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Title:
PROCESS OF PURIFYING VANCOMYCIN HYDROCHLORIDE
Document Type and Number:
WIPO Patent Application WO/2006/004238
Kind Code:
A1
Abstract:
Provided is a method of purifying a vancomycin from a fermentation broth of a microorganism containing vancomycin, including passing the fermentation broth of a microorganism containing vancomycin through a strong acid cation exchange resin, a weak base anion exchange resin, alumina and a hydrophobic absorbent resin sequentially.

Inventors:
LEE JU-WON (KR)
JUNG YUN-TAEK (KR)
SUH JUNG-WOO (KR)
LEE KWANG-SEOB (KR)
Application Number:
PCT/KR2004/002419
Publication Date:
January 12, 2006
Filing Date:
September 21, 2004
Export Citation:
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Assignee:
CJ CORP (KR)
International Classes:
C07K1/20; C07K9/00; (IPC1-7): C12P19/44; C07K1/14
Foreign References:
US4629781A1986-12-16
US4994555A1991-02-19
US5149784A1992-09-22
US5258495A1993-11-02
US6696412B12004-02-24
Attorney, Agent or Firm:
Y.P.LEE,MOCK & PARTNERS (Seocho-gu, Seoul 137-874, KR)
Download PDF:
Description:
Description

PROCESS OF PURIFYING VANCOMYCIN HY¬ DROCHLORIDE Technical Field [1] The present invention relates to a method of purifying highly pure vancomycin HCl from a fermentation broth of a microorganism. Background Art [2] Vancomycin is one of the glycopeptide antibiotics and produced by mi¬ croorganisms of the genus Actinomycetes , such as the strain Amycolatopsis orientalis (ATCC 19795). Vancomycin exhibits a strong inhibition effect on gram positive bacteria, such as Streptococci , Staphylococci, Clostridium difficile, and gram positive bacteria resistant to penicillin and cephalosporin antibiotics. Also, vancomycin is known to have high treatment effects on diseases derived from methiάllin-resistant Staphylococcus aureus (MRSA) which is fatal to post-operation, elderly patients, and persons having weak irπrunity. Vancomycin HCl is generally consimed as solution orally or in capsule form, or it is injected. [3] According to European Pharmacopeia , vancomycin be at least 93% pure and have impurities composing not more than 4% of the solution. [4] Conventional methods of purifying vancomycin are described in U.S. Patent Nos. 4 ,440,753, 4,845,194, 5,037,652, 5,149,784, 5,235,037, 5,258,495, 5,574,135 etc. In order to increase a yield of vancomycin, in these methods, the fermentation broth is adjusted to a pH of 11 and filtered, and then the pH of the obtained filtrate is adjusted to 8. Next, the filtrate is passed through an absorbent resin, and then the vancomycin is eluted from the absorbent resin. This operation is repeated two or three times. Sub¬ sequently, a basic salt of vancomycin is crystallized with a solvent, and the obtained crystals are acidified. The acidified product is recrystallized with acetone or alcohol to produce vancomycin HCl. Disclosure of Invention Technical Problem [5] The conventioal process is very complicated since vancomycin HCl crystals are obtained by adjusting its pH several times, and after obtaining crystals of the basic salt of vancomycin as intermediates, vancomycin HCl are recrystallized. In addition, vancomycin may be unstable at a high pH. Especially, due to a residual colorant, the obtained vancomycin may be reddish in color and it is difficult to obtain vancomycin having a purity of at least 93%. Thus, it is difficult to produce vancomycin which may satisfy a specification prescribed in European Antibiotic Drugs Standards. [6] The present inventors conducted vigorous research to overcome these problems and discovered a method of producing highly pure vancomycin HCl, which may directly recover vancomycin HCl without a process of obtaining an intermediate, i.e., a basic salt of vancomycin, which is performed due to an increased pH of the product containing vancomycin, thus resulting in a simplified process, and may provide a highly pure vancomycin and especially, eliminate most colorants contained in vancomycin. Technical Solution [7] The present invention provides a method of purifying a highly pure vancomycin HCl from a fermentation broth of a microorganism containing vancomycin in a