed, if desired, by adding to the organic phase obtained in step iii) an aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the phases to separate, separating them from each other and combining the aqueous phases, and if desired, purifying the solution obtained by any of steps i) - iii), precipitating the desired heparinate by adding a water-miscible solvent, and isolating the precipitated heparinate.

The term "heparin salts of pharmacopoeial quality" is used throughout the specification and claims and refers to heparin salts the purity of which meets the standards of USP XXI (The United States Phar-macopoea XXI).

The terms "heparin salts" and "heparinates" refer to pharmaceutically acceptable salts of heparin, e.g.

magnesium, potassium, lithium, ammonium and preferably calcium salts (heparinates). The terms "heparin salts" and "heparinates" are synonyms, e.g. heparin sodium salt is identical with sodium heparinate.

In the first step of the process according to the invention an aqueous solution of a quaternary ammonium halide which readily dissociates in water and forms with the heparinate anion a complex insoluble in water, is added to a pyrogen-free solution of the starting heparinate. The heparinate-solution generally contains sodium cation in association with the heparinate anion but other cations and amnions may be present as well, depending on the previous preparative steps.The process may be illustrated by the following simplified reaction equation: [Hepn-] + n[K,+] + n[Kr] + n[A-] < Hep(K2),+ n[Kl+]+n[A-] wherein [Hepn-] = heparinate anion, [K1+] = the original cation of heparin (generally sodium ion), [K2+] = quaternary ammonium cation forming a water-insoluble complex with the heparinate anion, [A-] = halide ion originally attached to the [K2+] quaternary ammonium cation (generally chloride or bromide ion), n = number of the negative charges of the heparinate anion.

Since the heparin-quaternary amine complex is insoluble in water, the reaction proceeds quantitatively in the direction of the arrow. The other cations and anions present in the reaction medium have no influence on the progress of the reaction. The precipitated heparin-quaternary amine complex is separated and washed with ion- and pyrogen-free water.

In contrast to the slow equilibrium processes, in the process according td the invention the ion exchange takes place instantaneously and the other cations may be eliminated by simple separation and washing techniques instead of lengthy and cumbersome dialysis.

Of the salts forming water-insoluble complexes with heparin, the quaternary ammonium halides. e.g.

cetyl-pyridinium chloride, cetyl-trimethylammonium bromide, HyamineR 1622 (benzyl-dimethyl-[2-(2-(p 1,1 ,3,3-tetramethyl-butyl-phenoxy) ethoxy)-ethyl] ammonium chloride) and its derivatives (United States Patent Specifications Nos. 2,989,438; 3,05 & 884; 3,160,563 and 3,342,683) and the quaternary ammonium halides disclosed in Hungarian Patent Specification No. 188,537 such as carbotetradecyl-oxymethyl-trimethyl ammonium chloride and related compounds proved to be the most suitable for the purpose of the invention.

The process preferably comprises using as a quaternary ammonium halide a compound of formula (I) or (II)

wherein R is a straight- or branched chain alkyl having from 8 to 18 carbon atoms, phenyl or phenyl-Cl 3-alkyl R1, R2 and R3 are identical or different and represent a straight-chain alkyl having from 1 to 4 carbon atoms, or R, and R2 together form a pentamethylene group and R3 is hydrogen, R. is hydrogen or a group identical with R, A represents a group

B and D are identical or different and represent a valency bond or a group identical with A, N+ is a quaternary nitrogen atom, X- is a monovalent inorganic anion, m is an integer from 1 to 4, n is an integer from 0 to 4, k is 1, 2 or 3, Quaternary ammonium halides have already been used in the manufacture of heparin but for different purposes, namely for separating out crude sodium heparinate from dilute aqueous solutions of heparincontaining animal organs and tissues and isolating these. In addition to the advantages described hereinabove, the use of quaternary amine halides is advantageous since most of them have bactericidal properties; hence, when using these compounds the risk of reinfection, (i.e. pyrogenity) is negligible compared to other, known techniques for the preparation of calcium heparinate.

