FR2582657A1 - PROCESS FOR PREPARING HEPARIN SALTS - Google Patents

Abstract

THE INVENTION CONCERNS A NEW PROCESS FOR THE PREPARATION OF HEPARIN SALTS OF PHARMACOPEIC PURITY, PREFERENCE OF CALCIUM HEPARINATE, IN WHICH IS PROCESSED BY: ADDITION OF AN AQUEOUS SOLUTION OF A QUATERNARY AMMONIUM HALOGENIDE CAPABLE OF FORMING A QUATERNARY HEPARINE-AMINE COMPLEX HAS AN AQUEOUS SOLUTION OF A PHARMACOPEIC QUALITY HEPARINATE OR AN AQUEOUS APYROGENIC HEPARINATE SOLUTION CONTAINING POSSIBLE HEPARINATE SALTS AND INACTIVE HEPARINOID FRACTIONS; -SEPARATION OF THE PRECIPITATED QUATERNARY HEPARINE-AMINE COMPLEX AND ITS WASHING WITH DEIONIZED APYROGEN WATER, AND DISSOLUTION OF THE PURIFIED HEPARINE-QUATERNATE AMINE COMPLEX: I. IN AN AQUEOUS SOLUTION OF A SALT CONTAINING THE CATION OF HEPARINATE TO BE PREPARED, OR II.IN AN ORGANIC SOLVENT NOT MISCIBLE TO WATER, AND ADDITION TO THE SOLUTION OBTAINED FROM AN AQUEOUS SOLUTION OF A SALT CONTAINING THE CATION OF THE HEPARINATE TO BE PREPARED, OR III.IN AN ORGANIC SOLVENT NOT MISCIBLE OR MISCIBLE WATER RESTRICTED, AND ADDITION TO THE SOLUTION OBTAINED FROM AN AQUEOUS SOLUTION OF A SALT CONTAINING THE CATION OF HEPARINATE TO BE PREPARED, WAITING FOR THE SEPARATION OF THE TWO PHASES AND SEPARATION OF THE PHASES FROM ONE OF THE OTHER; -AND IF DESIRED, ADDITION TO THE ORGANIC PHASE OBTAINED IN STEP III. OF AN AQUEOUS SOLUTION OF A SALT CONTAINING THE CATION OF THE HEPARINATE TO BE PREPARED, WAITING FOR THE SEPARATION OF THE PHASES, SEPARATION OF THE PHASES FROM ONE ANOTHER AND COMBINATION OF THE AQUEOUS PHASES, AND IF DESIRED, PURIFICATION OF THE SOLUTION OBTAINED BY ANY OF STEPS I., III., PRECIPITATION OF THE DESIRED HEPARINATE BY ADDITION OF A SOLVENT, MISCIBLE TO WATER, AND ISOLATION OF THE PRECIPITATED HEPARINATE IN A KNOWN MANNER.

Description

The present invention relates to a novel method of

  preparation of heparin salts of pharmacopoeial quality.

  Heparin was discovered in 1913 and since that date,

  it is widely used in clinical therapy and prophylaxis.

  Until recently, heparin was almost exclusively used in the form of sodium salt and the standards of the pharmacopoeia also concerned the quality of sodium heparin. The experiences accumulated during the application of sodium heparin for several decades and the results of certain clinical trials carried out in recent years show, however, that in certain fields other heparin salts have more interesting properties. The duration of activity

  for example, calcium heparinate is longer than that of hepa-

  sodium rinate, and therefore the therapeutic dose can be maintained by less frequent administration of heparin to patients; moreover hemorrhages. observed during the administration of sodium heparinate can be avoided by using heparin in the form of calcium salt / Kakkar, V.V .: Clinical use of Heparin. Present and future trends, dir. publ. M. Verstraete, S. J. Machin, Munsgaard, Copenhagen, 1980, p. 158;

  Kakkar V.V .: Heparin Chemistry and Chemical Use, Academic Press 1976 /.

  As a result, more and more manufacturers are offering various heparinates that do not contain sodium as a cation (eg, calcium, magnesium, lithium, potassium, ammonium heparinates) for clinical purposes.

and diagnostics.

