DE1195010B - Method for purifying heparin by the chromatographic method - Google Patents

Description

Method for purifying heparin by the chromatographic method The invention relates to a method for purifying raw heparin products and for further purification of pure heparin products.

The pure heparin products that are commonly available on the market have a titer of 100 to 120 U / mg, rarely do they reach a titer of 130 to 135 U / mg. In addition, some of these products contain traces of nucleic acids (more or less changed by cleaning and decolorization processes), which are also responsible for the viscosity that the concentrated heparin solutions often show.

The process according to the invention consists of a chromatographic treatment of heparin with an anionic resin of the cellulose type of the general formula in which cellulose stands for the cellulose carrier, R 1, AZ and R3 are H or alkyl radicals with 1 to 4 carbon atoms and R4 is -CH, or -CH, and X is OH or an acid radical (-Cl, -Br or SO "H ) represents.

The present process leads to products with a titer that is higher than what was found for the products that are usually cleaned, and these products are free from impurities such as nucleic acids and the like coloring substances that are not easily separated by other methods.

Preliminary tests have been made in this direction, albeit under different conditions and using different techniques and materials, however, they have all produced unsatisfactory results.

Various resins are useful for this purpose; actually adsorbed any anionic resin heparin easily. However, the adsorbability with anionic Cellulose type resins higher (triethylaminoethyl cellulose or diethylaminoethyl cellutose). In particular, diethylaminoethyl cellulose was used because of its comparatively high levels Resistance and its high adsorption capacity selected, in fact 10 g of diethylaminoethyl cellulose, which has an exchange capacity of 0.70 to 0.95 meq / g own, adsorb 1.5 to 2.5 g of heparin. The easy availability of this resin in the market was another reason for its selection. Assuming pure heparin from (110 to 120 U / mg), fractions with 160 to 180 U / mg and such are obtained at 25 to 50 U / mg. The overall yield of anticoagulant activity was with 75 to 100% found, the yield of more active fraction reached values of 65 to 85% of the total activity of the starting material.

From crude heparin 20 to 65 U / mg, fractions with 110 to 130 U / mg and fractions with 8 to 15 U / mg were obtained. The total yield of the anticoagulant activity was found to be 80 to 950 /, the yield of the more active fraction reached values of 70 to 800 /, the total activity of the starting material.

Two different techniques were used: 1. Column or column adsorption and elution, 2. adsorption with stirring and elution (batch process).

Both techniques can be used both for the further purification of the pure products, which are usually used therapeutically (110 to 120 U / mg), as well as for the purification of raw products (20 to 65 U / mg); for the latter purpose, however, the second type of technique (batch process) is more advisable because in general the amounts of raw products to be purified are considerable and the first technique (column adsorption) would cause various difficulties. When using the second type of technique, large amounts of raw products can be processed and a pure heparin with a titer of 110 to 130 U / mg is obtained. This product can then be purified to a titer of 160 to 180 U / mg with the aid of column adsorption. As stated above, a fraction with low anticoagulant activity (8 to 15 U / mg) is obtained from the crude products. This faction often retains notable "clearing" activity.

The following is a brief description of the chromatographic processes: Raw or pure heparin is in a buffer solution at pH 5.5 to 8.5 in amounts from 10 to 20 mg / cc dissolved. The buffering salt is in the buffer solution with a Concentration not higher than 0.2n included.

Heparin is made from an anionic cellulose resin, preferably diethylaminoethyl cellulose, adsorbed, which was previously buffered at the same pH as the initial amount.

The amount by weight of the resin to be used is five to twelve times as much as the product to be adsorbed. After the adsorption has ended, the Resin, which now contains all of the anticoagulant activity of the starting material, washed with some adsorption buffer solution. Then the first elution follows with a saline solution of suitable concentration.

Usually has - if the salt of a strong acid is used (e.g. chloride, bromide, sulfate, nitrate, etc.) - the concentration of the eluting solution for this first elution 0.4 to 0.5n and possibly above, but less than to be 0.8n; however, a salt of a weak acid (acetate, formate, borate, Sulphite or the like) is used, the concentration of the eluting solution must be 0.5 to 0.7n and possibly above, but less than 0.9n. The eluate contains a Heparin fraction with low anticoagulant activity (25 to 50 U / mg).

For the second elution, salts of strong acids of one concentration can be used of not more than 1.3n, preferably 1.0 to 1.2n, can be used. Instead, you can also salt solutions of weak acids with a concentration of not more than 1.8n, preferably from 1.5 to 1.7n *), can be used. The eluate contains a heparin fraction with a high anticoagulant activity (160 to 180 U / mg).

If column adsorption and elution is used, it will Resin first buffered at pH 5.5 to 8.5 with a buffer solution that has a salt concentration of not more than 0.2n. The resin is then filled into the column so that its Layer thickness is not less than 7 cm.

