KR19980039737A - Crystallization Method of Vancomycin - Google Patents

Abstract

The present invention relates to a method for crystallizing purified vancomycin from microbial fermentation broth using ethanol under a conductivity of 15 to 25 ms / cm and a pH of 2.0 to 4.0.

Description

Crystallization Method of Vancomycin

The present invention relates to a method for directly obtaining vancomycin hydrochloride by crystallizing purified vancomycin from microbial fermentation broth using ethanol under conditions of desired conductivity and pH.

Vancomycin is a glycopeptide antibiotic produced under controlled fermentation conditions of Amycolatopsis orientalis (formerly known as Norcardia orientalis). It is prepared by combining with a molecular weight of about 1449. This substance binds to mucopeptide precursors ending in D-ala-D-ala and exhibits antimicrobial activity as a mechanism for inhibiting cell wall synthesis. It is mainly used for the treatment of Gram-positive bacterial diseases, especially penicillin and cephalosporin antibiotics. Roh is known to be highly effective against untreated severe infections.

Conventional purification methods of vancomycin are expensive and multi-step processes with low yields in each step. Particularly, the final crystallization process is very low in yield, requiring a simpler and improved yield method. do.

Using a conventional crystallization method to precipitate crystals with phosphate after purification (US Pat. No. 4,440,753) or to crystallize vancomycin in the copper salt state (US Pat. No. 4,845,194) and replace it with hydrochloride; Precipitate crystals at the pi point of vancomycin by adding alkali and inorganic salts between pH 5 and 9 to produce vancomycin free salts, which are then recrystallized with hydrochloride or dissolved in appropriate distilled water, and the pH is dried under vacuum drying. Mention may be made of the method (see US Pat. No. 5,235,037). However, these prior arts had the disadvantage of being composed of complex multi-step processes. On the other hand, another crystallization method is a method of forming an imidazole / vancomycin complex by filtration (see US Patent No. 4,868,285), which requires subsequent dissociation of the imidazole / vancomycin complex.

Accordingly, the present inventors conducted intensive research to develop a method for crystallizing vancomycin with simpler and higher recovery rate by making up for the shortcomings of the prior art as described above, and using ethanol at specific pH and conductivity conditions. Crystallization of vancomycin has found that this problem can be solved and the present invention has been completed. Hereinafter, the configuration of the present invention will be described in detail.

In order to crystallize vancomycin purified and purified from the fermentation broth of microorganisms according to the present invention, the vancomycin solution is crystallized using ethanol after controlling the conductivity to 15 to 25 ms / cm and pH 2.0 to 4.0. In the crystallization method according to the present invention, if the conductivity and the pH of the solution are adjusted outside the above-mentioned ranges, it is difficult to obtain a high yield as desired. In particular, when the crystallization is performed by adjusting the pH to 4.0 or more, The gelation may occur, making recovery impossible. In addition, it is preferable that the concentration of vancomycin solution is 150 to 200 g / l, and the amount of ethanol added is 1.5 to 2.0 vol. In the case of ethanol, when used in excess of 2.0 times by volume, gelation may also occur and recovery may not be possible.

More specifically, the crystallization method of the present invention is as follows. Concentrate the vancomycin solution purified to vancomycin B factor of 94% or more (USP method) until it is 150 to 200 g / l, and the conductivity of the solution is 15 to 25 ms / cm using an inorganic salt such as ammonium chloride or sodium chloride. After adjusting so that 1.5-2.0 volume times of ethanol are added, the vancomycin hydrochloride crystal is precipitated by standing at 4 ° C. for about 15 to 24 hours. Once the precipitation of the crystals is complete, vancomycin is recovered by a filter while washing with 1-2 volumes of ethanol based on the final concentrate volume. The recovered vancomycin hydrochloride is dried for one day while maintaining the temperature of 20-40 ° C. under vacuum. The water content of the powdered vancomycin obtained through this process was found to be less than 4%, the purity of vancomycin B factor (Vn B) was 94% or more, and the antibacterial effect was 1030 mcg / mg (based on anhydride).

Hereinafter, the present invention will be described in more detail based on the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention in any sense is not limited to these examples.

Example 1 Optimal pH Conditions

Sodium chloride was added to 100 ml of purified vancomycin solution (180 g / L) to adjust the conductivity to 20 ms / cm, followed by adjusting the pH using hydrochloric acid as described in Table 1 below. 1.8 volume times of ethanol was added thereto, and the resultant crystals were filtered after being left at 4 ° C. for one day to recover vancomycin hydrochloride. The experimental results are shown in Table 1.

Table 1.

As can be seen from the results of Table 1, the vancomycin hydrochloride was precipitated when the pH of the solution was maintained at 2.0 to 4.0, in particular the highest yield at pH 2.5 to 3.0.

Example 2 Optimal Conductivity Conditions

Ammonium chloride was added to 100 ml of purified vancomycin solution (160 g / L) to adjust the conductivity as described in Table 2 below, and the pH was adjusted to 2.5 or 3.0, respectively. To this was added 1.9 volume times of ethanol and left at 4 ° C. for 1 day, after which the resulting crystals were filtered to recover vancomycin hydrochloride. The experimental results are shown in Table 2.

Table 2.

As can be seen from the results in Table 2, when the conductivity of the vancomycin solution was maintained in the range of 15 to 25 kPa / cm, a desirable yield was obtained and vancomycin having excellent color value was obtained.

Example 3: Optimal Temperature Conditions

Sodium chloride was added to 100 ml of purified vancomycin solution (160h / L) to adjust the conductivity to 20 kV / cm, and the pH was adjusted to 2.5 using hydrochloric acid. To this was added 1.9 volume times of ethanol and left for one day at the temperature conditions as shown in Table 3, and the resulting crystals were filtered to recover vancomycin hydrochloride. The experimental results are shown in Table 3.

Table 3.

As a temperature condition for crystallizing vancomycin, 4 ° C. was most appropriate. If the temperature was too low, crystals did not precipitate and gels were formed, thus making it impossible to recover.

Example 4 Optimal Ethanol Amount Condition

Ammonium chloride was added to 100 ml of purified vancomycin solution (160 g / L) to adjust the conductivity to 20 ms / cm, and then pH was adjusted to 2.5 using hydrochloric acid. Ethanol was added thereto in an amount as shown in Table 4 below, and the resulting crystals were filtered for 1 day at 4 ° C. to recover vancomycin hydrochloride. The experimental results are shown in Table 4.

Table 4.

From the results in Table 4, it can be seen that the amount of ethanol added to crystallize vancomycin may be 1.5 to 2.0 times by volume, but the highest crystallization yield when the amount of ethanol is 1.8 to 2.0 times by volume.

Claims (7)

Isolation of purified vancomycin from microbial fermentation broth and crystallization using ethanol under conditions of 15 to 25 ms / cm conductivity and pH 2.0 to 4.0. The method of claim 1 wherein the pH conditions are 2.5 to 3.0. The method of claim 1 or 2, wherein the pH is adjusted using hydrochloric acid. The method of claim 1, wherein the conductivity is adjusted using sodium chloride or ammonium chloride. The method of claim 1 wherein ethanol is used at 1.5 to 2.0 volume times. The method of claim 1, wherein the isolated, purified vancomycin is concentrated to a concentration of 150 to 200 g / l prior to crystallization. The process of claim 1 wherein the crystallization is carried out at a temperature of 4 ° C.