EP B1 (19) (11) EP B1 (12) EUROPEAN PATENT SPECIFICATION

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1 (19) (11) EP B1 (12) EUROPEAN PATENT SPECIFICATION (4) Date of publication and mention of the grant of the patent: Bulletin 12/18 (21) Application number: (22) Date of filing: (1) Int Cl.: C07D 7/06 (06.01) A61K 31/31 (06.01) A61P 31/04 (06.01) (86) International application number: PCT/EP07/00747 (87) International publication number: WO 08/0234 ( Gazette 08/) (4) PREPARATION AND PURIFICATION OF MUPIROCIN CALCIUM HERSTELLUNG UND REINIGUNG VON MUPIROCIN-CALCIUM PRÉPARATION ET PURIFICATION DE MUPIROCINE CALCIQUE (84) Designated Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR () Priority: GB (43) Date of publication of application: Bulletin 09/31 (73) Proprietor: Syngen Biotech Co., Ltd. Hsin-Ying T ai nan 7 (TW) (72) Inventor: AASSVEEN, Lene 0171 Oslo (NO) (74) Representative: Onsagers AS Universitetsgaten 7 P.O. Box 6963 St. Olavs plass 01 Oslo (NO) (6) References cited: WO-A-06/ US-A US-A US-A US-A EP B1 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). Printed by Jouve, 7001 PARIS (FR)

2 Description Field of the Invention [0001] The present invention relates to improved processes for the preparation and purification of mupirocin calcium. Background [0002] Mupirocin is an antibacterial agent produced as pseudomonic acid A by fermentation of Pseudomonas fluorescens. It is active against susceptible strains of Staphylococcus aureus and Streptococcus pyogenes, and is typically utilized as a topical solution. Mupirocin has the molecular formula C 26 H 44 O 9 (molecular weight = 00.63) and the chemical name (E)-(2S,3R,4R,S )--[(2S,3S,4S,S)-2,3-Epoxy--hydroxy-4-methylhexyl]tetra-hydro-3,4-dihydroxy-(beta)-methyl-2H-pyran-2-crotonic acid, ester with 9-hydroxynonanoic acid. The chemical structure of mupirocin is shown below: [0003] The calcium salt of pseudomonic acid A, mupirocin calcium or calcium pseudomonate, provides higher thermal stability, especially in crystalline state (U.S. Pat. No.,191,093). This stability is particularly favorable in formulations that involve higher temperatures. [0004] Several methods for purifying mupirocin and derivatives thereof are known in the art. For example, US,191,093 describes a process for preparing crystalline calcium pseudomonate or a hydrate thereof which process comprises reacting pseudomonate ions with calcium ions in solution in an aqueous solvent, recovering a crystalline calcium pseudomonate hydrate from the solution and therafter optionally removing water of crystallization. The method provides a simple means of converting mupirocin acid to the calcium salt. However, the purification effect of the operation is limited and a pure starting material is required to obtain a product that is acceptable for pharmaceutical use. U.S. Pat. No. 3,977,943 relates to purification from culture broth by chromatography on Amberlite XAD-2 polystyrene resin and elution using a series of low molecular weight acids. German patent and U.S. Pat. No. 4,289,703 relate to a purification process using sodium barium and methyl isobutyl ketone (MIBK). Belgian Pat. No. 870,8 relates to a process involving extraction of mupirocin from fermentation broth using MIBK and sodium hydrogen carbonate and crystallization from a MIBK-n-heptane mixture. U.S. Pat. No. 4,222,942 relates to a process involving extraction in a polar water immiscible organic solvent, and dilution in a non-polar solvent to effect crystallization. U.S. Pat. No. 6,24,921 relates to a process of extraction using a chlorinated aliphatic hydrocarbon or isobutyl acetate and evaporation of organic solvent. [000] Like US,191,093, WO03/0697 A2 provides a process for preparing mupirocin calcium from mupirocin in a two phase system by using an organic carboxylate and isolating solid mupirocin calcium from the aqueous phase, or by first precipitating amorphous mupirocin calcium from a C 1 -C 4 alcohol, and then convert the amorphous product to the crystalline form. [0006] Several methods for purification of Mupirocin Acid are known in the art, e.g. U.S. Pat. No. 4,222,942 (as noted above), these methods generally relates to partitioning of mupirocin in acidified/alkaline aqueous solution and polar water immiscible organic solvents such as methyl isobutyl ketone (MIBK) and isobutyl acetatate, followed by crystallization from an organic solvent such as MIBK. According to U.S. Pat. No. U.S. 4,289,703 A, soluble barium salts are added to the fermentation broth, then the microorganism cells with the insoluble inactive agents are separated by centrifugation and finally the antibiotics are extracted by MIBK. The antibiotics are then removed from the methyl isobutyl ketone extract by alkaline water and the resulting alkaline aqueous extract is cleaned by re-extraction with MIBK. The crude product obtained is subjected to chromatography and an ester derivative is prepared from the pseudomonic acid antibiotic complex and purified with preparative thin layer chromatography. The acid form of the pure antibiotic is obtained by hydrolysis. [0007] These methods involve high volumes of hazardous agents, and there still remains a need for a novel method for the manufacture of mupirocin calcium, which combines purification and conversion of the mupirocin to the calcium salt. Such a novel process would lead to a shorter production process, and as such be commercially and environmentally more attractive. [0008] There is a need in the art for more efficient processes for producing purified preparations of mupirocin derivatives, 2