We have further found that the precipitation of the heparin-quaternary amine complex can be monitored instrumentally, permitting an increase in the specific activity of heparin during ion exchange. By measuring the electrode potential of the aqueous heparin solution with a combined glass electrode the progress of precipitation can be monitored potentiometrically, and the quantity of the aqueous solution of the added quaternary ammonium halide can be adjusted to optimum. During the additon of aqueous quaternary ammonium halide solution, first the fractions having a high anticoagulant activity are precipitated from the heparin mixture, while the inactive fractions are the last to precipitate. By monitoring the potentiometric curve the optimum ratio at which the precipitation process should be terminated to prevent the precipitation of inactive fractions can be easily determined.In this way the activity of sodium heparinate starting material having a pharmacopoeial quality can be increased by 10 to 15 %.

From the heparin-quaternary amine complexes the desired salt of heparin can be easily prepared by a further irreversible cation exchange. The heparin-quaternary amine complexes can be dissolved in aqueous salt solutions, e.g. in at least 2 molar aqueous sodium chloride solutions. From the solution obtained, the corresponding salt, such as the sodium heparinate, can be isolated by precipitation with a solvent, since the quaternary ammonium halides are soluble in the organic solvents used for precipitation. Since the heparinates separate out, the anion exchange is irreversible.

As mentioned before, the heparin-quaternary amine complexes have so far been used merely for the isolation of crude sodium heparinate from aqueous sodium heparinate solutions, i.e. no ion exchange has taken place during the procedure. We have now unexpectedly found that by the process according to the invention any desired heparin salt can be prepared with pharmacopoeial purity, starting from any pyrogen-free aqueous heparinate solution containing a different cation, e.g. from sodium heparinate high-purity calcium heparinate can be easily obtained.

If the ion exchange is carried out according to variant i), i.e. the heparin-quaternary amine salt is dis solved in an aqueous solution of a salt containing the desired cation, due to the high salt concentrations a repeated dissolution of the precipitated heparin salt in water and a further precipitation with the se lected organic solvent may be necessary to obtain the product in satisfactory purity. For the complete elimination of the quaternary ammonium compound the product is thoroughly washed with an appropri ate solvent.

We have found that if the heparin-quaternary amine complex is first dissolved in an organic solvent conventionally used for the precipitation of heparin (e.g. methanol, ethanol, acetone) instead of a concen trated aqueous salt solution, the ion exchange of the dissolved heparin-quaternary amine complex may be carried out with a considerably less concentrated aqueous salt solution (variant ii)). By proper selec tion of-the solvent ratio the desired heparin salt is precipitated practically without any accompanying inorganic salt impurity. The quaternary ammonium halide should be eliminated from the product by washing with an appropriate solvent as in the case of the previous variant.

According to the most preferred variant of the process according to the invention (variant iii) the hepa rin-quaternary amine complex is first dissolved in an organic solvent immiscible or only partially miscible with water (e.g. isopropanol, n-butanol, chloroform), the ion exchange is carried out by adding an aqueous solution of a salt containing the desired cation to the first solution, the phases are separated and the desired heparin salt is precipitated from the aqueous phase by adding an organic solvent (e.g.

acetone, ethanol, methanol). In this variant the irreversibility of the ion exchange is already ensured by the presence of the immiscible liquid phases, since the quaternary ammonium halide remains in the or ganic phase, while the heparin salt formed with the new cation will be present in the aqueous phase. A major advantage of thins process is that in this way the quaternary ammonium compound is eliminated without any additional purification step, simply by separating the phases.

According to a preferred embodiment of the process according to the invention, a 1-20 % (w./v.), pref erably 5-15 % (w./v.) aqueous solution of a quaternary ammonium compound, preferably HyamineR 1622 or carbotetradecyl-oxymethyl-trimethyl ammonium chloride is added to an aqueous solution sodium heparinate of pharmacopoeial purity or to a pyrogen-free aqueous sodium heparinatesolution optionally containing heavy metal salts and inactive heparinoid fractions (heparin concentration 500-10 000 I.U./ml., - preferably 1000-5000 I.U./ml: ph 2-10, preferably 4-8), under stirring (I.U. = International Unit).The quan tity of the quaternary ammonium salt used is about 1.5 to 3.5 g.n05 I.U.. If in addition to the ion ex change the specific activity is also to be increased, the optimum ratio is determined using the following method: A 10 % (w./v.) aqueous solution of the selected quaternary ammonium salt is added to an aliquot of the-starting aqueous sodium heparinate solution under stirring, and the electrode potential of the solu tion is measured using a combined glass electrode. The electrode potential measured in mV is plotted against the quantity of the added quaternary ammonium salt solution and from the curve obtained the volume of quaternary ammonium salt solution required for the precipitation of the active anticoagulant fraction is determined.The optimum ratio can be determined without knowing the heparin concentration of the aqueous heparin solution, since the-ratio of the reactants can be expressed in ml. of quaternary ammonium solution used up in the titration/ml of heparin solution.

