MXPA00002620A - Method for purification of antithrombin iii - Google Patents

Abstract

The invention relates to a method for purification of antithrombin III from a starting material containing a heparin/AT III or heparinoid/AT III complex, whereby said complex is adsorbed by an anion-exchange material, after which it is cliven by elution of AT III.

Description

METHOD FOR PURIFYING ANTITHROMBIN III FIGURE OF THE INVENTION The invention relates to a new process for the purification of antithrombin III. Background of the Invention Antrithrombin III (AT III) is a plasma protein, which acts by preventing coagulation, inhibiting thrombin, factors IXa, Xa, Xla and Xlla. The lack of AT III or hereditary thrombophilia is an inherited autosomal dominant disease with a tendency to thrombosis and embolisms as a result of the lower formation of AT III. The lack of acquired AT III can occur, for example, in the case of coagulopathies (IC), sepsis, liver cirrhosis or in the case of nephrotic syndrome. Appearances of absence of AT III may also occur in the case of heart valve prostheses, in post-operative thromboembolic complications, in the case of estrogen therapy or asparaginase therapy. AT III has a high affinity to heparin and heparinoids, therefore in the preparation of pure preparations of antithrombin III a separation of heparin or heparinoids is regulated. According to EP 0 307 002-Al, the separation of these AT III / heparin or AT III / heparinoid complexes is performed with immobilized protamine, where the heparin is bound to immobilized protamine and AT III is obtained from the residue. The AT III / heparin or AT III / heparinoid complexes before treatment with the immobilized protamine is purified by adsorption in an ion exchanger and elution of the unwanted proteins by means of a saline solution at a pH of 7.5. It was shown that with the ion exchange chromatography a separation of the complex was not achieved, but a separation of additional undesirable proteins, where the complex remains as such adsorbed without changes. Another purification possibility for AT III is represented by affinity chromatography through heparin sepharose. For example in Prep. Biochem 13 (1) (1983), pages 1 to 20, describes a process for the purification of AT III, in which AT III is first purified by means of affinity chromatography with heparin sepharose and subsequently with exchange chromatography of ions and gel chromatography, performing the elution of AT III of the heparin sepharose at a pH of 7.4. The ion exchange chromatography was carried out in DEAE-Sepharose, where AT III is linked to a pH of 8.6. In Thrombosis Research 5 (1974), pages 431 to 452 the purification of AT III through heparin sepharose is also described, where adsorption is achieved at pH 8.5 and desorption, also a separation of the immobilized heparin complex and AT III, at a pH of 7.5. With the subsequent ion exchange chromatography on DEAE-Sephadex, AT III was bound to pH 8.0 and eluted at pH 7.4 A similar procedure is described in DE 2 243 688, here also the purification of AT III in heparin gel is presented. Agarose crosslinked, where the adsorption of the heparin gel is performed at a pH of 8.5, and the desorption, also the separation of the AT III from the immobilized heparin, at a pH of 7.3. Detailed Description of the Invention The present invention proposes the task of presenting a new process to produce preparations of antithrombin III with a high purity and yield, wherein an AT III should be obtained as free as possible of heparin or heparinoids. This task is solved according to the invention by means of a process for the purification of AT III from a starting material, which contains an AT III / heparin complex or AT III / heparinoids, which is characterized by the AT III / heparin complex. or AT III / heparinoids is adsorbed to an anion exchange material and subsequently AT III is separated from the adsorbed complex and eluted. The heparin remains during this separation in the anion exchanger, an elution of AT III is also carried out. Preferably the separation according to the invention is carried out with a buffer in a pH range of 8.5 to 10.5. Surprisingly, it has been determined that the separation of the AT III / heparin complex or AT III / heparinoids is carried out in the course of anion exchange chromatography and this at a pH which is greater than 8.5. It has been found that AT-III is eluted from the anion exchanger selectively against heparin or the complex formation between AT III and heparin or heparinoids can be dissolved., while simultaneously linking (not covalently opposite to heparin sepharose) the heparin to the adsorbent material remains unchanged. In the state of the art, the affinity of AT III to heparin has been considered very important, especially at such a pH (see Thrombosis Research (1974) or DE 2 243 688) and the separation of the complex (this is the separation of AT III of immobilized heparin) even in the case of heparin with covalent binding, always performed at low pH values, it was shown that by means of anion exchange chromatography, selective elution of AT III of the AT III complex is possible / heparin or AT III / heparinoids. In the context of the present application it has been determined that with such high pH values a separation of the complex can be obtained and in spite of that, heparin remains essentially bound to the anion exchanger. Most preferably in the process according to the invention, the elution is carried out with a buffer having a conductivity between 15 and 50 mS. With this conductivity the optimal specificity of the elution is achieved, that is, on the one hand it is sufficient to allow an essentially complete separation of the complex, but on the other hand it is not too high that it also elutes the heparin or the heparinoids. The adsorption or desorption conditions depend in general on the anion exchange material used and are essentially a function of the conductivity and the pH value of the buffer. In particular, the relationship between pH and conductivity is such that in the case of a lower conductivity, for example of approximately 20 mS, the pH value of the buffer solution can also be lower, for example 8.5, and vice versa. As a buffer, solutions containing Tris, phosphates or