3 such as mupirocin calcium. Such processes and products produced thereby are provided herein, as discussed below. Summary 1 [0009] The present invention relates to methods for preparation and purification of mupirocin calcium. In one embodiment, the process comprises adsorbing a cell-free preparation of mupirocin (soluble salt or acid, preferably in the form of a soluble salt e.g. pseudomonate) to a solid support, exposing the bound mupirocin to a calcium-containing solution, washing impurities from the solid support, and eluting purified mupirocin calcium from the solid support. Also provided are preparations of mupirocin calcium prepared and purified using such methods. [00] In another embodiment, a cell-free preparation of mupirocin is prepared, following adsorption of a pseudomonate to a solid support. The bound pseudomonate is thereafter contacted with a calcium-containing solution and mupirocin calcium is formed on the solid support. Impurities are thereafter washed out followed by selective elution of the target molecule from the solid support. Surprisingly, a complete salt swap to calcium is achieved on the adsorbed pseudomonate and the correct molecular equivalency between mupirocin and calcium is obtained. [0011] The method provides high purification efficiency due to selective removal of impurities during processing. Detailed Description [0012] Mupirocin is an antibacterial agent produced as pseudomonic acid A by fermentation of Pseudomonas fluorescens. It is active against susceptible strains of Staphylococcus aureus and Streptococcus pyogenes, and is typically utilized as a topical solution. Mupirocin has the molecular formula C 26 H 44 O 9 (molecular weight = 00.63) and the chemical name (E)-(2S,3R,4R,S )--[(2S,3S,4S,S)-2,3-Epoxy--hydroxy-4-methylhexyl]tetra-hydro-3,4-dihydroxy-(beta)-methyl-2H-pyran-2-crotonic acid, ester with 9-hydroxynonanoic acid. The chemical structure of mupirocin is shown below: As used herein, the term "mupirocin" may refer to pseudomonic acid A, mupirocin acid, mupirocin calcium, pseudomonate, or any mupirocin derivate. The processes described herein are particularly useful for preparing and purifying mupirocin calcium. The processes described herein are particularly useful for preparing and purifying mupirocin calcium. [0013] Within this application, a method for preparation of mupirocin calcium comprising adsorbing mupirocin(in the form of a soluble salt or acid, preferably a soluble salt e.g. pseudomonate) to a solid support, typically a hydrophobic adsorbent resin, exposing the bound mupirocin to a calcium-containing solution, washing impurities from the solid support and eluting purified mupirocin calcium from the solid support, is provided. [0014] One method comprises adsorption of a pseudomonate to a solid support, typically an adsorbent resin, contacting the bound pseudomonate with a calcium-containing solution, washing out impurities, and eluting purified mupirocin calcium from the solid support. The eluted product can thereafter be crystallized and pure mupirocin calcium isolated by a method known in the art, e.g. as described in U.S. Pat. No.,191,093. [001] A cell-free preparation of mupirocin is utilized. This is prepared by fermenting a mupirocin-producing culture of Pseudomonas fluorescens, obtaining the fermentation broth thereof, and removing the biomass (clarifying the broth) by filtration. The ph of the solution is adjusted so as to assure that all mupirocin is deprotonated, i.e. a pseudomonate. Ammonium sulfate may be added to the mupirocin preparation prior to adsorption to the solid support in order to increase the hydrophobic interactions between the molecule and the adsorbent, and thereby increase the binding capacity of the resin for the pseudomonate. [0016] A method for preparing a mupirocin preparation is described in U.S. Patent No. 3,977,943. Briefly, a suitable strain of Pseudomonas fluorescens, such as strain NCIB 86, is grown in submerged culture at C in a medium containing corn steep liquor and glucose in a basic salts solution for approximately 24 hours. Several methods of preparing the fermentation broth are known in the art and would be suitable for use herein. [0017] A solid support may be any material capable of reversibly adsorbing to mupirocin acid. The solid support is a material to which mupirocin binds through hydrophobic interactions in such a way that the acid group of mupirocin is available for binding to other agents, compounds, or moieties such as calcium ions. Another important characteristic of the solid support is that binding of mupirocin to the support is reversible at the option of the skilled artisan. In certain embodiments, the solid support is a resin and in certain others it is a hydrophobic adsorbent resin. Suitable adsorbent 3