  According to the methods known hitherto, the sodium cation of heparin was replaced by another cation, either by bringing an aqueous solution of sodium heparinate into contact directly with a water-soluble salt of the chosen cation (below "reversible" process), or by isolating heparin as a free acid by means of a resin

  acid ion exchanger and then transforming it into the desired salt (hereinafter

  below: "irreversible" process).

  In the "reversible" process it is virtually impossible to replace the sodium cation with the other desired cation so that the product contains less than 1% by weight of sodium cation. Even if we increase the concentration of the new cation, that is to say if we use its salt in large excess, we can not shift the balance of the reversible process in the direction of the formation of the new salt. heparin desired only to a certain extent. It is also possible to shift the equilibrium by isolating the "mixed" heparin salt (i.e., the heparin salt containing the sodium cation in addition to the desired new cation) by precipitation with a solvent and repeating again several times the process. It is clear that this multistep procedure is tedious, expensive in labor and consumes a lot of solvent, and that the yield is

  - significantly reduced by the multiplicity of treatments.

  According to the "irreversible" process the cation exchange is carried out by means of a cation exchange resin. In the first step of this process, heparin is formed as a free acid, which is likely to decompose and has anticoagulant activity of approximately

20 to 30% lower.

  An improved variant of the "reversible" process is described in German Patent No. 2,417,859, in which the equilibrium of the reversible process is displaced in the desired direction not only

  by isolating the mixed salt formed but also by eliminating the heparin cation.

  starting sodium nate by dialysis. According to this process, for example, calcium heparinate is prepared starting from sodium heparin of pharmacopoeial purity, by adding calcium chloride in an aqueous solution of sodium heparin, stirring the mixture for 16 hours and then stirring. dialysing the solution in a small volume (300 ml) against deionized and pyrogen-free water for 6 hours. The dialysed solution is diluted to about twice its original volume and the mixed heparin sodium and calcium heparin salt is precipitated with an organic solvent. The isolated product is dissolved in distilled water and the entire process is repeated. Isolated calcium heparinate contains less than 0.1% by weight of sodium ion and its specific activity is approximately

  same as that of the starting material.

  In fact, the process is advantageous because, by combining the

  With dialysis, cation exchange can be performed in two stages instead of previous multi-stage treatments. However, dialysis has several disadvantages: the reaction time is increased, there is a greater risk of pyretogeny and infection,

  dialysis performed in small volumes is impractical, and the

  are therefore increased. The process is not suitable for the purification of heparin or for increasing its specific activity, because already the

  starting material is of pharmacopic purity.

  In summary, according to known methods, the cation of sodium heparin is replaced by another cation, either by using a cation exchange resin, that is to say by an "irreversible" process or by the succession of processes. "reversible", where the equilibrium state achieved in the various stages is fixed by precipitating the "mixed" salt of heparin and efforts are made to further achieve a displacement of the state

  of equilibrium to the desired extent from this fixed equilibrium.

  The object of the invention is to provide a process by which - the exchange of the original cation of heparin is rendered irreversible by preparing an insoluble intermediate in water (without formation of heparic acid which is likely to to decompose), from which the cation exchange can be carried out quantitatively, in a single step,

  - cation exchange can be conducted not only from hepatic

  pharmacopoeia quality, but also from any aqueous solution of heparin obtained after the removal of pyrogens during the production of heparin, and - and which is suitable, in addition to the exchange of cations, for a purification more

  heparin and to increase its specific activity.

  It has surprisingly been found that the quaternary ammonium quaternary ammonium halide cations conventionally used for other purposes during the preparation of heparin, in certain

  conditions are also suitable for the exchange of heparinate cations.

  Given the insoluble nature in the water of the heparin-

  quaternary amine, the process is irreversible and therefore the exchange of

  cations can be done quantitatively.