Following the precipitation of the heparin-quaternary amine complex the mixture is centrifuged and the mother liquor is removed. The centrifuged precipitate is suspended in deionized, pyrogen-free water and subjected to further centrifuging.

After centriguging the precipitate obtained is dissolved in a 4- to 10-fold, preferably 6-to 8-fold volume of an organic solvent, preferably immiscible or only partially miscible with water, such as chloroform, isopropanol, ethyl acetate or preferably n-butanol. To the organic, preferably butanolic solution a 1 to 10 % (w./v.), preferably2 to 5 % (w./v.) solution of a water-soluble salt of the desired cation, preferably-con taining chloride ion in an amount of 20 to 50 % (v./v.), preferably 30 to 40 % v./v.) is added. The mixture is stirred for 5-15 minutes, preferably 15 minutes and the phases are separated. The aqueous-phase is filtered through a Seitz filter plate or a membrane filter and is precipitated by adding 50-150 % (v./v.), preferably 60-80 % (v./v.) of a water-miscible lower alcohol, e.g. methanol, ethanol, or ketone, e.g. ace tone.

The precipitated heparinateis isolated in a manner known per se and dried. The product obtained meets the requirements of USP XXI and contains less than 0.1 % (w./w.) of sodium ion.

The invention-is described in detail by the following non-limiting Examples.

Example t From sodium heparinate of pharmacopoeial quality having a specific activity of 165 I.U./mg. a 4 % (w./ v.) solution is prepared with pyrogen-free distilled water. To100 ml. of the solution 165 ml. of a 10 % (w./ v.) aqueous cetyl-pyridinium-chloride solution are added under stirring. The precipitate obtained (hepa ,rin-quáternary amine complex is separated by centrifuging.The wet precipitate obtained by ceritrifuging weighs 16.5 g. The precipitate is suspended in 40 ml. of pyrogen-free distilled water and the suspension is centrifuged.

The wet cetyl-pyridinium-heparinate obtained by the second centrifuging is dissolved in 200 ml. of a 2 molar calcium heparinate solution under stirring. The solution is purified by filtration and the heparincalcium salt is precipitated from the filtrate with 150 % (v./v.) of ethanol. The calcium heparinate precipitate separated by centrifuging is suspended in ethanol and is then dissolved in 200 ml. of pyrogen-free distilled water. To the solution 100 % (v./v.) of ethanol are added under stirring. The precipitated calcium heparinate is centrifuged, washed with ethanol and subsequently with acetone, and dried.

The specific activity of the calcium heparinate obtained is 162 I.U./mg., its sodium content is less than 0.1 % by mass and the product meets the requirements of USP XXI.

Example 2 To 100 ml. of the sodium heparinate solution according to Example 1160 ml. of a 10% (w./v.) aqueous solution of cetyl-trimethyl-ammonium-bromide are added. The precipitate (heparin quaternary amine salt) is separated by centrifuging suspended in 40 ml. of pyrogen-free distilled water and again centrifuged.

15.8 g. of a wet precipitate are obtained. The precipitate is dissolved in 100 ml. of ethanol, and the solution obtained is added to 200 ml. of a 0.5 molar aqueous calcium chloride solution. The calcium heparinate obtained is purified by filtration and 120 ml. of ethanol are added to the filtrate with stirring. The calcium heparinate precipitate obtained is suspended twice in ethanol, filtered, washed with acetone and dried in vacuo.

The specific activity of the calcium heparinate obtained is 167 I.U./mg., its sodium content is less than 0.1 % by mass and the product meets the requirements of USP XXI.

Example 3 The procedure described in Example 2 is followed except that the heparin-quaternary amine complex is dissolved in methanol instead of ethanol.