4 1 2 3 resins include but are not limited to modified agaroses, modified silica, polystyrenic or acrylic/metacrylic materials. Examples of adsorbent resins include, but are not limited to, the acrylic adsorbents XAD7HP (Rohm & Haas) or HP2MG (Diaion), and the polystyrene divinylbenzene adsorbents XAD1600, XAD4 (both being available from Rohm and Haas) or HP (Diaion). [0018] Suitable hydrophobic adsorbent resins include but are not limited to modified silica, polystyrene or acrylic materials and include, for example, the acrylic adsorbent XAD7HP (Rohm & Haas) - or HP2MG (Diaion), and the polystyrene divinylbenzene adsorbents XAD1600, XAD4. [0019] In a first method, mupirocin (e.g pseudomonate or mupirocin acid, preferable pseudomonate) is adsorbed to a solid support, exposed to a calcium-containing agent and eluted from the column, thus providing a solution of mupirocin calcium. In preferred embodiments, the calcium-containing agent is calcium-based soluble salt. Exemplary calciumcontaining agents include but are not limited to calcium chloride, calcium acetate, calcium nitrate and calcium propionate. Elution may be carried out using an organic solvent for example methanol. Elution may be carried out using other suitable agents which include but are not limited to ethanol, iso-propanol and acetonitrile or any mixtures of these, including aqueous mixtures. [00] The methods described above also include the addition of ammonium sulfate preparation of mupirocin prior to adsorption to the solid support. Ammonium sulfate is added prior to adsorption to increase the hydrophobic interactions between the mupirocin molecule and the adsorbent, and thereby increase the binding capacity. In other embodiments, the preparation of mupirocin acid is purified mupirocin. [0021] The methods described above are useful in preparing purified mupirocin calcium. In certain embodiments, purified mupirocin calcium is purified to at least 0%; at least 6%; at least 70%; at least 80%; or at least 90%. [0022] Mupirocin calcium purified using the methods described herein may be prepared as a composition for use in treating mammals using standard techniques in the art. The composition(s) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients (i.e., a "pharmaceutical composition"), including humans and other mammals. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of purified mupirocin, alone or in combination with another active agent, typically in conjunction with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of mupirocin as a pharmaceutical composition. The pharmaceutical compositions may also be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, and the like. [0023] A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but can be determined using routine methods known by those skilled in the art. For topical administration, a suitable topical dose of a composition may be administered one to four, and preferably two or three times daily. The dose may also be administered with intervening days during which no dose is applied. Suitable compositions may comprise from % to %, less than %, 1-2%, or 0.1-1% mupirocin of the formulation. Formulations suitable for topical administration include, for example, liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes). Other modes of administration are known in the art and are encompassed by the embodiments described herein. [0024] A better understanding of the present invention and of its many advantages will be gained from the following examples, given by way of illustration. EXAMPLES 4 0 Example 1 Cell-free solution of mupirocin acid in water [002] A fermentation broth containing mupirocin acid was obtained by fermenting a mupirocin producing culture of Pseudomonas fluorescens in a manner known per se, i.e. in line with the procedure of example 1 in US patent 3,977,943. Five liters mupirocin-containing whole culture fermentation broth from a laboratory fermentor was adjusted to ph = 8.3 (by addition of 3M NaOH) and filtered through a 144 m metal screen at room temperature, followed by ultrafiltration (Millipore Pellicon-2 Biomax-). About 70% of the mupirocin present in the fermentation broth was recovered in the final clarified broth. Optionally, the mupirocin containing permeate was concentrated by NF (Osmonic Desal DK membrane). 4