glycine are used for example. Heparin or heparinoids are used more preferably before adsorption in an amount between 30 and 3000 U / ml. With this measure it is guaranteed that all the AT III is in the output material in complex form and with this there is no loss of performance caused by free AT III. The process according to the invention is preferably carried out in a two-step chromatographic purification, wherein in a first step the complex is adsorbed on an anion exchange material and at a pH in the range of 6.0 to 7.5 the stable complex is eluted. The adsorption is then carried out according to the invention of the complex and the separation or elution of AT III from the complex. At a pH value between 8.5 and 10.5, a higher conductivity of the buffer is conveniently adjusted, for example 10-60 mS, preferably between 15-50 mS, most preferably between 20-35 mS, during the elution. Since AT III is preferably used as a therapeutic, viral inactivation is most often reported. This treatment is carried out in the stage of the AT III / heparin complex or AT III / heparinoids, since AT III in complex form is more stable than in free form. Viral inactivation is preferably carried out in two stages, and this is with two viral inactivation procedures independent of each other. This inactivation treatment for viral inactivation is preferably guaranteed by means of a surfactant and / or thermal treatment, for example by means of a solid-state heat treatment, in particular a steam treatment according to EP-0 159 311, EP-0 519 901 or EP-0 674 531. Other treatments for virus inactivation also include treatment with chemical or chemical / physical methods, for example with chaotropic materials according to W094 / 13329, DE 44 34 538 or EP- 0 131 740 (solvent) or photoinactivation. Nanofiltration or nanofiltration with antibody amplification also represents a preferred method for virus reduction within the framework of the present invention. As anion exchangers, all anion exchangers having an affinity for heparin or heparinoids are in principle considered., as for example anion exchanger based on cellulose with dimethylaminoethyl groups (DEAE-Sphacell®, DE32, DE53 among others or Express Ion D; all from the firm Whatman) or with groups CH2N * (CH3) 3 (QA52 or Express Ion Q; Signature Whatman), anion exchanger based on dextran with diethylaminoethyl groups (DEAE-Sephadex®), anion exchanger based on agarose with diethylaminoethyl group (DEAE-Sepharose CL6B®, DEAE-Sepharose Fast Flow®), cross-linked cross-linked dextran exchanger with diethyl [2-hydroxyproyl] aminoethyl group (QAE-Sephadex®), anion exchanger based on agarose with CH2N * (CH_) 3 (Q-Sepharose Fast Flow®, Q-Sepharose High Performance®, Q-Sepharose Big Beads®) or agarose and dextran copolymers (Q-Sepharose XL) (all from Pharmacia), gels spherical chromatography, prepared by means of copolymerization of N-acryloyl-2-amino-2-hydroxymethyl-l, 3-propanediol and an acrylic functional derivative with diethylamino-ethyl groups as functional anion exchangers (DEAE-Tris-Acril® ), non-compressible silica-dextran matrices, in which the gel d The porous silica is introduced in a transversely cross-linked dextran matrix, with diethylaminoethyl anion exchange groups (DEAE-Spherodex®), rigid polystyrene particle gels, the porous of which are filled with a hydrogel, which carries quaternary amino groups with a strong anion exchange effect (Q-Hyper-D®) (all signed Sepracor), rigid macroporous hydrophilic surfaces with N * (C2H5) 2 or N * (CH3) 3 groups (Macroprep DEAE®, Macroprep Q® (all from BioRad), anion exchangers with diethylaminoethyl diethyl (2-hydroxypropyl) aminoethyl and CH2N * (CH3) 3 groups (DEAE-Toyopearl®, QAE-Toyopearl®, Toyopearl Super-Q® (all Tosohaas), anion intercalation resins consisting of porous polymethacrylate / polyacrylate gel (Protein PAK DEAE®, Waters Signature); ion exchangers based copolymers consisting oligoethylene-dimethyl acrylate, glycidyl methacrylate and pentaerythritol-dimethyl acrylate with a hydrophobic surface (Fractogel EMD-TMAE®, Fractogel EMD-DEAE®, Fractogel EMD-DMAE®) exchanger silica gel-based anions with pressure-stable porous spherical chromatography particles (Licrospher 1000 TMAE®, Licrospher 1000 DEAE® and Licrospher 4000 DMAE®) (all from MERCK). In a particularly preferred embodiment of the process according to the invention, the human plasma or a plasma fraction containing AT II is mixed with heparin or a heparinoid, thus forming an AT III / heparin or AT III / heparinoid complex, and this complex it is optionally subjected to the presence of stabilizing organic polyvalent salts, such as citrate and / or ammonium sulfate, and preferably at a temperature in the range of 40 to 70 ° C for a period of between 3 and 30 hours; wherein the treatment at a temperature of about 60 ° C for about 10 hours is particularly preferred. Preferably in the method according to the invention, the AT III obtained from human plasma or a plasma (human) fraction, preferably from low plasma in cryoprecipitate or from a Cohn fraction, preferably from a Cohn IV fraction. The present invention will be explained with the help of the following examples to which, however, it should not be limited. Example 1 93.2 1 of low plasma in Critoprecipitate were mixed with 7.5 x 106 U of heparin, stirred for half an hour and 1 g of DEAE-Sephadex A50 (Pharmacia) was added per liter. By means of gel separation and subsequent separation of the unbound proteins through a NaCl solution buffered with citrate, 1 g / 1 NaCl, pH 7.5), the AT 111 / heparin complex was obtained by means of elution with a buffer solution, which had a conductivity of 44 mS and a pH value of 7.5. The solution was then heated for 10 hours at 60 ° C in the presence of stabilizing organic polyvalent salts (160 g / 1 Na citrate), to inactivate the possible pathogenic microorganisms. The formed precipitate was separated by means of centrifugation or filtration and discarded. The transparent solution was desamortiguo to a Ph of 9.0 and 12.2mS of conductivity. The AT III / heparin complex is ligated into a chromatographic column consisting of 1000 ml of Q-Sepharose Fast Flow® (Fa, Pharmacia) and the AT III is eluted selectively by means of a buffer solution at a pH of 9.0 and a conductivity of 26. mS.