The specific activity of the calcium heparinate obtained is 165 I.U./mg., its sodium content is lower than 0.1 % by mass and its quality meets the requirements of USP XXI.

Example 4 The procedure described in Example 2 is followed except that the heparin-quaternary amine complex is dissolved in acetone.

The specific activity of the calcium heparinate obtained is 161 I.U./mg., its sodium content is lower than 0.1 % by mass and its quality meets the requirements of USP XXI.

Example 5 To 100 ml. of an aqueous sodium heparinate solution prepared according to Example 1130 ml. of a 10 % (w./v.) aqueous solution of HyamineR are added. The heparin-quaternary amine complex precipitated is separated by centrifugation, suspended in 45 ml. of water and again centrifuged.

The wet precipitate is dissolved in 110 ml. of isopropanol, and thereafter 40 ml. of a 10 % (w./v.) aqueous calcium chloride solution are added. After vigorous stirring for 5 minutes the mixture is allowed to stand. The phases are separated and the aqueous phase is passed through a membrane filter, and the dissolved calcium heparinate is precipitated by means of 100 % (v./v.) of ethanol.

The calcium heparinate precipitate is centrifuged, washed with ethanol and subsequently with acetone and dried.

The calcium heparinate obtained meets the requirements of USP XXI, its specific activity is 163.6 I.U./ mg., and its sodium ion content is less than 0.1 % by mass.

Example 6 The procedure described in Example 5 is followed, except that the heparin-quaternary amine complex is dissolved in n-butanol instead of isopropanol.

The product obtained meets the requirements of USP XXI, its specific activity is 167.5 I.U./mg., its sodium content remains below 0.1 % by mass.

Example 7 The procedure described in Example 5 is followed, except that the heparin-quaternary amine complex is dissolved in chloroform instead of isopropanol.

The product obtained meets the requirements of USP XXI, its specific activity is 165.6 I.U./mg., and its sodium content is below 0.1 % by mass.

Example 8 The procedure described in Example 5 is followed, except that the precipitate is formed using carbotetradecyloxomethyl-trimethyl-ammonium-chloride instead of HyamineR 1622, and the precipitate is dissolved in n-butanol instead of isopropanol.

The product obtained meets the requirements of USP XXI, its specific activity is 169.3 I.U./mg. and its sodium content is lower than 0.1% by mass.

Example 9 From sodium heparinate of pharmacopoeial quality (specific activity: 158 I.U./mg.) a 1 % (w./v.) solu tion is prepared with pyrogen-free distilled water.

50 ml. of the solution are titrated with a 10 % (w./v.) aqueous HyaminR 1622 solution, by means of a Radiometer titrator (Type TT 2), using a combined glass electrode. Using this equipment the electrode potential expressed in mV is recorded as a function of the volume of the reactant added (Figure 1).

On the basis of the curve obtained the point of inflection of the first titration step corresponds to 11.45 ml of the 10 % (w./v.) aqueous HyamineR 1622 solution.

On the basis of the titration 229 mi. of a 10 % (w./v.) aqueous HyamineR 1622 solution are added to 1000 ml. of the sodium heparinate solution, under stirring. The precipitate is centrifuged, suspended in pyrogen-free water and again centrifuged.

The wet precipitate is dissolved in 250 ml. of n-butanol and 95 ml. of a 0.3 molar aqueous calcium chloride solution are added to the first solution. After stirring for 5 minutes the mixture is allowed to stand. Following the separation of the phases from the aqueous phase calcium heparinate is precipitated by the addition of 100 % (v./v.) ethanol. The precipitate is centrifuged, washed with ethanol and then with acetone and is dried.

The specific activity of the product obtained is 177 I.U./mg., and its quality meets the requirements. of USP XXI. The yield amounts to 98.9 % and the sodium content of the product is below 0.1 % by mass.

Example 10 The procedure described in Example 9 is followed, except that instead of a calcium chloride solution an equivalent amount of a lithium chloride solution is employed.

The specific activity of the product obtained is 181 I.UJmg., the yield is 97,8 % and the product con- tains less than 0.1 % of sodium.

Example 17 The procedure described in Example 9 is followed, except that instead of the calcium chloride solution an equivalent amount of a magnesium chloride solution is employed.

The specific activity of the product obtained is 173 I.U./mg., the yield is 99.2 % and the sodium content is below 0.1 %.