simpli fied process. [8] According to another aspect of the present invention, there is provided a method of purifying vancomycin HCl from a fermentation broth of a microorganism containing vancomycin, comprising: passing the fermentation broth of a microorganism containing vancomycin through a strong acid cation exchange resin at pH 1-3 and eluting vancomycin with a Q05 to Q2 N ammonium hydroxide solution in water having a pH of 9-11 ; adjusting a pH of the eluted solution to 3-5, passing the solution through a weak base anion exchange resin and subsequently alumina, and then washing the weak base anion exchange resin and alimina with water to elute vancomycin, and decoloriang vancomycin; passing the eluted solution through a hy¬ drophobic absorbent resin and eluting vancomycin with an aqueous solution of C 1-4 alcohol; and adding hydrochloric acid to the eluted solution to adjust the pH of the eluted solution to 2-5 and adding an water-soluble organic solvent selected from the group consisting of a C 1-4 alcohol, acetonitrile, acetone, and methyl isobutyl ketone to the eluted solution to crystallize vancomycin HCl. Advantageous Effects [9] According to the method in an embodiment of the present invention, highly pure vancomycin can be obtained in a simplified purification process. Description of Drawings [10] The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which: [11] FIG. 1 is a view illustrating an HPLC analytical result of vancomycin HCl purified using a method according to an embodiment of the present invention; and [12] FIG. 2 is a view illustrating an HPLC analytical result (based on European Antibiotic Drugs Standards) of vancomycin HCl available from Lilly, Japan. Best Mode [13] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are given for the purpose of il¬ lustration and are not intended to limit the scope of the invention. [14] EXAMPLE [15] Example 1: Purification using a strong acid cation exchange resin (DOWEX 50WX2-100) [16] Sulfuric acid was added to 6,000 ml of a fermentation broth of Amycolatopsis orientalis (ATCC 19795) containing vancomycin in a concentration of 5,000 mg/L to adjust the pH to 2. The adjusted culture was left for 30 min, and then filtered through a filter of diatomaceous earth to obtain 7,500 ml of a filtrate. [17] The concentrated filtrate was passed through 2,000 ml of a strong acid cation exchange resin DOWEX 50WX2-100(Dow chemical) at a flow rate of 2,000 ml/hr and adsorbed thereto. Next, the resin was washed with 4,000 ml distilled water at a flow rate of 2,000 ml/hr. After washing, the vancomycin was eluted from DOWEX 50WX2-100 using 5,000 ml of a Q 1 N NH OH aqueous solution to obtain 4,500 ml of 4 the eluted solution (yield: 88 %). As soon as the eluted solution was obtained, 2 N HCl was added to the eluted solution to adjust the pH to 3.5. [18] Example 2: Purification using a weak base anion exchange resin and alimina [19] 4,500 ml of the eluted solution obtained in Example 1 was passed through 1,000 ml of a weak base anion exchange resin DCAl 1 (Mitsubishi, Japan) and subsequently 800 ml of activated carbon (ALDRICH, U.S.A.) at a flow rate of 1,000 ml/hr. Then, DCAl 1 and alumina were washed with 2,000 ml of water at a flow rate of 1,000 ml/hr. [20] Next, 15g of activated carbon was placed into the obtained eluted solution, stirred for 20 minutes and then filtered off. The volume of the eluted solution was 4,800 ml (yield: 85%). [21] Example 3: Purification using a hydrophobic absorbent resin (Amberchrom CG- 161M) [22] 4,800 ml of the eluted solution obtained in Example 2 was passed through 500 ml of a hydrophobic absorbent resin Amberchrom CG-161M (Rohm & Haas) at a flow rate of 1,000 ml/hr and adsorbed thereto. Then, the resin was washed with 1,000 ml of water at a flow rate of 1,000 ml/hr. After washing, the vancomycin was eluted at a flow rate of 1,000 ml/hr using an increasing concentration of isopropanol containing 50 niM anhydrous sodium phosphate (NaH PO ) in the water eluant, to 8 % for 2.5 2 4 hours at a constant rate. At this time, fractions of 400 ml were obtained and subjected to HPLC analysis based on European Antibiotic Drugs Standards to determine the purity