5 Example 2 Adsorption of pseudomonic acid to a solid support and conversion to mupirocin calcium [0026] A. A filtrate obtained as in Example 1 (363 ml, 1.3 g mupirocin) was added (NH 4 ) 2 SO 4 (24 g). The ph was adjusted to ph 7. by addition of 1 M NaOH. The acrylic adsorbent XAD7HP (Rohm & Haas) was packed into a chromatography column (24 ml) and the prepared mupirocin solution (in the form of pseudomonate solution) was added to the column (1 ml/min). The column was washed with 0.1M Tris-buffer containing 0.M (NH 4 ) 2 SO 4. ph 7. (1 ml). A 0.1M calcium acetate solution was then added to the column (72 ml), followed by water (48 ml). Mupirocin calcium was eluted from the column with 80% methanol. 0.9 g mupirocin calcium was recovered in the elution pool (60 ml, 70% yield). [0027] B. A filtrate obtained as in Example 1 (293 ml, 1. g mupirocin), was added (NH 4 ) 2 SO 4 (19 g). The ph was adjusted to ph 7. by addition of 1 M NaOH. The polystyrene divinylbenzene adsorbent XAD1600 (Rohm & Haas) was packed into a chromatography column (22 ml) and the prepared mupirocin solution (in the form of pseudomonate solution) was added to the column (1 ml/min). The column was washed with a 0.1M Tris-buffer containing 0.M (NH 4 ) 2 SO 4, ph 7. (1 ml). A 0.1M calcium acetate solution was subsequently added to the column (66 ml), followed by water (66 ml). Mupirocin calcium was eluted from the column with 80% methanol. 1.3 g mupirocin calcium was recovered in the elution pool ( ml, 87% yield). [0028] C. A filtrate obtained as in Example 1 (3 ml,.8 g mupirocin), was added (NH 4 ) 2 SO 4 (68 g). The ph was adjusted to ph 7. by addition of 1 M NaOH. The acrylic adsorbent XAD7HP (Rohm & Haas) was packed into a chromatography column (191 ml) and the prepared mupirocin solution (in the form of pseudomonate solution) was added to the column (1.9 ml/min). The column was washed with 0.1 M Tris-buffer containing 0.M (NH 4 ) 2 SO 4, ph 7. (98 ml). A 0.1M calcium acetate solution was added to the column (7 ml), followed by water (73 ml). Mupirocin calcium was eluted from the column with 60% methanol..0 g mupirocin calcium was recovered in the elution pool (216 ml, 83% yield). [0029] A fraction of the elution pool (4 ml, 1.2 g mupirocin calcium) was evaporated (90 mbar, 0 C). The resulting methanol free solution (1 ml) was allowed to crystallize with stirring at room temperature. After approximately hr the crystalline material was filtered off and the resulting filter cake was washed with water ( ml) and dried in a vacuum tray dryer (<0 mbar, C). 1.1 g product was recovered with a specific activity of 91.4% ( as is ). [00] D. Purified mupirocin acid ( g, containing 94% active substance) was dissolved in a 0.M (NH 4 ) 2 SO 4 -solution (1L). The solution was adjusted to ph 7. with 1 M NaOH. The polystyrene divinyl benzene adsorbent XAD4 (Rohm & Haas) was packed into a chromatography column (22 ml) and the prepared mupirocin solution (in the form of pseudomonate solution) was added to the column (88 ml, 2 ml/min). The column was washed with 0.1 M phosphate-buffer ph 7. containing 0.M (NH 4 ) 2 SO 4 (33 ml), followed by 0.1M phosphate-buffer, ph 7. (1 ml), and finally water (66 ml). A 0.1M CaCl 2 solution was added to the column (66 ml). Surplus of calcium was washed out by water (66 ml), followed by % methanol (176 ml). Mupirocin calcium was eluted from the column with 60% methanol (176 ml). 0. g mupirocin calcium was recovered in the elution pool (121 ml, 1% yield). [0031] The eluted product was evaporated under vacuum in a laboratory rotary evaporator (80 mbar, 0 C). The concentrated and methanol free solution (2. ml) was allowed to crystallize with stirring at room temperature. After hr the crystalline material was filtered off, and the filter cake was dried in a tray vacuum dryer (<0 mbar, 0 C). 0.3 g product was recovered with a specific activity of 92% ( as is ) and melting point of 12 C. The FT-IR spectrum for the product corresponds to the reference spectrum for mupirocin calcium. 4 0 Claims 1. A method for purifying mupirocin calcium comprising adsorbing a preparation of mupirocin to a hydrophobic adsorbent resin to produce bound mupirocin, exposing the bound mupirocin to a soluble calcium-containing agent to produce mupirocin calcium, washing the bound mupirocin calcium, and eluting purified mupirocin calcium from the hydrophobic adsorbent resin. 2. The method of claim 1 wherein the preparation of mupirocin is prepared by fermenting a mupirocin-producing culture of Pseudomonas fluorescens, obtaining the fermentation broth produced thereby, and optionally clarifying the broth by filtration. 3. The method of claim 2 wherein the resin is selected from the group consisting of modified silica, a polystyrene and an acrylic material.

