Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-06-21
2003-02-11
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S412000, C530S418000, C530S419000
Reexamination Certificate
active
06518406
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for purification of antithrombin-III (AT-III) by precipitation of impurities. The invention also relates to pharmaceutical compositions obtainable by the said methods.
BACKGROUND ART
Antithrombin III (AT-III) is a plasma glycoprotein that inhibits serine proteases in the coagulation cascade and thus plays a major role in the regulation of blood clotting. A small decrease of the AT-III content in the blood is associated with increased risk of thromboembolism. AT-III concentrates are used in the prophylaxis and treatment of thromboembolic disorders in patients with acquired or hereditary antithrombin deficiency.
Generally, AT-III is isolated from human plasma and administered to the bloodstream of the patient. Consequently, virus inactivation of AT-III concentrates is desirable. Precipitation with polyethylene glycol (PEG) has been widely used in AT-III purification for concentrating the protein and for precipitating viruses (see e.g. Wickerhauser et al. (1979) Vox Sanguinis 36, 281). In addition to PEG, also barium sulfate, ethanol, trichloroacetic acid, dextran- and ammonium sulfate have been used as precipitating agents during purification of AT-III. Many of these precipitating agents would be harmful if present in the final AT-III formulation. Consequently, subsequent removal of these agents is necessary, and the recovery of AT-III is thereby reduced.
PEG precipitation alone is not sufficient to ensure complete removal of hepatitis virus. AT-III concentrates for therapeutical use are therefore normally pasteurized, normally at +60° C. for 10 h. In general, plasma proteins lose activity during heat treatment. For this reason, stabilizing agents are used during pasteurization.
Citrate and carbohydrates, such as sucrose, have been used as stabilizing agents for AT-III during pasteurization (Mitra et al. (1982) Biotechnology and Bioengineering vol. XXIV, 97-107; Tengborn et al. (1987) Thrombosis Research 48, 701-711; Einarsson et al. (1989) Transfusion 29, 148-152). However, there is no indication in these documents that citrate and saccharides can be used as precipitation agents in the purification of AT-III.
As a final step in the purification of AT-III, the formulation is often lyophilized. Many attempts have been made to prolong the shelf life of lyophilized AT-III.
U.S. Pat. No. 4,340,589 (Uemura et al.) discloses a lyophilized preparation of AT-III, stabilized with at least one substance selected from amino acids, saccharides, polysaccharides, etc., more specifically albumin, urokinase, gelatin, mannitol, heparin, glycine and lysine.
Ashizawa et al. (Japanese Patent Application No. 1994-199566) discloses lyophilized preparations of modified AT-III stabilized with one or more elements selected from organic acid salts, saccharides, amino acids and sodium chloride. The said organic salts could be sodium succinate or sodium citrate. The said saccharides could be D-mannitol, lactose, glucose and D-sorbitol. There is no indication in this document of lyophilized AT-III preparations comprising citrate in combination with sucrose.
A more or less known problem with liquid pharmaceutical preparations, is the formation of particles (approximately 2-75 um) after filling the product in vials or after reconstitution of freeze-dried products with a solution. Especially when working with larger molecules, like proteins, this is a phenomenon which relatively often occurs and the mechanism behind this is not clearly understood. Probably it is a combination of factors which affects in which amount and size the particles occurs, including molecular size of product, excepients of product, material of product container and solution in which the product is reconstituted in. It has not been shown that it should be of any danger having this relatively small amounts of particles in the products. However, it is obviously, that every pharmaceutical producer is aiming to reduce the amount of particles as much as possible in the products, especially the visible particles.
DISCLOSURE OF THE INVENTION
It has surprisingly been found that a combination of a saccharide, such as sucrose, and citrate can advantageously be used for precipitating impurities, including viruses and proteins other than AT-III, in the purification of AT-III. This procedure involves several advantages:
The precipitation step according to the invention assures high viral inactivation, high purity and minimal AT-III deactivation.
The obtained AT-III solution can be subjected directly to pasteurization without further addition of stabilizing compounds. Thus the combination of citrate and saccharide serves the dual purposes as precipitating agents and stabilizing agents during pasteurization.
There is no need to remove the pharmaceutically acceptable combination of saccharide and citrate during further purification of AT-III. Instead the precipitating agents are also useful as stabilizers of a lyophilized preparation of AT-III.
Consequently, in a first aspect this invention provides a process for purifying antithrombin-III comprising the steps of:
(a) adding, to a solution comprising antithrombin-III, a saccharide and citrate, in an amount sufficient for impurities in the said solution to precipitate while antithrombin-III essentially remains in solution;
(b) allowing impurities to precipitate; and
(c) removing the precipitated impurities, thereby obtaining a solution comprising purified antithrombin-III.
In the present context, the term “impurities” is intended to mean undesired substances, including substances used during purification of AT-III (cf. Example 1, below). Impurities include e.g. histidine-rich glycoproteins, hemopexine, lipoproteins, gammaglobulins, Triton-X-100, tri-n-butyl-phosphate, virus, and prions.
The AT-III for use in the process according to the invention can be obtained by any suitable process and be of mammalian, e.g. bovine, porcine or, preferably, human origin. The AT-III may also be obtained by genetic engineering, such as by recombinant DNA techniques or from transgenic animals, e.g. from the milk of sheep producing AT-III in its milk.
Irrespective of origin, the AT-III can be of any isoform, e.g. &agr;-AT-III or &bgr;-AT-III, or any derivative of AT-III. The difference between &agr;-AT-III or &bgr;-AT-III is described e.g. by Brennan et al. (FEBS Letters 219(2), 431-436, 1987). The term “derivative of AT-III” refers e.g. to polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of AT-III.
The said saccharide is preferably a disaccharide such as sucrose or trehalose, or a monosaccharide such as glucose, sorbitol, mannitol, glyconic acid, or maltose. Sucrose is preferred because of its advantageous bioavailability in the body.
For precipitation of impurities, the concentration of saccharide can range from about 10% (w/v) to about 30% (w/v). Preferably the saccharide concentration is from between 15% and 25% (w/v), most preferably from about 20% (w/v).
The source of citrate can be citric acid or a pharmaceutically acceptable salt thereof, e.g. alkali metal citrates and alkaline earth metal citrates. Examples of alkali metal citrates are sodium and potassium citrate, while examples of alkaline earth metal citrates are magnesium and calcium citrate. For reasons of bioavailability, low cost and easy handling, sodium citrate is preferred.
For precipitation of impurities, the concentration of citrate can range from about 0.1 to about 3 M. Preferably the citrate concentration is from between 0.5 and 1.5 M, most preferably from between 1 M and 1.25 M.
During precipitation of impurities, pH can range from about 6 to about 9. Preferably, pH is from between 7 and 8, and most preferably neutral (around 7.5). The precipitation is suitably performed at a temperature from between +10° C. and +40° C., preferably at room temperature, such as from between +15° C. and +25° C.
Preferably, citrate and saccharide are added contin
Octapharma AG
Sterne Kessler Goldstein & Fox P.L.L.C.
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