Example 12 The procedure described in Example 9 is followed, except that instead of calcium chloride, ammonium chloride is employed.

The specific activity of the product obtained is 183 I.U./mg., the yield is 97.0 % and the sodium content is below 0.1 %.

Example 13 The procedure described in Example 9 is followed, except that instead calcium chloride, potassium chloride is- employed.

The specific activity of the product obtained is 174 I.U./mg., the yield is 96.8 % and the sodium content is below 0.1 %.

Example 14 From crude sodium heparinate containing 150 ey/g. of heavy metal ions (crude-, protein-free product; specific activity: 130 I.U./mg.) following the elimination of pyrogenic substances in a conventional man ner 200 lit. of an aqueous solution is obtained with a specific activity of 3250 I.U. heparin/ml. To this solution 104 lit. of a 10 % (w./v.) aqueous HyamineR 1622 solution are added. The required amount of the HyamineR 1622 was determined by means of the potentiometric titration described in Example 9.

The precipitate obtained is centrifuged on a settling centrifuge, whereupon it is suspended in 30 lit. of pyrogen-free distilled water and again centrifuged.

The wet precipitate is dissolved in 90 lit. of n-butanol and 30 lit. of a 4 % (w./v.) aqueous potassium chloride solution are added to the first solution. The mixture is stirred for 5 minutes and is then filtered into a 150-lit. glass flask equipped with a stirrer and a lower draining tap. After separation of the phases the lower aqueous phase is eliminated and to the butanolic phase 10 lit. of a 4 % (w./v.) aqueous calcium chloride solution are added. After stirring for 5 minutes the aqueous phase is separated and the two -aqueous phases are combined.

The combined aqueous phase is filtered and the dissolved calcium heparinate is precipitated by adding 100 % (v./v.) of ethanol. The precipitate is washed with.ethanol and then with acetone and dried in vacuo.

3760 g. of calcium heparinate are obtained having a specific activity of 170 I.U./mg. The product meets the requirements laid down in USP XXI, and its sodium content slower than 0.1 % by mass.

If instead of calcium heparinate another heparinate containing a different cation is to be prepared instead of calcium chloride other salt solutions of equal molarity are employed.

Claims

1. A process for the preparation of heparin salts of pharmacopoeial purity which comprises adding an aqueous solution of a quaternary ammonium halide capable of forming a heparin-quaternary amine complex to an aqueous solution of a heparinate of pharmacopoeial quality or to a pyrogenfree aqueous heparinate solution optionally containing heavy metal salts and inactive heparinoid fractions; and separating the precipitated heparin-quaternary amine complex formed and washing it with pyrogenfree, deionized water and dissoliving the purified heparin-quaternary amine complex either i) in an aqueous solution of a salt containing the cation of the heparinate to be prepared; or ii) in a water-immiscible organic solvent, and adding to the solution obtained an aqueous solution of a salt containing the cation of the heparinate to be prepared, or iii) in an organic solvent immiscible or partially miscible with water, and adding to the solution obtained an aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the two phases to separate and separating them from each other, followed, if desired, by adding to the organic phase obtained in step iii) an aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the phase to separate, separating them from each other and combining the aqueous phases, and, if desired, purifying the solution obtained by any of steps i) - iii), precipitating the desired heparinate by adding a water-miscible solvent, and isolating the precipitated heparinate.

2. A process as claimed in claim 1, which comprises adding to an aqueous solution of a heparinate of pharmacopoeial quality or of a pyrogen-free heparinate optionally containing also heavy metal salts and inactive heparinoid fractions having a concentration of 500 to 10,000 I.U./ml., preferably 1000 to 5000 I.U./ ml. a 1-20 % (w./v.), preferably 5-15 % (w./v.) aqueous solution of a quaternary amine halide capable of forming a heparin-quaternary amine salt, in an amount of 1.5 to 3.5 gd10 I.U., separating the precipitated heparin-quaternary amine complex formed and washing it with pyrogenfree water, and dissolving the purified, centrifuged wet heparin-quaternary amine complex either i) in a 1.5 to 2.5 molar aqueous solution of a salt corresponding to the cation of the heparinate to be prepared, or ii) in a 4-10 fold amount of a water-miscible organic solvent, and adding to the solution obtained a 0.3 to 1.0 molar aqueous solution of a salt corresponding to the cation of the heparinate to be prepared, or iii) in a 4-10 foid amount of an organic solvent immiscible or partially miscible with water, adding to the solution obtained a 0.3 to 1.0 molar aqueous solution of a salt corresponding to the cation of the heparinate to be prepared, allowing the two phases to separate and separating them from each other, and if desired, adding to the organic phase obtained in step iii) a 0.3-1.0 molar aqueous solution of a salt containing the cation of the heparinate to be prepared, allowing the phases to separate, separating them from each other and combining the aqueous phases, and if desired, purifying the solution obtained by any of the steps i)-iii), precipitating the desired heparinate by adding a water-miscible solvent, and isolating the precipitated heparinate.

