Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
  • C07K14/8121—Serpins
  • C07K14/8128—Antithrombin III
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to a new process for the purification of antithrombin III.
• Antithrombin III is a plasmatic protein that has an anticoagulant effect by inhibiting thrombin, the factors IXa, Xa, XIa and Xlla.
• AT III deficiency or hereditary thrombophilia is an autosomal dominant hereditary disease with a tendency to thrombosis or embolism, which means that AT III has less formation.
• Acquired AT III deficiency can e.g. with consumption coagulopathy (DIC), sepsis, cirrhosis of the liver or with nephrotic syndrome.
• DIC consumption coagulopathy
• sepsis sepsis
• cirrhosis of the liver or with nephrotic syndrome.
• AT III deficiency symptoms can occur with heart valve prostheses, postoperative, thromboembolic complications, with estrogen therapy or with asparagine therapy.
• AT III has a high affinity for heparin and heparinoids, which is why it is necessary to separate the heparin or heparinoid when producing pure antithrombin III preparations.
• this AT III / heparin or AT III / heparinoid complex is cleaved with immobilized protamine, heparin being bound to the immobilized protamine and AT III being obtained from the supernatant.
• the AT 111 / heparin or AT III / heparinoid complex is cleaned before treatment with the immobilized protamine by adsorption on an ion exchanger and elution of unwanted proteins using a salt solution at pH 7.5. It was found that no ion cleavage of the complex was achieved by ion exchange chromatography, but rather a separation of undesired accompanying proteins, the complex as such remaining adsorbed unchanged.
• a further purification option for AT III is affinity chromatography using heparin sepharose.
• pages 1 to 20 described a process for the purification of AT III, in which AT III was first purified via affinity chromatography with heparin sepharose and then with ion exchange chromatography and gel chromatography, the elution of AT III from the heparin sepharose pH 7.4 has occurred. Ion exchange chromatography was performed on the DEAE-Sepharose, with AT III bound at a pH of 8.6.
• AT III via heparin sepharose is also described in Thrombosis Research 5 (1974), pages 431 to 452, the adsorption at pH 8.5 and the desorption, ie the cleavage of the complex from immobilized heparin and AT III, at pH 7 , 5 was achieved.
• AT III was bound at pH 8.0 and eluted at pH 7.4.
• the present invention is based on the object of producing a new process for the preparation of antithrombin III preparations with high purity and yield, as a result of which a heparin or heparinoid-free AT III should be obtained.
• This object is achieved according to the invention by a process for the purification of AT III from a starting material containing an AT III / heparin or AT III / heparinoid complex, which is characterized in that the AT III / heparin or AT 111 / heparinoid complex Complex is adsorbed on an anion exchange material and then AT III is cleaved from the adsorbed complex and eluted. During this cleavage, heparin remains on the anion exchanger, so there is a selective elution. t ion of AT III.
• the cleavage according to the invention is preferably carried out with a buffer at a pH range from 8.5 to 10.5.
• AT III can be selectively eluted from an anion exchanger for heparin or the complex bond between AT III and heparin or heparinoid can be released, while at the same time the (in contrast to heparin sepharose not covalent) binding of heparin to the adsorbent material remains.
• affinity of AT III for heparin was considered to be very high, especially at such a pH (cf.
• the elution is preferably carried out with a buffer which has a conductivity between 15 and 50 mS. With this conductivity, an optimal specificity of the elution is achieved, ie that it is sufficient on the one hand to enable an essentially complete cleavage of the complex, but on the other hand not so high that the heparin or heparinoid also co-elutes .
• the conditions for the adsorption or desorption generally depend on the anion exchange material used are essentially a function of the conductivity and pH of the buffer.
• the relationship between pH and conductivity is such that at low conductivity, e.g. by 20 mS, the pH of the buffer solution can also be lower, e.g. 8.5, and vice versa.
• solutions containing tris, phosphate or glycine are used as buffers.
• Heparin or heparinoid is preferably added in an amount between 30 and 3000 U / ml before the adsorption. This measure ensures that all AT III is present in the starting material as a complex and thus there is no loss of yield due to free AT III.
• the process according to the invention is preferably carried out in a two-stage chromatographic purification, the complex being adsorbed on an anion exchange material in a first stage and the stable complex being eluted at a pH in the range from 6.0 to 7.5. This is followed by the adsorption of the complex according to the invention and the cleavage or elution of AT III from the complex.
• a higher conductivity of the buffer is preferably set, e.g. 10-60 mS, preferably between 15-50 mS, most preferably between 20-35 mS, in the elution.
• AT III is preferably used as a therapeutic agent
• treatment for virus inactivation is usually necessary. This treatment is preferably carried out at the level of the AT III / heparin or AT III / heparinoid complex, since AT III is more stable in complex form than in free form.
• the virus inactivation treatment is preferably also carried out in two stages, namely with two mutually independent virus inactivation methods.
• This inactivation treatment is preferably ensured by a tenside and / or heat treatment, for example by a heat treatment in the solid state, in particular one Steam treatment according to EP-0 159 311, EP-0 519 901 or EP-0 674 531.
• inactivating viruses also include treatment with chemical or chemical / physical methods, e.g. with chaotropic substances according to W094 / 13329, DE 44 34 538 or EP-0 131 740 (solvent) or photo inactivation.