  The invention relates to a process for the preparation of heparin salts of pharmacopoeial quality, preferably calcium heparin by addition of an aqueous solution of a quaternary ammonium halide capable of forming a heparin-quaternary amine complex to a aqueous solution of a heparin of pharmacopoeial quality or a pyrogen-free aqueous heparin solution optionally containing heavy metal salts and inactive heparinoid fractions, separation of the precipitated heparin-quaternary amine complex and its washing with deionized pyrogen-free water, and dissolving the purified quaternary heparin-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 it to the resulting solution. an aqueous solution of a salt containing the cation of the heparinate to be prepared, or iii) in an immiscible organic solvent or miscible with in water is limited, and adding to the solution obtained a solution

  aqueous solution of a salt containing the cation of the heparinate to be prepared, and separated

  ration of the two phases, and if desired, addition to the organic phase obtained in step iii) of an aqueous solution of a salt containing the cation of the heparinate to be prepared, waiting for phase separation, separation phases of each other and combination of the aqueous phases, and if desired, purification of the solution obtained by any one of steps i) -iii), precipitation of the desired heparinate by addition of a water-miscible solvent, and isolation of the precipitated heparinate in a manner known per se, the term "pharmacopoeial-grade heparin salts"

  is used throughout the description and claims to refer to

  heparin salts whose purity meets USP XXI standards (Pharma-

  American cop - The United States Pharmacopoea XXI).

  The terms "heparin salts" and "heparinates" refer to pharmaceutically acceptable salts of heparin, e.g. ex. salts of magnesium, potassium, lithium, ammonium and preferably calcium (heparinates). The terms "heparin salts" and "heparinates" are synonymous;

  thus the sodium salt of heparin is identical to sodium heparinate.

  In the first step of the process according to the invention, an aqueous solution of a quaternary ammonium halide is easily dissociated in water and, with the heparinate anion, forms a water-insoluble complex with a pyrogen-free solution. starting heparinate. The heparinate solution generally contains the sodium cation associated with the heparin anion, but other cations and anions may also be present, according to the prior art steps. The method can be specified by the following simplified reaction equation: [Hepr + + [Kl +] + n [K2 + '+ n [A -] Hep (K2) i + n [l +] + n. [A-] oQ [Hep 11] - heparin anion, [KX1 + 3 the original cation of heparin (usually the sodium ion), [K +] quaternary ammonium cation forming an insoluble complex 2 in water with the heparin anion, [A '] = halide ion originally attached to the quaternary ammonium cation [K2 +] (usually the chloride or bromide ion),

  n - = number of negative charges of the heparin anion.

  Since the heparin-quaternary ammonium complex is insoluble in water, the reaction proceeds quantitatively in the direction of the arrow. Other cations and anions that may be 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 deionized and pyrogen-free water.

  In contrast to slow equilibrium processes, in the process according to the invention the ion exchange takes place instantaneously, and the other cations can be removed by simple separation techniques.

  and washing instead of long and inconvenient dialysis.

  Among the salts forming water-insoluble complexes with heparin, quaternary ammonium halides, e.g. ex. cetyl-pyridinium chloride, cetyltrimethylammonium bromide, Hyamine R 1622

  (benzyl-dimethyl- / 2- (2- (p-1,1,3,3-tetramethyl-butyl-phenoxy) chloride)

  ethoxy) -ethyl / ammonium) and its derivatives (US Pat. Nos. 2,989,438; 3,058,884; 3,160,563 and 3,342,683) and the quaternary ammontum halides described in the Hungarian patent N 188,537 as the chloride

  carbotetradecyloxymethyltrimethylammonium and the compounds

  the most appropriate for the purposes of the invention.

  The quaternary ammonium halide used is preferably a compound of formula (I) or (II) R - A - (CE 2) - R 2 x () I

(CH2) 2

  R | B1 (CH2) m7I1 '- (CH2) 2 -N - (CH2) 1A D-RD 2VX

(CH'2) 2

  klc, 2, 3 o R is a straight or branched chain alkovyl having 8 to 18 carbon atoms, a phenyl or a phenyl-lower alkyl

C1 to C3,

  R1, R2 and R3 are the same or different and represent a straight chain alkyl having 1 to 4 carbon atoms, or R1 and R2 together form a pentamethylene group and R3 is hydrogen, Rn is hydrogen or a group identical to R ,

  A represents a group -O-C- or -C-O-

He I1l

O O

  Bet D are the same or different and represent a valence bond or a group identical to A, N is a quaternary nitrogen atom, X is a monovalent inorganic anion, m is an integer ranging from 1 to 4,

  n is an integer ranging from 0 to 4.