Example 2: (Currently according to the applicant's point of view the best way to carry out the invention) 24.5 i plasma after separating the cryoprecipitate and the prothrombin complex was mixed with 1.85 x 106 U heparin. 15 g DE 52 of cellulose (Whatman signature) per liter were used for the adsorption of the AT III / heparin complex and the product was obtained by means of elution with a buffer solution, which had a conductivity of 45 mS and a pH of 8.09 . After pasteurization (60 ° C 10 h) the solution was desamortiguo and treated with Triton® X 1000 (polyethylene glycol octylphenyl ether tert, Tween® 80 (polyoxyethylene monooleate) and tri- (n-butyl) phosphate (TNBP) at 25 ° C according to US-PS 4 540 573. The AT III was chromatographically purified through a Q-Sepharose Fast-Flow® column (Pharmacia) and was obtained by means of elution with a solution . buffer at pH 9.8 and conductivity of 23 mS through ultrafiltration and diafiltration aT III was adjusted to 100 U / ml, then packaged by sterile filtration and optionally freeze-dried results after freeze drying.: AT III: 96 U / ml Heparin 0.8 U / ml AT III / protein: 6.2 U / ml Triton, Tween, TNBP: <; determinable limits Heparin-bound AT III: > 95% *) *) according to Europ. Pharmacopeia Example 3: Example 2 was repeated, but Toyopearl® Q-650 Th® (Toso Haas) was used instead of Q-Sepharose. Results (eluate): AT III: 4.5 U / ml Heparin: 0. l U / ml AT III / protein: 3.2 u / ml Example 4: Example 2 was repeated, but instead of Q-Sepharose, Express Ion was used Q (Whatman). Results { eluate): AT III: 8.0 U / ml Heparin: 0.8 U / ml AT III / protein: 5.5 U / ml It is noted that in relation to this date, the best method known by the applicant to carry out said invention , which is clear from the present description of the invention.

Claims (8)

1. CLAIMS Having described the invention as above, property is claimed as contained in the following: 1. - Procedure for the purification of antithrombin III from a starting material containing a complex of AT III / heparin or AT III / heparinoids, characterized in that the complex is adsorbed on an anion exchange material, after which the AT III complex is separated by elution.
2. 2. Method according to claim 1, characterized in that the elution is carried out in a buffer in a pH range of 8.5 to 10.5.
3. 3. Method according to claim 1 or 2, characterized in that the buffer for the elution has a conductivity between 10 and 60 mS, preferably between 15 and 50 mS; most preferably between 20 and 35 mS.
4. 4. Method according to one of claims 1 to 3, characterized in that a heparin or a heparinoid in an amount between 30 and 3000 U / ml is added to a starting material containing AT III, after which it is formed a complex of AT III / heparin or AT III / heparinoids. 5. - Method according to one of claims 1 to 4, characterized in that the complex of AT III / heparin or AT III / heparinoids is subjected to a two-step chromatographic purification, where in a first stage the complex is adsorbed in an anion exchange material and elutes at a pH in the range of 6.0 to 7.
5. 5.
6. 6. - Method according to one of claims 1 to 5, characterized in that a stage of viral inactivation is provided.
7. 7. - Method according to one of claims 1 to 6, characterized in that heparin or a heparinoid is added to the human plasma or to the plasma fraction containing AT III for the formation of a complex of heparin / AT III or heparinoids / AT III and complex is subjected to a heat treatment for the inactivation of infectious agents, possibly in the presence of polyvalent stabilizing organic salts, preferably citrate and / or ammonium sulfate, at a temperature in the range of 40 to 70 ° C during the 3 to 30 hours, preferably at a temperature of about 60 ° C for a period of 10 hours.
8. 8. Method according to one of claims 1 to 7, characterized in that part of human plasma or a plasma fraction containing AT III, preferably low cryoprecipitate plasma.