CS216189B2 - Method of gaining the heparin - Google Patents

Abstract

Heparin is recovered from intestinal brine formed by treatment, with aqueous sodium chloride solution or solid sodium chloride in the presence of water, of animal intestines from which intestinal mucus has been removed. The heparin recovered is almost completely free of by-products, and heparin of good quality is obtained.

Description

(54) A method for obtaining heparin

The invention relates to a process for recovering heparin from brine, which is omitted in the treatment of animal intestines (intestinal brine).

Heparin has long been available as a natural anticoagulation material in medicine, as it has been indispensable in modern medicine as well as in the past and is increasingly used.

A number of methods have been known in recent years, all of which aim to isolate the active ingredient heparin in a gentle manner from animal tissues such as lungs, liver and more recently mainly from intestinal mucus (mucosa) of swine, cattle and sheep (GB 754 885, DE 1 228 241, U.S. Pat. No. 3,058,884, DE 1 253 868). Such raw materials, because they are perishable, have the disadvantage that they can only be stored for a limited time, although this limited shelf life is possible. / prolonged by freezing, which, in addition to the increased technical cost, also leads to an increase in financial costs. During processing, both operating personnel and residents are often faced with unexpected problems due to the odor load associated with this insulation.

DE 1 253 8 8 describes the recovery of heparin in several conventional ways. According to him, animal speech is initially contacted with a hot aqueous salt solution to release heparin from the cells. The proportion of heparin in the medium thus obtained is minimal.

It has now been found that brine that falls off slaughterhouses and intestinal mucosa processing plants contains heparin. This brine is formed when animal casings that have been previously mucus-free are placed in a salt solution for preservation and drainage.

Thus, with regard to DE 1 253 868, it is expressly surprising that the brine which results from the insertion of animal intestines into a solution of table salt contains a greater amount of heparin. Nor could it be expected that this heparin was present almost completely free of chemically related by-products, so that the brine, previously known only as a waste product burdening the environment, constitutes a new source of heparin raw materials, obtainable due to the narrow raw material profile only in very modest quantities. Due to the minimal amount of contaminants present, the treatment of crude heparin can be carried out in a particularly gentle manner, eliminating the costly isolation methods known from the prior art (see U.S. Pat. Nos. 3,451,996 and GB 1221,784).

Finally, the quality of heparin is reached, which is far from being achieved with the heparin preparations available on the market. Crude heparins isolated by known intestinal brine treatment methods used as raw material according to the invention are already presented in such purity that subsequent purification results in heparins exceeding far 200 USP units / mg. Heparin preparations obtainable so far in the shop generally have a heparin quality of 150 USP units / mg, and in a few cases 155 USP units / mg.

Here, the USP unit / mg is understood to be the specific activity which results from the USP test, which measures the inhibition of the formation of lumps of preserved sheep plasma. 1 USP unit corresponds to about 1.1 International Units (UI). Since the activity value of heparin preparations found in the shop is unsatisfactory despite numerous improvements in procedures, the USP, for example, prescribes that heparin preparations (from intestinal mucus) must contain at least 140 USP units. So far, only L. W. Kananagh and L. B. Jaques [Azneimittelforschung (Drug. Res.) 24, Nr. 1942 (1974)] to isolate heparin by repeated crystallization as a barium salt with a maximum efficiency of 175 USP units / mg.

According to the invention, the problem discussed above has been solved by improving the method of obtaining heparin from saline solutions of animal tissue which are diluted with water at a ratio of 1: 3 to 1:10 by brine, and from which heparin is obtained by methods known per se. An improvement is that intestinal brine is used as the 'starting material', which results from the deposition of intestinal mucus-depleted animal casings in edible (cooking) salt solutions at a temperature of from 0 to 30 ° C.

For reasons of economy, sodium chloride is generally used in food grade grades. Therefore, in the description of the present invention, sodium salt of the trivial name uses common salt or its corresponding standardized food salt to indicate that the normal use of the invention, for example in a slaughterhouse, is sufficient for this commercial grade of sodium chloride, although, of course, sodium chloride of the highest degree of purity could be used.