of the vancomycin. Then, the fractions with a purity of at least 93% were combined to recover the vancomycin. The combined fractions had a volime of 1,400 ml and contained at least 95% of vancomycin with most colorants removed therefrom. [23] Example 4: Recovery and analysis of the vancomycin crystals [24] The eluted solution obtained in Example 3 was concentrated using reverse osmosis (R/O) to a volime of 400 ml. The same volime of water was added to the concentrated solution and then, the solution was again concentrated using reverse osmosis (R/O) to a volime of 400 ml. The silimar procedure was repeated 12 times to concentrate the solution to a volime of 100 ml. The concentrated solution was adjusted to a pH of 3.2 using 2 N HCl. Then, acetone was added dropwise slowly to the solution, a volime of acetone added being a 5 fold volime of the solution. The solution was left at 4 ° C overnight, and then filtered off. The filtered precipitate was dried in a vacuum dryer at 40 ° C or below. The dried vancomycin HCl had a weight of 13,500 mg and determined by HPLC analysis based on European Pharmcopeia , 2001. In addition, a commercial vancomycin HCl (Lilly, Japan) was used as a control. [25] The results are illustrated in FIGS. 1 and 2. As illustrated in FIG. 1, using a method according to an embodiment of the present invention, highly pure vancomycin HCl was obtained, which had a vancomycin content of 95.5 % and an antimicrobial activity of at least 1,024 rrcg/mg. Meanwhile, as illustrated in FIG. 2, the commercial vancomycin HCl had a vancomycin content of about 93 % and had many colorants therein compared to the vancomycin purfied according to the present invention. [26] Thus, vancomycin HCl obtained using the purification method according to an embodiment of the present invention had a purity of at least 95% and had a remarkably decreased content of colorants. [27] While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims. Mode for Invention [28] According to an embodiment of the present invention, there is provided a method of purifying vancomycin HCl from a fermentation broth of a microorganism containing vancomycin, comprising: passing the fermentation broth of a microorganism containing vancomycin through a strong acid cation exchange resin with a pH of 1-3 and eluting vancomycin with a Q05 to Q2 N ammonium hydroxide solution in water having a pH of 9-11 ; adjusting a pH of the eluted solution to 3-5, passing the solution through a weak base anion exchange resin and subsequently alumina, and then washing the weak base anion exchange resin and alimina with water to elute vancomycin, and decoloriang vancomycin; passing the eluted solution through a hy¬ drophobic absorbent resin and eluting vancomycin with an aqueous solution of C 1-4 alcohol; and adding hydrochloric acid to the eluted solution to adjust the pH of the eluted solution to 2-5 and adding an water-soluble organic solvent selected from the group consisting of a C 1-4 alcohol, acetonitrile, acetone, and methyl isobutyl ketone, preferably acetone, to the eluted solution to crystallize vancomycin HCl. [29] In the present embodiment, the fermentation broth of a microorganism containing vancomycin is passed through a strong acid cation exchange resin at pH 1-3 and an aqueous solution of ammoniim hydroxide is eluted through the strong acid cation exchange resin. The microorganism may be any microorganism that produces vancomycin and accunulates the produced vancomycin in a culture. For example, a shaking culture of Amycolatopsis orientalis, more specifically Amycolatopsis orientalis (ATT 19795) may be used. The pH of the culture may be adjusted to 1-3 using an acid, such as a strong inorganic acid, such as hydrochloric acid and sulfuric acid. After the culture is left for a predetermined time, for example, 30 minutes, the microorganism and solid impurities are filtered off using a filtration funnel and the filtrate can be purified in the following process. The strong acid cation exchange resin is a compound which can adsorb a cation material in a strong acidic condition, preferably at a pH of 1-3.D0WEX 50WX2-100 (50-100 mesh, a sulfone resin, available from Dow