The starting product is dissolved in the same buffer solution that was used for Serves to buffer the resin, then the solution is allowed to trickle through the resin column, which is then washed with further adsorption buffer solution. To this Way, the entire anticoagulant activity of the starting product is ensured by the Resin retained.

The elution is then performed with a used for the first elution buffer solution and the eluate with the following added: 1.0 to 1.5 parts by volume of acetone, 1.2 to 1.7 parts by volume 95o / ethyl cent ') salts with monovalent anion are generally used (e.g. NaCl, CH, COONa, etc.), the expression »molar concentrationw is equivalent to the expression» normal concentrations and is often used in its place. In the event that salts with polyvalent anions are used (e.g. NaaHP04, KH.P04, NaHS04, etc.), the concentration is expressed on a »normal basis, the valency of the anion must be taken into account in the calculation draw. alcohol or 2 to 3 parts by volume of methyl alcohol. The first heparin fraction with a low titer precipitated in this way.

Another elution is then carried out with the buffer solution for the second elution is carried out and the following is added to the eluate: 0.7 to 1.0 parts by volume acetone, 1.0 to 1.2 parts by volume 95% ethyl alcohol or 2 to 3 parts by volume of methyl alcohol. In this way, a heparin fraction with higher Titer (160 to 180 U / mg) precipitated.

When using adsorption with stirring (batch adsorption) it is advisable to make two adsorptions, adding half for each of the two of the total amount of resin to be used (namely on 1 g of starting material 3 to 5 g resin for the first adsorption and the same amount for the second adsorption).

Each adsorption lasts 30 minutes and is followed by filtration through a Büchner funnel.

Usually the resin is mixed with the adsorption buffer solution twice washed so as to remove the inactive mother liquor.

The first elution is carried out and then twice with the chosen one Repeated eluent, stirred for 30 minutes and each time through the Buchner funnel filtered.

The less active part of the heparin is removed from the resin in this way and can be obtained by the above-described precipitation (titre: 6 to 15 U / mg).

The second elution is carried out in the same way and repeated twice with the selected eluent, stirred for 30 minutes and each time filtered through the Buchner funnel. The three eluates are combined and treated in the manner described above for the purpose of precipitation. In this way, the more active part, which is contained in the starting material, is precipitated, the precipitates are collected by filtration or centrifugation, washed with anhydrous methanol, ethanol or ethyl ether and finally dried in vacuo (titer: 110 to 130 U / mg).

The technique just described is used for cleaning raw products before. In that case the active eluates are often dark brown and the end product shows a dark hue. To a substantial part of the colored substance too The precipitate can be eliminated in water or 10% NaCl solution pH value - if necessary - adjusted to 7.0 to 9.5 and the resulting solution treated with activated carbon and a filter aid. The loss of activity during the first bleaching process - if it is within the stated pH values running - not high.

The final discoloration, however, must be carried out by one of the usual oxidation methods (with H, 02, KMn04, NaC10 and the like) to be completed; heard of this discoloration however, not the subject of the invention. Example 1 1 g heparin (titer: 110 U / mg) is dissolved in 100 ccm of a 0.1 molar acetate solution containing the same amount of NaCl solution, dissolved at pH 6.2. A column (diameter: 24 mm) is then filled with 7 g of diethylaminoethyl cellulose (Exchange capacity: 0.95 meq / g), which is buffered at pH 6; 2 with the same buffer solution is how they dissolve the heparin was used. The resulting Resin layer is 8.5 cm thick. The heparin solution is allowed to percolate through them and followed by two washes with 50 cc buffer solution each. The first elution is carried out with 0.5 molar NaCl solution, so as to reduce the amount of the less active fractions and remove the traces of nucleic acids; this is followed by a second elution with 1.2 molar NaCl solution. A total of 180 cc of eluate is obtained and is precipitated by addition of 1.2 parts by volume of 95% ethanol. After standing overnight at room temperature the precipitate is collected by centrifugation, washed twice with a small amount of methanol and dried in vacuo: 560 mg of heparin are obtained with a titer of 165 U / mg, the recovery of the activity is 85 ° / a. The less active fraction has a titer of 35 U / mg. example 2 20g of raw heparin (titer: 60U / mg) are dissolved in 1000 ccm of an O, lmolar acetate buffer solution, which contains the same molar amount of NaCl, dissolved at pH 6.0. Diethylaminoethyl cellulose is in two separate amounts of 60 g each at pH 6.0 with the same buffer solution, which served to dissolve the heparin, buffered. The first adsorption is with the one half of the buffered resin performed: - After stirring for 30 minutes The resin is separated off on the Buchner funnel at room temperature. One submits the filtrate of a second adsorption with the second part of the buffered resin and stir again for 30 minutes at room temperature. Then it is filtered, The two resins are combined, washed twice with 1200 ccm of buffer solution each time and filtered via a Büchner funnel.