3. A process as claimed in claim 1, which comprises using as a quaternary ammonium halide a compound of formula (I) or (II)

wherein R is a straight- or branched chain alkyl having from 8 to 18 carbon atoms, phenyl or phenyl -C1.3-alkyl R1,R2 and R3 are identical or different and represent a straight-chain alkyl having from 1 to 4 carbon atoms, or Rl and R2 together form a pentamethylene group and R2 is hydrogen, R, is hydrogen or a group identical with R, A represents a group

B and D are identical or different and represent a valency bond or a group identical with A, N+ is a quaternary nitrogen atom, X- is a monovalent inorganic anion, m is an integer from 1 to 4, n is an integer from 0 to 4, k is 1, 2 or 3,

4. A process as claimed in any one of claims 1 to 3, which comprises using as a quaternary ammonium halide carbotetradecyl-oxymethyi-trimethyl-ammonium chloride in an amount of 1.0 to 2.0 9./105 I.U. heparin.

5. A process as claimed in any one of claims 1 to 3, which comprises using as a quaternary ammonium halide benzyl-dimethyl-[2-(2-(p-1 ,1 ,3,3-tetramethyl-butyl-phenoxy) ethoxy-ethyl]ammonium chloride (HyamineR 1622) in an amount of 1.0 to 2.0 g/105 I.U. heparin.

6. A process as claimed in any one of claims 1 to 3, which comprises using as a quaternary ammonium halide cetyl-pyridinium chloride or cetyl-trimethyl-ammonium bromide in an amount of 1.0 to 2.0 g/ 105 I.U. heparin.

7. A process as claimed in any one of claims 4 to 6, wherein the quaternary ammonium halide is used in an amount corresponding to the point of inflection on the potentiometric titration curve.

8. A process as claimed in any one of claims 1 to 7, which comprises dissolving the purified, centrifuged wet heparin-quaternary amine complex in 20 to 50 ml. of a 1.5 to 2.5 molar aqueous calcium chloride related to g/105 I.U. of heparin.

9. A process as claimed in any one of claims 1 to 6, which comprises dissolving the purified, centrifuged wet heparin-quaternary amine complex in 4- to 6-fold amount of an organic solvent partially miscible with water, isopropanol or n-butanol.

10. A process as claimed in any one of claims 1 to 6, which comprises dissolving the purified, centrifuged wet heparin-quaternary amine complex in a 4- to 6-fold amount of a water-immiscible organic solvent.

11. A process as claimed in Claim 10 wherein the water-immiscible organic solvent is chloroform.

12 A process as claimed in any one of claims 1 to 6, which comprises dissolving the purified, centrifuged wet heparin-quaternary amine complex in 4- to 10-fold amount of n-butanol, and adding to the solution 20 to 50 % by mass of a 0.3 to 0.5 molar aqueous calcium chloride solution.

13. A process as claimed in any one of claims 1 to 12, which comprises precipitating the formed heparinate by means of a water-miscible organic solvent.

14. A process as claimed in claim 13, wherein the water-miscible organic solvent is an alkanol or an alkanone.

15. A process as claimed in any one of claims 1 to 14, which comprises precipitating the heparinate formed with methanol, ethanol or acetone.

16. A process as claimed in any one of claims 1 to 6, which comprises determining the amount of quaternary ammonium halide required for the formation of heparin quaternary amine salt by potentiometric titration.

17 A process substantially as herein described with reference to the Examples.

18. A process substantially as herein described.