  Quaternary ammonium halides have already been used

  in the preparation of heparin but for different purposes, namely to separate the raw sodium heparin from dilute aqueous solutions of organs and animal tissues containing heparin and isolate them. In addition to the advantages described above, the use of quaternary amine halides is advantageous because most of them have bactericidal properties, and therefore when using these compounds the risk

  reinfection, that is to say of pyretogeny, is negligible by comparison

  its with the other known techniques of preparation of calcium heparinate.

  It has been found, moreover, that one can control with an instru-

  precipitation of the heparin-quaternary amine complex, which increases the specific activity of heparin during the exchange.

  ion. By measuring the electrode potential of the aqueous solution of

  parine with a combined glass electrode, one can control potentio-

  metrically the progress of the precipitation, and it is possible to adjust to the optimum the quantity of the aqueous solution of the ammonium halide

  0 quaternary added. During the addition of the aqueous halide solution

  quaternary ammonium nitrate, firstly the fractions having an activity

  high anticoagulant precipitate from the heparin mixture

  many fractions, while the inactive fractions are the last to precipitate. By controlling the potentiometric curve, one can easily determine the optimum ratio at which the precipitation process must be completed to prevent the precipitation of inactive fractions. In this way, the activity of the sodium heparin product having a pharmacopoeial quality can be increased by 10 to 15%. From the heparin-quaternary amine complexes, the desired heparin salt can be easily prepared by another exchange of

  irreversible cations. We can indeed dissolve the heparin-

  quaternary amine in aqueous salt solutions, e.g. ex. in aqueous solutions of sodium chloride at least 2 molar, and from

  of the resulting solution, the corresponding salt, such as hepatic

  sodium rinate, by precipitation with a solvent, since the quaternary ammonium halides are soluble in the organic solvents used for the precipitation. As heparinates separate, the exchange

anions is irreversible.

  As stated above, heparin-quaternary amine complexes have heretofore been used simply for the isolation of crude sodium heparin from aqueous solutions of sodium heparin, i.e. No ion exchange occurred during the process. It has now been unexpectedly found that by the process according to the invention any desired heparin salt can be prepared at pharmacopoeial purity, starting from any apyrogenic heparin-containing aqueous solution containing a different cation, p. ex. from sodium heparin one can easily obtain

  calcium heparinate of high purity.

  If the ion exchange is conducted according to variant i), that is

  that is, if the quaternary heparin-amine salt is dissolved in a solution

  aqueous solution of a salt containing the desired cation, given the concentrations

  high salt levels repeated dissolution of the heparin salt precipitated in water and further precipitation with the chosen organic solvent

  may be necessary to obtain the product with satisfactory purity

  tory. For the complete elimination of the quaternary ammonium compound,

  the product is washed thoroughly with a suitable solvent.

  It has been found that if the heparin-quaternary amine complex is first dissolved in an organic solvent conventionally used for the precipitation of heparin (eg methanol, ethanol, acetone) instead of a concentrated aqueous solution of salt. the ion exchange of the dissolved quaternary heparin-amine complex can be carried out with a solution

  aqueous salt considerably less concentrated (variant ii)). By choosing

  suitably the solvent ratio, the heparin salt is precipitated

  desired virtually without any accompanying salt impurity inor-

  ganic. The quaternary ammonium halide must be removed from the product by washing with a suitable solvent as in the case of the previous variant. According to the most preferred variant of the process of the invention

  (variant iii) the first step is to dissolve the heparin-amine complex quater-

  in an immiscible or only slightly water-immiscible organic solvent (eg isopropanol, n-butanol, chloroform), the ion exchange is conducted 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 addition of an organic solvent (eg acetone, ethanol, methanol). In this variant, the irreversibility of the ion exchange is already ensured by the presence of immiscible liquid phases, since the quaternary ammonium halide remains in the organic phase, while the heparin salt formed with the new cation is present in the aqueous phase. A great advantage of this process is that in this way the quaternary ammonium compound is removed without any additional purification step, simply

separating the phases.