The resin that now has all of the anticoagulant activity of the starting material is eluted three times with 1200 cc of a 0.5 molar NaCl solution, each time Stirred for 30 minutes and filtered. The three eluates are mixed with 1.5 parts by volume of acetone added, the resulting precipitate is allowed to stand for a few hours, collected remove it by centrifugation, wash with methanol and dry in vacuo. The received Product has a very low anticoagulant activity (9 U / mg) and a considerable "clearing" activity.

The resin is then eluted three times with 1200 cc 1.2 molar NaCl solution, stirred for 30 minutes and each time filtered. The combined eluates are precipitated by adding 1 part by volume of acetone, the resulting precipitate is left to stand overnight, centrifuged, washed with methanol and dried in vacuo. The resulting product is dark in color. It is therefore dissolved in 300 cc of distilled water and the solution is brought to a pH of 7.8 by adding 0.1 nN & OH solution. Then 20 g, activated charcoal and 50 g of a filter aid (Hylfo-Supercel) are added and the mixture is filtered several times. A slightly yellow-brown colored solution is obtained. By adding NaCl, its concentration is brought to 10 /, and then it is precipitated with 1.2 parts by volume of ethanol.

7.9 g of heparin with a titer of 125 U / mg are obtained, its anticosgulatory Activity is around 80%.

Example 3 1.4 g of heparin (titer: 108 U / mg) are dissolved in 100 cc of 0.05 molar phosphate solution which contains 0.1 mol of CH3COONa and has a pH of 7.0. A column (diameter: 30 mm) is then filled with 13 g of diethylaminoethyl cellulose (exchange capacity: 0.80 meq / g) which has been buffered at pH 7.0 with the aforementioned buffer solution which was used to dissolve the heparin. The thickness of the resin layer in the column is 10 cm. The heparin solution is allowed to slowly seep through it and is washed twice with 80 cc of the above-mentioned buffer solution each time. The first elution is carried out with 0.7 molar sodium acetate solution and a precipitate is obtained from the eluate by adding 1.2 parts by volume of 95% ethanol. After drying, this fraction shows a titer of 30 U / mg. The second elution is carried out with a 1.7 molar sodium acetate solution. The eluate is then mixed with 1 part by volume of 95% ethanol, centrifuged, washed with ethanol and ethyl ether and dried in vacuo. 680 mg of heparin are obtained with a titer of 172 U / mg, the anticoagulant activity of which is 77.3 % . Example 4 15 g of crude heparin (titer: 48 U / mg) are dissolved in 600 cc of 0.1 mol of acetate buffer solution, pH 6, which contains 0.1 mol of NaCl. 90 g of TEAE cellulose are prepared, which are treated at pH 6 with the same buffer that is used for the heparin solution.

First, adsorption with half the amount of resin below 30 Minutes stirring and carried out at room temperature. After filtering on the Book funnel becomes the solution of a second adsorption with the other half of the buffered resin, being stirred for 30 minutes and at room temperature is being worked on. After filtering, the resin is mixed together with the first half and washed twice with the adsorption buffer (900 cc each time). After filtration on the Büchner funnel is the resin, which still has the same anticoagulant activity of the starting product, which three times with 900 ccm of a 0.5 molar KCl solution eluted, stirring for 30 minutes at room temperature and filtering off each time.

The three eluates are combined and treated with 3 volumes of methanol. After standing for a few hours, the precipitate is centrifuged and washed with methanol and concentrated in the dry. The product has a very low (6 U / mg) anticoagulant Effect.

The resin is three times with 900 ccm of a KCl solution 1.1 mol under30 Minutes of agitation eluted and filtered off each time. The combined eluates are with 3 volumes of methanol precipitated. The precipitate is centrifuged after standing one night, washed with alcohol and concentrated in the dryer. The titer is 125 U / mg.

The yield is 4.6 g of purified heparin. The recovery of activity is about 800 / ,.

Claims (1)

Claim: Method for purifying heparin into a product with a titer of 160 to 180 U / mg using column adsorption and elution or adsorption with stirring and elution, characterized by the following process steps: a) Adsorption in the presence of a buffer solution from pH 5.5 to 8.5, its salt concentration does not exceed 0.2 n, with an anionic cellulose resin, preferably from diethylaminoethylcellulose or triethylaminoethyl cellulose type, in amounts of 6 to 12 g, based on 1 g of starting material; b) a first elution with solutions of salts of strong acids (e.g. chlorides, Sulfates, nitrates, bromides and the like) a concentration of 0.4 to 0.5 n and possibly above, but below 0.8 n or with solutions of salts of weak Acids (e.g. acetates, borates, formates, sulfites, etc.) in one concentration from 0.5 to 0.7n and optionally above, but below 0.9 n; c) a second elution with solutions of salts of strong acids (e.g. chlorides, sulfates, nitrates, bromides and the like) a concentration of 1.0 to 1.2 N and possibly above, but below 1.3 n or with solutions of salts of weak acids (e.g. acetates, borates, Sulphites, formates and the like) with a concentration of 1.5 to 1m7 n and optionally above, but below 1.8 n.