• chemical or chemical / physical methods e.g. with chaotropic substances according to W094 / 13329, DE 44 34 538 or EP-0 131 740 (solvent) or photo inactivation.
• Nanofiltration is also a preferred method for depleting viruses in the context of the present invention.
• anion exchanger in principle, all anion are suitable, which have an affinity for heparin or heparinoids, such as anion exchangers based on cellulose with di- ethylaminoethyl groups (DEAE-Sephacell ®, DE32, DE52, inter alia, or Express Ion D; all Fa. Whatman) or CH 2 N + (CH 3) 3 groups (QA52 or Express Ion Q; Fa.
• anion exchangers based on cross-linked dextran with diethylaminoethyl groups (DEAE-Sephadex ®), agarose-based anion exchangers with diethylaminoethyl Groups (DEAE-Sepharose CL6B ® , DEAE-Sepharose Fast Flow ® ), anion exchangers based on cross-linked dextran with diethyl [2-hydroxypropyl] aminoethyl groups (QAE-Sephadex ® ), anion exchangers based on agarose with CH 2 N + (CH 3 ) 3 groups (Q-Sepharose Fast Flow ® , Q-Sepharose High Performance ® , Q-Sepharose Big Beads ® ) or copolymers of agarose and dextran (Q-Sepharose XL) (all from Pharmacia), spherical chromatographic gels , forth by copolymerization of N-acrylate,
• Anion exchanger with diethylaminoethyl-diethyl (2-hydroxypropyl) aminoethyl and CH 2 N + (CH 3 ) 3 groups (DEAE-Toyopearl ® , QAE-Toyopearl ® , Toyopearl Super-Q ® (all from Tosohaas), anion exchange exchanger resins consisting of porous polymethacrylate / polyacrylate gel (protein PAK DEAE ® , from Waters);
• Anion exchanger based on copolymers consisting of oligoethylene glycol dimethylacrylate, glycidyl methacrylate and pentaerythritol dimethylacrylate with a hydrophobic surface Fractogel EMD-TMAE ® , Fractogel EMD-DEAE ® , Fractogel EMD-DMAE ®
• anion base exchanger with silica gel porous spherical pressure-stable chromatography particles Lirospher 1000 TMAE ® , Licrospher 1000 DEAE ® and Licrospher 4000 DMAE ® ) (all from MERCK).
• heparin or a heparinoid is added to human plasma or a plasma fraction containing AT III, an AT III / heparin or AT III / heparinoid complex being formed, and this complex is one Heat treatment to inactivate infectious agents.
• This treatment is optionally carried out in the presence of stabilizing, organic polyvalent salts, such as e.g. 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, with treatment at a temperature around 60 ° C for about 10 hours being particularly preferred.
• the process according to the invention is preferably based on human plasma or a [human] plasma fraction containing AT III, preferably low-cryoprecipitate plasma, or a Cohn fraction, preferably a Cohn fraction IV.
• Example 1 The present invention is explained in more detail by the examples below, but without restricting it thereto.
• Example 1 The present invention is explained in more detail by the examples below, but without restricting it thereto.
• Example 1 Example 1 :
• the resulting precipitate was separated by centrifugation or filtration and discarded.
• the clear solution was buffered to pH 9.0 and to 12.2 mS conductivity.
• a T 111 / heparin complex was eluted on a chromatographic column consisting of 1000 ml Q-Sepharose Fast Flow ® (Fa. Pharmacia), and AT III selectively by means of a buffer solution at pH 9, 0 and a conductivity of 26 mS.
• the AT III was purified by chromatography on a 25 ml Q-Sepharose Fast- Flow® (Pharmacia) column and obtained by elution with a buffer solution at pH 9.8 and 23 mS conductivity.
• the AT III was adjusted to 100 U / ml by ultrafiltration and diafiltration, then filled sterile by filtration and optionally freeze-dried.
• Example 2 was repeated, but 650 Q-Th ® (Toso Haas) was used instead of Q-Sepharose ® Toyopearl.
• Q-Th ® Toso Haas
• Example 2 was repeated, but instead of Q-Sepharose Express Ion Q (Whatman) was used. Result (eluate)

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• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

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• 1997
  • 1997-09-19 AT AT0159497A patent/AT405739B/de not_active IP Right Cessation
• 1998
  • 1998-09-17 EP EP98944885A patent/EP1015491A1/de not_active Withdrawn
  • 1998-09-17 HU HU0003671A patent/HUP0003671A1/hu unknown
  • 1998-09-17 BR BR9812229-0A patent/BR9812229A/pt not_active Application Discontinuation
  • 1998-09-17 US US09/509,052 patent/US6395880B1/en not_active Expired - Fee Related
  • 1998-09-17 WO PCT/AT1998/000223 patent/WO1999015562A1/de not_active Application Discontinuation
  • 1998-09-17 CA CA002303924A patent/CA2303924A1/en not_active Abandoned
  • 1998-09-17 JP JP2000512865A patent/JP2001517679A/ja active Pending
  • 1998-09-17 AU AU92448/98A patent/AU742861B2/en not_active Ceased
  • 1998-09-17 SK SK401-2000A patent/SK4012000A3/sk unknown
• 2000
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