6 4. The method of claim 3 wherein the resin is selected from the group consisting of the acrylic adsorbent XAD7HP, and the polystyrene divinylbenzene adsorbents XAD1600 and XAD4.. The method of any one of claims 1-4 wherein the mupirocin calcium is further purified by crystallization. 6. The method of any one of claims 1- wherein the mupirocin calcium is purified to at least 0%. 7. The method of any one of claims 1- wherein the mupirocin calcium is purified to at least 6%. 8. The method of any one of claims 1- wherein the mupirocin calcium is purified to at least 90%. 9. A method of any one of claims 1-8 wherein a base is added to the preparation of mupirocin to aid adsorption to the hydrophobic adsorbent resin. 1. The method of any one of claims 1-9 wherein the calcium-containing agent is either calcium acetate, calcium chloride, calcium nitrate or calcium propionate. 11. The method of claim 1 wherein the preparation of mupirocin is in the form of pseudomonate, ammonium sulfate solution is added to the preparation of mupirocin to aid adsorption of the mupirocin to the hydrophobic adsorbent resin, the hydrophobic adsorbent resin is XAD7HP or XAD1600, the calcium containing agent is calcium acetate, and the purified mupirocin calcium is eluted using an organic solvent such as methanol The method of claim 1 wherein the preparation of mupirocin is in the form of mupirocin acid, ammonium sulfate solution is added to the preparation of mupirocin to aid adsorption of the mupirocin to the hydrophobic adsorbent resin, the hydrophobic adsorbent resin is XAD4, the calcium-containing agent is calcium chloride and the purified mupirocin calcium is eluted using an organic solvent such as methanol. 13. A method for preparing mupirocin calcium comprising: 3 a. adsorbing purified mupirocin to a hydrophobic adsorbent resin; b. exposing the adsorbed mupirocin to a calcium-containing agent; c. removing excess calcium-containing solution; and, d. eluting mupirocin calcium from the hydrophobic adsorbent resin. 14. The method of claim 13 wherein the calcium-containing solution is selected from the group consisting of calcium chloride, calcium acetate, calcium nitrate and calcium propionate. 1. The method of claim 14 wherein the calcium-containing solution is calcium acetate. 16. The method of claim 14 wherein the calcium-containing solution is calcium chloride. 17. The method of any one of claims wherein mupirocin calcium is eluted from the hydrophobic adsorbent resin using an organic solvent The method of claim 17 wherein the organic solvent contains methanol. 19. The method of claim 13 wherein the resin is selected from the group consisting of a modified silica precipitate, a polystyrene and an acrylic material. 0. The method of claim 19 wherein the resin is selected from the group consisting of the acrylic adsorbent XAD7HP, and the polystyrene divinylbenzene adsorbents XAD1600 and XAD The method of any one of claims 13- wherein the mupirocin calcium is further purified by crystallization. 22. The method of any one of claims 13- wherein the mupirocin calcium is purified to at least 0%. 23. The method of any one of claims 13- wherein the mupirocin calcium is purified to at least 6%. 6

7 24. The method of any one of claims 13- wherein the mupirocin calcium is purified to at least 90% The method of any of claims wherein a base, such as ammonium sulfate is added to the purified mupirocin to aid adsorption to the hydrophobic adsorbent resin. 26. The method of claim 13 wherein the purified mupirocin is in the form of pseudomonate, ammonium sulfate solution is added to the purified mupirocin to aid adsorption of the mupirocin to the hydrophobic adsorbent resin, the hydrophobic adsorbent resin is XAD7HP or XAD1600 and the mupirocin calcium is eluted using an organic solvent such as methanol. 