  According to a preferred embodiment of the process according to the invention, it is added to an aqueous solution of sodium heparinate.

  pharmacopoeia or a pyrogen-free aqueous solution of heparin

  sodium nate optionally containing heavy metal salts and inactive heparinoid fractions (heparin concentration: 500-10,000 IU / ml, preferably 1000-5000 IU / ml, pH 2-10, preferably 4-8) aqueous solution at 1-20% (w / v), preferably 5-15% (w / v) of a quaternary ammonium compound, preferably HyamineR 1622 or carbotetradecyloxymethyltrimethylammonium chloride , all in

  waving (U.I. = International Unity). The amount of ammonium salt quater-

  In addition to the ion exchange, the specific activity must also be increased, the optimum ratio is determined by the following method: An aqueous solution is added to the aqueous solution. 10% (w / v) ammonium salt

  quaternary selected to an aliquot sample of the aqueous solution of

  starting sodium parinate while stirring, following the electrode potential through a combined glass electrode. The electrode potential measured in mV is plotted as a function of the amount of solution

  added quaternary ammonium salt and from the resulting curve

  mine the amount of quaternary ammonium salt required for

  fraction of the active anticoagulant. The optimal ratio can be determined without knowing the heparin concentration of the aqueous heparin solution, since the ratio of reactants can be expressed in ml of solution

  of quaternary ammonium used for titration / ml of heparin solution.

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

to another centrifugation.

  After the centrifugation, the precipitate obtained is dissolved in a volume 4 to 10 times higher, preferably 6 to 8 times greater, of an organic solvent, preferably immiscible or only slightly miscible with water, such as chloroform, isopropanol, ethyl acetate or preferably n-butanol. To the organic solution, preferably butanol, is added a solution of 1 to 10% (w / v), preferably 2 to 5% (w / v) of a water-soluble salt of the desired cation, preferably containing the chloride ion as anion in an amount of 20 to 50% (v / v), preferably 30 to 40% (v / v). The mixture is stirred for 5 to 15 minutes, preferably 15, and the phases are separated. The aqueous phase is filtered through a Seitz filter plate or membrane filter and precipitated by adding 50150% (v / v), preferably 60-80% (v / v) of a miscible lower alkanol. water, p. ex. methanol, ethanol, or an alkanone, e.g. ex. acetone. The precipitated heparinate is 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 sodium ion.

  The invention is specified in detail using the examples

following non-limiting

EXAMPLE 1

  From sodium heparin of pharmacopoeial quality before a specific activity of 165 U.I./mg a 4% (w / v) solution is prepared with pyrogen-free distilled water. To 100 ml of the solution

  165 ml of a 10% (w / v) aqueous solution of cetyl chloride

  pyridinium while stirring. The precipitate obtained (quaternary heparin-amine complex) is separated by centrifugation. The wet precipitate obtained by centrifugation weighs 16.5 g. The precipitate is suspended in

  ml of pyrogen-free distilled water and the suspension is centrifuged.

  The wet cetyl-pyridinium heparinate obtained is dissolved

  by the second centrifugation in 200 ml of a 2 molar solution of hepatic

  calcium rinate while stirring. The solution is purified by filtration and the heparin calcium salt is precipitated from the filtrate with% (v / v) ethanol. We suspend in

  ethanol the precipitate of calcium heparinate separated by centrifugation

  then dissolved in 200 ml of pyrogen-free distilled water. To the solution is added 100% (v / v) ethanol with stirring. The precipitated calcium heparinate is centrifuged, washed with ethanol and then with acetone

and dry.

  The specific activity of calcium heparin obtained is 162 IU / mg, its sodium content is less than 0.1% by weight

  and the product meets the requirements of USP XXI.

EXAMPLE 2

  To 100 ml of the sodium heparin solution according to the example

  160 ml of a 10% aqueous solution of cetyl bromide are added.

  trimethyl-ammonium. The precipitate (quaternary amine salt of hepatic

  by centrifugation, it is suspended in 40 ml of pyrogen-free distilled water and centrifuged again. 15.8 g of a wet precipitate are obtained. The precipitate is dissolved in 100 ml of ethanol, and the resulting solution is added to 200 ml of a 0.5 molar aqueous solution of calcium chloride. The obtained calcium heparinate was purified by filtration and 120 ml of ethanol was added to the filtrate with stirring. The resulting calcium heparinate precipitate is suspended twice in ethanol.

  filtered, washed with acetone and dried under vacuum.