Preparation of intestinal brine is usually carried out in slaughterhouses, for example as follows:

First, the intestines are cleared of mucus, washed with water and placed in a hazel. The loading of the intestines into the edible (cooking) salt solution is normally carried out by pouring the edible salt solution into the hazel-filled intestine until the hazel is filled. After two hours to - two days, the desired intestinal brine is formed.

In particular, edible (kitchen) salt solutions having a salt content of 0.5 M to a molar amount at saturation are suitable for preserving and draining animal intestines.

Particularly preferred are those solutions of edible (table salt) having a table salt content of 3 M to a molar amount at saturation, as molar amount at saturation being the molar amount containing a concentrated aqueous solution at room temperature of the slaughterhouse relative to the edible dissolved. (kitchen) salt (concentrated aqueous sodium chloride solution is about 26.4%).

As a rule, mucus-free animal casings, edible salt solutions - - 2 hours to 3 days are processed. Most often, the processing of animal intestines is completed after 3 to 24 hours. After removal of the preserved and drained animal intestines from the edible (cooking) salt solution, a saline intestine is obtained which has a surprisingly high heparin content.

By using such intestinal brine as raw material, according to the invention - heparin has been isolated on an industrial scale - in such purity that it is available as an almost colorless substance. On the other hand, all crude heparins which have hitherto been obtainable from other raw materials have to be discolored in a costly and lossy manner only (U.S. Pat. No. 3,179,566).

In addition, intestinal brine is a storable, almost odorless raw material that does not degrade quality for long periods of storage. This eliminates chemical and other preservation methods.

The invention thus relates to the use of intestinal saline obtained by introducing mucus-free animal intestines into common salt solutions for the production of pure heparin, and to a method for isolating pure heparin from saline solutions of animal tissues by methods known per se, characterized in that The method according to claim 1, characterized in that intestinal saline is used as the starting material, which has been obtained by introducing the mucus-free animal intestines into solutions of common salt - at a temperature of - 0 to 30 ° C.

Example 1

600 L of brine was added in a 1500 L mixer at room temperature with stirring for - half an hour with 600 L of methanol. It was stirred for the same time, and then the mixture was allowed to stand for about one hour.

Now the cloudy top of the mixture solution was decanted into the - second vessel - and the precipitated salt cake was removed. The suspension obtained now stands for two days. It was then decanted again, the upper part discarded and the precipitate collected by centrifugation. After vacuum drying, 1.44 kg of product was obtained having about 10 USP units / mg. It still consists of about 45% - kitchen salt. An aliquot of the crude heparin thus obtained corresponding to about 425,000 USP E and 42.5, respectively

21 g of 18 grams, was extracted three times with 200 ml of a 2 molar solution of common salt, and was stirred at 60 ° C for one hour. The combined extracts were diluted with water until the solution contained about 0.9 moles of chloride ions per liter. Now 200 ml of anion exchanger (Lewatit CA 9249) in chloride form was added and stirred for one hour.

It was then aspirated and additionally washed with about 200 ml of 0.9 molar sodium chloride solution. The ion exchanger was then stirred for five hours with a two molar solution of table salt at 40 ° C and then aspirated and washed again with a two molar table solution of table salt. The elution solution was combined with the wash solution and precipitated with 1.5 times the volume of methanol. The precipitate was centrifuged, first washed with about 50 ml of a mixture of water and methanol (1 - (~ 1.5 volumes) then with about 50 ml of methanol and dried.

198 g of sodium heparinate was obtained at 195 USP units per mg.

15.5 g of sodium heparinate obtained as described above with about 200 USP units per mg were dissolved in 200 ml of a 2 molar sodium salt solution. The solution was suction filtered and the residue was washed first with 30 ml of a 2 molar sodium salt solution, then with about 250 ml of perfectly deionized water. The combined solutions were now treated with perfectly deionized water to a chloride molarity of 0.9 M. Now, as before, purification with a liter of Lewatit CA 9249 was repeated.