27. The method of claim 13 wherein the purified mupirocin is in the form of mupirocin acid, ammonium sulfate solution is added to the purified mupirocin to aid adsorption of the mupirocin to the hydrophobic adsorbent resin, the hydrophobic adsorbent resin is XAD4, the calcium-containing solution is calcium chloride and the mupirocin calcium is eluted using an organic solvent such as methanol. Patentansprüche Verfahren zum Reinigen von Mupirocin-Calcium, das das Adsorbieren eines Präparats von Mupirocin an einem hydrophoben Adsorptionsharz, um gebundenes Mupirocin zu erzeugen, das Aussetzen des gebundenen Mupirocins einem löslichen calciumhaltigen Mittel, um Mupirocin-Calcium zu erzeugen, das Waschen des gebundenen Mupirocin-Calciums und das Eluieren des gereinigten Mupirocin-Calciums aus dem hydrophoben Adsorptionsharz aufweist. 2. Verfahren nach Anspruch 1, wobei das Präparat von Mupirocin durch Fermentieren einer Mupirocin produzierenden Kultur von Pseudomonas fluorescens, Gewinnen der dadurch erzeugten Fermentationslösung und gegebenenfalls Reinigen der Lösung durch Filtration hergestellt wird. 3. Verfahren nach Anspruch 2, wobei das Harz aus der Gruppe ausgewählt ist, die aus modifizierter Kieselerde, einem Polystyrol- und einem Acrylmaterial besteht. 4. Verfahren nach Anspruch 3, wobei das Harz aus der Gruppe ausgewählt ist, die aus dem Acryl-Adsorptionsmittel XAD7HP und den Polystyrol- Divinylbenzol-Adsorptionsmitteln XAD1600 und XAD4 besteht. 4. Verfahren nach einem der Ansprüche 1 bis 4, wobei das Mupirocin-Calcium ferner durch Kristallisieren gereinigt wird. 6. Verfahren nach einem der Ansprüche 1 bis, wobei das Mupirocin-Calcium bis zu mindestens 0 % gereinigt wird. 7. Verfahren nach einem der Ansprüche 1 bis, wobei das Mupirocin-Calcium bis zu mindestens 6 % gereinigt wird. 8. Verfahren nach einem der Ansprüche 1 bis, wobei das Mupirocin-Calcium bis zu mindestens 90 % gereinigt wird Verfahren nach einem der Ansprüche 1 bis 8, wobei dem Präparat von Mupirocin eine Base zugesetzt wird, um die Adsorption am hydrophoben Adsorptionsharz zu unterstützen.. Verfahren nach einem der Ansprüche 1 bis 9, wobei das calciumhaltige Mittel entweder Calciumacetat, Calciumchlorid, Calciumnitrat oder Calciumpropionat ist. 11. Verfahren nach Anspruch 1, wobei das Präparat von Mupirocin in Form von Pseudomonat vorliegt, dem Präparat von Mupirocin eine Ammoni- 7

8 umsulfatlösung zugesetzt wird, um die Adsorption des Mupirocins am hydrophoben Adsorptionsharz zu unterstützen, das hydrophobe Adsorptionsharz XAD7HP oder XAD1600 ist, das calciumhaltige Mittel Calciumacetat ist und das gereinigte Mupirocin-Calcium unter Verwendung eines organischen Lösungsmittels, wie Methanol, eluiert wird Verfahren nach Anspruch 1, wobei das Präparat von Mupirocin in Form von Mupirocinsäure vorliegt, dem Präparat von Mupirocin eine Ammoniumsulfatlösung zugesetzt wird, um die Adsorption des Mupirocins am hydrophoben Adsorptionsharz zu unterstützen, das hydrophobe Adsorptionsharz XAD4 ist, das calciumhaltige Mittel Calciumchlorid ist und das gereinigte Mupirocin-Calcium unter Verwendung eines organischen Lösungsmittels, wie Methanol, eluiert wird. 13. Verfahren zum Herstellen von Mupirocin-Calcium, das folgendes aufweist: a. Adsorbieren von gereinigtem Mupirocin an einem hydrophoben Adsorptionsharz; b. Aussetzen des adsorbierten Mupirocin einem calciumhaltigen Mittel; c. Entfernen der überschüssigen calciumhaltigen Lösung; und d. Eluieren von Mupirocin-Calcium aus dem hydrophoben Adsorptionsharz. 14. Verfahren nach Anspruch 13, wobei die calciumhaltige Lösung aus der Gruppe ausgewählt ist, die aus Calciumchlorid, Calciumacetat, Calciumnitrat und Calciumpropionat besteht Verfahren nach Anspruch 14, wobei die calciumhaltige Lösung Calciumacetat ist. 16. Verfahren nach Anspruch 14, wobei die calciumhaltige Lösung Calciumchlorid ist. 17. Verfahren nach einem der Ansprüche 13 bis 16, wobei das Mupirocin-Calcium unter Verwendung eines organischen Lösungsmittels aus dem hydrophoben Adsorptionsharz eluiert wird Verfahren nach Anspruch 17, wobei das organische Lösungsmittel Methanol enthält. 