  The specific activity of the calcium heparinate obtained is 167 IU / mg, its sodium content is less than 0.1% by weight

  and the product meets the requirements of USP XXI.

EXAMPLE 3

  The process described in Example 2 is followed except that it is dissolved

  the heparin-quaternary amine complex in methanol instead of ethanol.

  The specific activity of the calcium heparinate obtained is 165 IU / mg, its sodium content is less than 0.1% by weight

  and its quality meets the requirements of USP XXI.

EXAMPLE 4

  The process described in Example 2 is followed except that it is dissolved

  the heparin-quaternary amine precipitate in acetone.

  The specific activity of calcium heparin obtained is 161 IU / mg, its sodium content is less than 0.1% by weight

  and its quality meets the requirements of USP XXI.

EXAMPLE 5

  To 100 ml of an aqueous solution of sodium heparinate prepared according to Example 1 is added 130 ml of a 10% (w / v) aqueous solution of Hyamine. The heparin-amine complex is separated by centrifugation

  obtained quaternary, it is suspended in 45 ml of water and

fuge again.

  The wet precipitate is dissolved in 110 ml of isopropanol and then 40 ml of a 10% (w / v) aqueous solution of calcium chloride are added. After stirring vigorously 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 calcium heparinate is precipitated.

with 100% ethanol (v / v).

  The precipitate of calcium heparinate is centrifuged,

  wash with ethanol and then with acetone and dry.

  The calcium heparinate obtained meets the requirements of USP XXI, its specific activity is 163.6 U.I./mg, and its

  sodium is less than 0.1% by weight.

EXAMPLE 6

  The process described in Example 5 is followed, except that it is dissolved

  the heparin-quaternary amine complex in n-butanol instead of iso-

i propanol.

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

0.1% by weight.

EXAMPLE 7

  The process 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 U.I./mg, and its sodium content is lower than

] at 0.1% by weight.

EXAMPLE 8

  The process described in Example 5 is followed except that

  the precipitate with carbotetradecyloxomethyltrimethyl chloride

  ammonium instead of HyamineR 1622, and the precipitate

  n-butanol instead of isopropanol.

  The product obtained meets the requirements of USP XXI, its specific activity is 169.3 IU / mg and its sodium content is lower than

0.1% by weight.

EXAMPLE 9

  From pharmacopoeial grade sodium heparin (specific activity: 158 IU / mg), a 1% solution (w / v) with

pyrogen-free distilled water.

  50 ml of the solution is titrated with a% (w / v) aqueous solution of Hyamine R 1622 using a Radiometer (TT 2 type) assayer using a combined glass electrode. With this equipment the electrode potential expressed in mV is recorded in

  depending on the reagent volume added (Fig. 1).

  On the basis of the curve obtained, the inflection point of the first titration step corresponds to 11.45 ml of the aqueous solution

10% (w / v) HyamineR 1622.

  On the basis of the titration, 229 ml of a 10% (w / v) aqueous solution of Hyamine R 1622 is added to 1000 ml of the heparin solution.

  sodium, while shaking. The precipitate is centrifuged, suspended in pyrogen-free water and centrifuged again.

  The wet precipitate is dissolved in 250 ml of n-butanol and 95 ml of a 0.3 molar aqueous solution of calcium chloride is added to the first solution. After stirring for 5 minutes, the mixture is allowed to stand. After phase separation from the aqueous phase

  the calcium heparinate is precipitated by the addition of 100% (v / v) ethanol.

  The precipitate is entrained and washed with ethanol and then with acetone.

and dry.

  The specific activity of the product obtained is 177 U.I./mg, and its quality meets the requirements of USP XXI. The yield is

  at 98.9% and the sodium content of the product is less than 0.1% by weight.

EXAMPLE 10

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

  a solution of lithium chloride.

  The specific activity of the product obtained is 181 IU / mg,

  the yield is 97.8% and the product contains less than 0.1% sodium.

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

  a solution of magnesium chloride.

  The specific activity of the product obtained is 173 IU / mg,

  the yield is 99.2% and the sodium content is less than 0.1%.