The combined elution and wash solutions were further processed in the same manner as described above. After washing and drying the precipitate, 11.2 g of sodium heparinate was obtained, which had an activity of 251 USP units per mg.

Example 2

Similar to British Patent 754 885, 3 L of brine was acidified with acetic acid to pH 3.1-3.2 (about 50 mL) with stirring. After a few hours, decanted and the residue was centrifuged. The precipitate was discarded, the combined solutions were neutralized with sodium hydroxide.

Now, with stirring, the solutions were charged with an equal volume of methanol. After 45 minutes, decanted salt was decanted. The suspension was allowed to stand for several hours, the upper part decanted and the residue centrifuged. The precipitate was dried in vacuo. 3.43 g of compound with 21 USP units per mg were obtained. An aliquot of the crude heparin thus obtained, corresponding to about 425,000 USP-E or 20.2 g, respectively, was purified analogously to Example 1. 10.65 g of sodium heparinate was obtained with an activity of 262 USP units per mg.

Example 1 a d 3

1 l of brine was diluted with 12 l of water to a chloride molarity of about 0.85 m. Then 50 g of diatomaceous earth as well as a solution of 6 g of benzethonium chloride (= Hyamin 1622, Rohm and Hass) in 100 ml of water were added. After a few hours, decanted and the residue was aspirated. After washing with water, the still wet precipitate was extracted three times in the manner described in DE patent 1 228 241 each with 200 ml of a 2 molar solution of common salt. The combined extracts were precipitated with 2 volumes of methanol. The isolated and dried precipitate weighed 465 mg and had an activity of 155 USP units / mg.

Example 4

1 l of brine was diluted with 120 l of water to a chloride molarity of about 0.85 ø (cf. DE patent 1 156 938). It was then acidified with acetic acid (about 50 mL) to pH 3.2. After a few hours, the precipitate settled, which was centrifuged after decanting the upper part. After drying, 111 g of crude heparin was obtained at 6.5 USP units / mg. An aliquot of the crude heparin thus obtained, with about 425,000 USP units or 65.5 g, was first purified analogously to Example 1. After a first purification step through an ion exchanger, 196 g of sodium heparinate was obtained with 198 USP -E per mg.

g of sodium heparinate obtained as described above with about 200 USP units per mg was dissolved in 200 ml of a 2 molar calcium chloride solution. The solution was suction filtered and the residue was washed first with 30 ml of 2 molar calcium chloride solution, then about 250 ml of perfectly deionized water. The combined solutions were now adjusted to 0.9 molarity with perfectly deionized water. They were then mixed, as mentioned above, with one liter of Lewatit CA 9249. Elution and additional washes were performed with a 2 molar calcium chloride solution instead of the sodium chloride solution.

The combined elution solution and wash solution were further treated as described above. After washing and drying the precipitate, 10.7 g of calcium heparinate with an activity of 260 USP units per mg were obtained.

Example 5

1 L of brine was diluted with 40 L of water to a chloride molarity of about 0.85 M. This solution was pumped through a filter through an ion exchange column with a diameter of 26 nm and a length of 380 mm (about 3 L / h). A medium basic macroporous anion exchange resin in Cl-form (Lewatit CA 9249) was used. Other anion exchange resins such as Dowex 1-X-1 described in German Patent 1,253,868 can also be used.

Then the resin was removed from the column, washed twice with 500 ml 0.9 molar sodium chloride solution and eluted with 400 ml 2 molar sodium chloride solution at 40 ° C for hours | with stirring. This solution was precipitated with 1.5 volumes of methanol. The isolated and dried precipitate weighed 1.2 grams and had activity of 149 USP / units / mg.

Claims (1)

I will invent the object A method for obtaining heparin from saline solutions of animal tissue which are diluted with water at a ratio of 1: 3 to 1:10 by brine, and from which heparin is obtained by methods known per se, characterized in that intestinal brine is used as the starting material, which is formed by the deposition of intestinal mucus-free animal intestines in edible salt solutions at a temperature of 0 to 30 degrees Celsius.