19. Verfahren nach Anspruch 13, wobei das Harz aus der Gruppe ausgewählt ist, die aus modifizierter Kieselerde, einem Polystyrol- und einem Acrylmaterial besteht.. Verfahren nach Anspruch 19, wobei das Harz aus der Gruppe ausgewählt ist, die aus dem Acryl-Adsorptionsmittel XAD7HP und den Polystyrol- Divinylbenzol-Adsorptionsmitteln XAD1600 und XAD4 besteht. 21. Verfahren nach einem der Ansprüche 13 bis, wobei das Mupirocin-Calcium ferner durch Kristallisieren gereinigt wird. 22. Verfahren nach einem der Ansprüche 13 bis, wobei das Mupirocin-Calcium bis zu mindestens 0 % gereinigt wird Verfahren nach einem der Ansprüche 13 bis, wobei das Mupirocin-Calcium bis zu mindestens 6 % gereinigt wird. 24. Verfahren nach einem der Ansprüche 13 bis, wobei das Mupirocin-Calcium bis zu mindestens 90 % gereinigt wird. 2. Verfahren nach einem der Ansprüche 13 bis 24, wobei dem gereinigten Mupirocin eine Base, wie Ammoniumsulfat, zugesetzt wird, um die Adsorption am hydrophoben Adsorptionsharz zu unterstützen. 8

9 26. Verfahren nach Anspruch 13, wobei das gereinigte Mupirocin in Form von Pseudomonat vorliegt, dem gereinigten Mupirocin eine Ammoniumsulfatlösung zugesetzt wird, um die Adsorption des Mupirocins am hydrophoben Adsorptionsharz zu unterstützen, das hydrophobe Adsorptionsharz XAD7HP oder XAD1600 ist, und das Mupirocin-Calcium unter Verwendung eines organischen Lösungsmittels, wie Methanol, eluiert wird. 27. Verfahren nach Anspruch 13, wobei das gereinigte Mupirocin in Form von Mupirocinsäure vorliegt, dem gereinigten Mupirocin eine Ammoniumsulfatlösung zugesetzt wird, um die Adsorption des Mupirocins am hydrophoben Adsorptionsharz zu unterstützen, das hydrophobe Adsorptionsharz XAD4 ist, die calciumhaltige Lösung Calciumchlorid ist und das Mupirocin und Verwendung eines organischen Lösungsmittels wie Methanol eluiert wird Revendications 1. Procédé de purification de mupirocine calcique comprenant l adsorption d une préparation de mupirocine sur une résine d adsorbant hydrophobe pour produire de la mupirocine liée, l exposition de la mupirocine liée à un agent soluble contenant du calcium pour produire de la mupirocine calcique, le lavage de la mupirocine calcique liée, et l élution de la mupirocine calcique purifiée de la résine d adsorbant hydrophobe. 2. Procédé selon la revendication 1, dans lequel la préparation de mupirocine est préparée en fermentant une culture de Pseudomonas fluorescens produisant de la mupirocine, en obtenant le bouillon de fermentation ainsi produit et en clarifiant facultativement le bouillon par filtration. 3. Procédé selon la revendication 2, dans lequel la résine est choisie dans le groupe constitué par la silice modifiée, un polystyrène et un matériau acrylique. 4. Procédé selon la revendication 3, dans lequel la résine est choisie dans le groupe constitué par l adsorbant acrylique XAD7HP, et les adsorbants en poly(styrène divinylbenzène) XAD1600 et XAD4.. Procédé selon l une quelconque des revendications 1 à 4, dans lequel la mupirocine calcique est en outre purifiée par cristallisation. 6. Procédé selon l une quelconque des revendications 1 à, dans lequel la mupirocine calcique est purifiée à au moins 0 %. 7. Procédé selon l une quelconque des revendications 1 à, dans lequel la mupirocine calcique est purifiée à au moins 6 % Procédé selon l une quelconque des revendications 1 à, dans lequel la mupirocine calcique est purifiée à au moins 90 %. 9. Procédé selon l une quelconque des revendications 1 à 8, dans lequel une base est ajoutée à la préparation de mupirocine pour faciliter l adsorption sur la résine d adsorbant hydrophobe.. Procédé selon l une quelconque des revendications 1 à 9, dans lequel l agent contenant du calcium est l acétate de calcium, le chlorure de calcium, le nitrate de calcium ou le propionate de calcium. 