EXAMPLE 12

  The process described in Example 9 is followed except that

  calcium chloride is used ammonium chloride.

  The specific activity of the product obtained is 183 IU / mg,

  the yield is 97.0% and the sodium content is less than 0.1%.

EXAMPLE 13

  The process described in Example 9 is followed except that

  calcium chloride is used potassium chloride.

  The specific activity of the product obtained is 174 IU / mg,

  the yield is 96.8% and the sodium content is less than 0.1%.

EXAMPLE 14

  From crude sodium heparinate containing 150 μg of heavy metal ions (raw product lacking protein, specific activity: 130 IU / mg) after the elimination of pyrogen-containing substances in the conventional manner, 200 liters of aqueous solution with a specific activity of 3250 IU heparin / ml. To this solution we

  added 104 liters of a 10% (w / v) aqueous solution of Hvamine R 1622.

  The required amount of Hyamine R 1622 is determined by titration

  potentiometric described in Example 9.

  The precipitate obtained is centrifuged in a sedimentation centrifuge, after which it is suspended in 30 liters of water.

  pyrogen-free distilled and centrifuged again.

  The wet precipitate is dissolved in 90 liters of n-butanol and 30 liters of a 4% (w / v) aqueous solution of potassium chloride are added to the first solution. The mixture is stirred for 5 minutes and then poured into a 150-liter glass flask equipped with a stirrer and a lower flow tap. After phase separation

  the lower aqueous phase is removed and added to the butanol phase

  10 liters of a 4% (w / v) aqueous solution of calcium chloride.

  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 liver is precipitated.

  dissolved calcium rinate by adding 100% (v / v) ethanol. The precipitate is washed with ethanol and then with acetone and dried under vacuum. 3760 g of calcium heparinate having a specific activity of 170 U.I./mg are obtained. The product meets the requirements laid down in

  USP XXI, and its sodium content is less than 0.1% by weight.

  If instead of calcium heparin one must prepare another heparinate containing a different cation, instead of

  calcium chloride other salt solutions of equal molarity.

Claims (13)