chemical) may be used as a strong acid cation exchange resin. An ordinary person in the art will arbitrarily control a flow rate at which the fermentation is loaded on the cation exchange resin. For example, the flow rate may be 05 to 2.0 volunes of column. Elution may be performed with a solution of ammonium hydroxide, for example, a Q05 to 02 N ammoniim hydroxide solution in water having a pH of 9-11. The obtained vancomycin has a purity of at least 85 %. As soon as the vancomycin solution is eluted, the pH of the vancomycin solution must be adjusted to 3-5 using an acid, for example, hydrochloric acid. [30] Then, the resultant solution is passed through a weak base anion exchange resin and subsequently alumina, and then the weak base anion exchange resin and alumina were washed with water to elute vancomycin and decoloriang of the eluted vancomycin by adsoption. For example, DCAl 1 (a porous acrylic resin, available from Mitsubishi) may be used as the weak base anion exchange resin and an activated alumina may be used as the alumina (-150 mesh, acidic activated, available from ALDRICH). [31] In an exemplary embodiment of the present invention, the eluted solution obtained after the decoloriang may be further mixed with activated carbon and then stirred, followed by filtration to further decolorize the vancomycin. The amount of activated carbon used may be, for example, 10% based on the amount of vancomycin. The purity of the decolorized vancomycin in water increases to about 90%. [32] The eluted solution obtained after the decoloriang may be passed through a hy¬ drophobic absorbent resin and eluting is performed with an aqueous solution of C 1-4 alcohol. Conventional hydrophobic resin used in reverse phase chromatography may be used as the hydrophobic absorbent resin, for example, Amberchrom CG- 16 IM (particle size: 75 βm , available from Rohm & Haas). The Cl-4 alcohol solution may be a solution of alcohol selected from the group consisting of methanol, ethanol, and isopropanol. T he Cl-4 alcohol solution may further contain inorganic salts, such as anhydrous sodium phosphate (NaH PO ), for example, in a concentration of 50 mM. 2 4 Gradient elution can be applied to increase the elution efficiency of vancomycin. An ordinary person in the art can easily determine a process of performing the gradient elution through a suitable preliminary experiment. The vancomycin obtained by eluting with the Cl-4 alcohol h as a purity of at least 95% as determined by an HPLC analysis based on European Pharmcopeia. [33] In another exemplary embodiment of the present invention, the eluted solution obtained by eluting the vancomycin with an aqueous solution of Cl-4 alcohol may be further concentrated. The eluted solution may be concentrated using conventional methods known in the art, such as reverse osmosis and vacuum drying, etc. [34] Hydrochloric acid may be added to the eluted solution obtained by eluting the vancomycin with an aqueous solution of Cl-4 alcohol to adjust the pH of the eluted solution to 2-5 and then, an water-soluble organic solvent selected from the group consisting of a Cl-4 alcohol, acetonitrile, acetone, and methyl isobutyl ketone, preferably acetone, may be added to the eluted solution to crystallize vancomycin HCl. For example, the eluted solution is concentrated using reverse osmosis and hy¬ drochloric acid is added thereto to adjust the pH of the eluted solution to 2-5, and then acetone is added to the eluted solution in an amount of 5 fold by volume of the eluted solution. The solution is then left at a temperature of about 2 to 8 ° C for a pre¬ determined time, for example, about 20 hours, to obtain vancomycin crystals. The obtained vancomycin crystals are filtered off and then dried in a vacuim dryer to recover vancomycin HCl. The resultant vancomycin HCl has a purity of at least 95% as determined by the HPLC analysis based on European Pharmcopeia and has an an¬ timicrobial thresho Id of at least 1,000 mcg/ml. Thus, according to the method in an embodiment of the present invention, highly pure vancomycin can be obtained in a simplified purification process.




 
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