11. Procédé selon la revendication 1, dans lequel la préparation de mupirocine est sous la forme de pseudomonate, une solution de sulfate d ammonium est ajoutée à la préparation de mupirocine pour faciliter l adsorption de la mupirocine sur la résine d adsorbant hydrophobe, la résine d adsorbant hydrophobe est XAD7HP ou XAD1600, l agent contenant du calcium est l acétate de calcium, et la mupirocine calcique purifiée est éluée à l aide d un solvant organique tel que le méthanol. 12. Procédé selon la revendication 1, dans lequel la préparation de mupirocine se présente sous la forme de mupirocine acide, une solution de sulfate d ammonium est ajoutée à la préparation de mupirocine pour faciliter l adsorption de la mupirocine sur la résine d adsorbant hydrophobe, la résine d adsorbant hydrophobe est XAD4, l agent contenant du calcium est le chlorure de calcium, et la mupirocine calcique purifiée est éluée à l aide d un solvant organique 9

10 tel que le méthanol. 13. Procédé de préparation de mupirocine calcique comprenant : a. l adsorption de mupirocine purifiée sur une résine d adsorbant hydrophobe ; b. l exposition de la mupirocine adsorbée à un agent contenant du calcium ; c. l élimination de l excès de solution contenant du calcium ; et d. l élution de la mupirocine calcique de la résine d adsorbant hydrophobe. 14. Procédé selon la revendication 13, dans lequel la solution contenant du calcium est choisie dans le groupe constitué par le chlorure de calcium, l acétate de calcium, le nitrate de calcium et le propionate de calcium. 1. Procédé selon la revendication 14, dans lequel la solution contenant du calcium est l acétate de calcium Procédé selon la revendication 14, dans lequel la solution contenant du calcium est le chlorure de calcium. 17. Procédé selon l une quelconque des revendications 13 à 16, dans lequel la mupirocine calcique est éluée de la résine d adsorbant hydrophobe à l aide d un solvant organique. 18. Procédé selon la revendication 17, dans lequel le solvant organique contient du méthanol. 19. Procédé selon la revendication 13, dans lequel la résine est choisie dans le groupe constitué par un précipité de silice modifiée, un polystyrène et un matériau acrylique Procédé selon la revendication 19, dans lequel la résine est choisie dans le groupe constitué par l adsorbant acrylique XAD7HP et les adsorbants en poly(styrène divinylbenzène) XAD1600 et XAD Procédé selon l une quelconque des revendications 13 à, dans lequel la mupirocine calcique est en outre purifiée par cristallisation. 22. Procédé selon l une quelconque des revendications 13 à, dans lequel la mupirocine calcique est purifiée à au moins 0 %. 23. Procédé selon l une quelconque des revendications 13 à, dans lequel la mupirocine calcique est purifiée à au moins 6 %. 24. Procédé selon l une quelconque des revendications 13 à, dans lequel la mupirocine calcique est purifiée à au moins 90 % Procédé selon l une quelconque des revendications 13 à 24, dans lequel une base, telle que du sulfate d ammonium, est ajoutée à la mupirocine purifiée pour faciliter l adsorption sur la résine d adsorbant hydrophobe. 26. Procédé selon la revendication 13, dans lequel la mupirocine purifiée se présente sous la forme de pseudomonate, une solution de sulfate d ammonium est ajoutée à la mupirocine purifiée pour faciliter l adsorption de la mupirocine sur la résine d adsorbant hydrophobe, la résine d adsorbant hydrophobe est XAD7HP ou XAD1600, et la mupirocine calcique est éluée à l aide d un solvant organique tel que le méthanol Procédé selon la revendication 13, dans lequel la mupirocine purifiée se présente sous la forme de mupirocine acide, une solution de sulfate d ammonium est ajoutée à la mupirocine purifiée pour assister l adsorption de la mupirocine sur la résine d adsorbant hydrophobe, la résine d adsorbant hydrophobe est XAD4, la solution contenant du calcium est le chlorure de calcium, et la mupirocine calcique purifiée est éluée à l aide d un solvant organique tel que le méthanol.

11 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description US 1993 A [0003] [0004] [000] [0014] US A [0004] [0016] [002] DE [0004] US A [0004] [0006] BE 8708 [0004] US A [0004] [0006] US B [0004] WO 0697 A2 [000] 11