  1. A process for the preparation of heparin salts of pharmacopoeial purity, preferably calcium heparinate, in which it is carried out by adding an aqueous solution of a quaternary ammonium halide capable of forming a heparin-quaternary amine complex to an aqueous solution of a heparin of pharmacopoeial quality or a pyrogen-free aqueous heparin solution optionally containing heavy metal salts and inactive heparinodide fractions, separation of the precipitated heparin-quaternary amine complex and its washing with pyrogen-free water deionized, and dissolving the quaternary purified heparin-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 from an aqueous solution of a salt containing the cation of the heparinate to be prepared, or iii) into an immiscible or miscible organic solvent of Restricted to water, and addition to the resulting solution of an aqueous solution of a salt containing the cation of the heparinate to be prepared, waiting for separation of the two phases and phase separation 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, waiting for phase separation, separation of the phases of the other and combination of aqueous phases, and if desired, purification of the solution obtained by any one of steps i) -iii), precipitation of the desired heparinate by addition of a solvent miscible with water, and   isolation of precipitated heparinate in a known manner.   2. The process as claimed in claim 1, in which an aqueous solution of a pharmacopoeial grade heparin or a pyrogen-free heparinate optionally containing also heavy metal salts and inactive heparinoid fractions having a concentration of 500 to 10,000 is added to an aqueous solution. IU / ml, preferably from 1000 to 5000 IU / ml, a 1-20% (w / v) aqueous solution, preferably 5-15% (w / v), of a quaternary amine halide capable of forming a quaternary heparin-amine salt, in an amount of 1.5 to 3.5 g / 10 5 IU, the precipitated heparin-quaternary amine complex formed is separated and washed with pyrogen free water, and the heparin-quaternary amine complex centrifuged, purified   i) in a 1.5 to 2.5 molar aqueous solution of a salt corresponding to   heparin cation to be prepared, or   (ii) in a quantity of 4 to 10 times higher, preferably   6 to 8 times higher, of a water-miscible organic solvent, and to the solution obtained is added 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 quantity of 4 to 10 times higher, preferably   6 to 8 times higher, an immiscible organic solvent or poorly miscible with water, is added to the solution obtained an aqueous solution   0.3 to 1.0 molar of a salt corresponding to the cation of heparin   The two phases are allowed to separate and separate from one another and, if desired, to the organic phase obtained in step iii), a 0.3-1.0 molar aqueous solution is added. of a salt containing the cation of the heparinate to be prepared, the phases are allowed to separate, they are separated from one another and the aqueous phases are combined, and if desired, the solution obtained is purified by any of steps i) -iii), the desired heparinate is precipitated by adding a water-miscible solvent, and   the precipitated heparinate is isolated in a known manner per se.   3. The process according to claim 1, wherein the halogenated   quaternary ammonium compound a compound of formula (I) or (II) R 1 (2 μl 2 X O (I) R -A - (CH 2) m R 2 (R 3 (cHz) 2 L 3 (CH2) 2 |   R-B- (CH2) mN - (CH2) 2 -14 - (CH2) - D-Rn 2ecX (CH2) 2   or R is straight or branched chain alkyl having 8 to 18 carbon atoms, phenyl or phenyl-alkyl having C1 to C3,   R1, R2 and R3 are the same or different and represent a straight chain alkyl having 1 to 4 carbon atoms, or R1 and R2 together form a pentamethylene group and R3 is hydrogen, Rn is hydrogen or a group identical to R ,   A represents a group -O-C- or -C-O- He He O O   B and D are the same or different and represent a valence bond or a group identical to A, N is a quaternary nitrogen atom, X is a monovalent inorganic anion, m is an integer from 1 to 4, n is a integer from 0 to 4 k is 1, 2 or 3.   The method of any one of claims 1 to 3, wherein   quaternary ammonium halide is used as the carbon dioxide   tetradecyloxymethyltrimethylammonium in an amount of 1.0 to 2.0 g / 10 IU heparin, preferably in a corresponding amount   at the point of inflection of the potentiometric titration curve.   The method of any one of claims 1 to 3, wherein   benzyl chloride is used as the quaternary ammonium halide.   dimethyl- / Z- (2- (p-1,1,3,3-tetramethyl-butyl-phenoxy) ethoxy) ethyl / ammonium (Hyamine R 1622) in an amount of 1.0 to 2.0 g / 105 IU d heparin, preferably in an amount corresponding to the point of inflection of the   Potentiometric titration curve.   The method of any one of claims 1 to 3, wherein   cetyl chloride is used as quaternary ammonium halide.   pyridinium or cetyltrimethylammonium bromide in an amount   1.0 to 2.0 g / 105 IU of heparin, preferably in a corresponding amount.   at the point of inflection of the potentiometric titration curve.   The method of any one of claims 1 to 6, wherein   the centrifuged, purified wet quaternary heparin-amine complex is dissolved in 20 to 50 ml of a 1.5 to 2.5 molar aqueous solution of chloride   of calcium, relative to 1 x 105 IU heparin.   The method of any one of claims 1 to 6, wherein   the centrifuged, purified wet quaternary heparin-amine complex is dissolved in a quantity of from 4 to 10 times, preferably from 6 to 8 times, of a water-insensitive organic solvent,   preferably isopropanol or n-butanol.   The method of any one of claims 1 to 6, wherein   the centrifuged, purified wet quaternary heparin-amine complex is dissolved in a quantity of from 4 to 10 times, preferably from 6 to 8 times, of a water-immiscible organic solvent, preferably chloroform.   The method of any one of claims 1 to 6, wherein   the centrifuged, purified wet quaternary heparin-amine complex is dissolved in a quantity of 4 to 10 times, preferably 6 to 8 times, n-butanol, and the solution is added to 50%, preferably from 30 to 40% by weight of an aqueous solution   0.3 to 0.5 molar sodium chloride.   The method of any one of claims 1 to 10, wherein   the heparinate formed is precipitated by means of a miscible organic solvent   with water, preferably an alkanol or an alkanone.   The method of any one of claims 1 to 11, wherein   the heparinate formed is precipitated with methanol, ethanol or acetone.   The method of any one of claims 1 to 6, wherein   the quantity of quaternary ammonium halide required is determined   for the formation of quaternary amine salt of heparin by potential titration metric.