KR100419707B1 - Purification method of vancomycin using prep-hplc - Google Patents

Abstract

PURPOSE: A purification method of vancomycin using Prep-HPLC is provided, thereby improving the purification yield and reducing the purification time, so that high quality and purity of vancomycin is obtained. CONSTITUTION: The purification method of vancomycin using Prep-HPLC comprises the steps of: culturing Amycolatopsis orientalis in a medium; regulating pH of the cultured medium to 2.0 to 4.0; sequentially passing the cultured medium through strong acidic cation exchange resin, strong basic anion exchange resin and porous strong acidic cation exchange resin; passing the active fraction to active alumina resin; and subjecting the active fraction to Prep-HPLC using the C-8 reverse phase column, wherein the mobile phase solvent used in the Prep-HPLC is 10 to 100 mM ammonium phosphate buffer solution or ammonium chloride buffer solution containing 10 to 20% methanol; and pH of the buffer solution is 2.0 to 3.0.

Description

Purification of Vancomycin by Preparative Reverse Phase High Pressure Liquid Chromatography (Prep-HPLC)

The present invention is to adjust the microbial fermentation broth containing vancomycin to pH 2.0 to 4.0 to continuously pass a strong acid cation exchange resin, a strong base anion exchange resin and a porous strong acid cation exchange resin and to activate activated alumina (Alumina activated) resin The present invention relates to a method for purifying vancomycin, characterized by rapidly separating and recovering vancomycin in high yield by application to preparative high pressure liquid chromatography (Prep-HPLC) using a C-8 reversed phase column.

Vancomycin is a glycopeptide antibiotic in which α-O-vancosamine-β-O-glucosyl is chemically bound to heptapeptide, and is known to exhibit antimicrobial activity by inhibiting the synthesis of cell walls. Vancomycin is mainly used for the treatment of Gram-positive bacterial diseases and is particularly effective for severe infections that are not treated with antibiotics of penicillin and cephalosporins.

Conventional methods for producing vancomycin may refer to the method described in EP 84308610, where the fermentation broth is filtered, the filtrate is eluted from cation exchange resins, treated with bleached coal, and the soluble vancomycins are separated into insoluble copper complexes. Vancomycin is isolated in free base form after dissolution by treatment with hydrogen sulfide under acidic conditions. However, this method sometimes produces odors, produces slightly colored vancomycin, and yields have not exceeded 30%.

On the other hand, US Patent No. 4,440,753, after the fermentation process is completed, the fermentation broth is filtered to remove solids and mycelia, and the filtrate containing soluble antibiotics is converted to cation exchange resin and nonionic adsorption resin, Diaion HP (High Porous). A method for preparing vancomycin phosphate is obtained by eluting with a resin, then concentrating, adding ethanol to perform a crystallization process, and then recovering and drying the same. However, this method requires a step of replacing the prepared vancomycin phosphate with vancomycin hydrochloride again, and the recovery rate of vancomycin is not high.

Accordingly, the present inventors studied various angles to develop a new purification method that can quickly obtain high purity and high quality vancomycin with high recovery rate by supplementing the problems of the previously developed vancomycin purification method, and various ion exchange resins and activities The present invention has been completed by discovering that preparative high pressure liquid chromatography (Prep-HPLC) using a C-8 reversed phase column as well as passing through an alumina resin can achieve the desired purpose.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention adjusts the microbial fermentation broth containing vancomycin to pH 2.0 to 4.0 to continuously pass strong acid cation exchange resin, strong base anion exchange resin and porous strong acid cation exchange resin, and pass through active alumina resin, followed by C-8 A method for separating and purifying vancomycin by applying to preparative high pressure liquid chromatography using a reversed phase column.

As the vancomycin fermentation broth in the present invention, a shake culture medium of Amy-colatopsis orientalis (formerly named Norcardia orientalis) is used. The fermentation broth was adjusted with sulfuric acid to an acidic range of pH 2.0 to 4.0 where vancomycin could be present in the most stable state, followed by removal of cells and solid impurities using a drum filter coated with diatomaceous earth to obtain a filtrate. do. Since the filtrate contains a large amount of various cations and anions, it is passed through a styrenic strong acid cation exchange resin, preferably SK1B, and a strong base anion exchange resin, preferably PA408, in order to remove the porosity, and then adsorbs vancomycin. It is adsorbed on a strongly acidic cation exchange resin, preferably PK208. At this time, the pretreatment process with the cation exchange resin and the anion exchange resin before the adsorption to the porous cation exchange resin is because the adsorption of vancomycin 30 to 35% more than the case of the pre-treatment filtrate, This is because the recovery rate increases.

Thus, after passing three types of resin columns continuously at a flow rate of 0.5-2.0 SV (multiple of the column volume based on PK208), the absorbance at 280 nm of the porous cation exchange resin to which vancomycin is adsorbed becomes 0.1 or less. Wash with water until. When the washing is completed, vancomycin adsorbed with an alkaline aqueous solution of pH 10.0 to 11.0 is eluted from the resin, and the resin eluate is immediately adjusted to 2.0 to 4.0 in which the vancomycin is stable with hydrochloric acid. The vancomycin-containing fraction is passed through the activated alumina resin again to remove impurities such as pigments, proteins and pyrogenic substances remaining in the aqueous solution. The results of HPLC analysis of the alumina resin passing solution according to the internationally recognized USP analysis method are shown in FIG. 1. After passing through the alumina resin, the aqueous vancomycin solution was concentrated using a polyamide reverse osmosis membrane under conditions of a temperature of 10 to 30 ° C. and a pressure of 10 to 40 kg / cm 2 until the concentration of vancomycin was 50 to 100 g / l. In this concentration, inorganic salts are also removed.

The concentrate is finally purified by applying preparative high pressure liquid chromatography (Prep-HLPC). In this case, a C-8 reverse phase column (Eka Novel, Kromasil KR 100-10-C8, 250 × 20 mm) is used as the HPLC column, and as a mobile phase, 10-100 mM ammonium phosphate buffer solution containing 10-20% methanol. Or ammonium chloride buffer solution to obtain a solution containing vancomycin B (Vn B) in a purity of 96-97% or more. In the purification process by Prep-HPLC, it is appropriate to adjust the pH of the mobile phase to 2.0 to 3.0, preferably pH 2.7. This is because when the pH of the mobile phase buffer solution is less than 2.0, there is a problem in stability of vancomycin, and when it exceeds 3.0, purity and yield are poor. In addition, in the present invention, a C-8 reversed phase column was preferably used, which is particularly effective in shortening the yield and processing time of the C-8 reversed phase column compared to the C-18 reversed phase column, especially when purifying vancomycin in large quantities. It is the result of confirming.

The solution is passed through a strong base anion exchange resin again to remove phosphoric acid and salts, the pH is adjusted, and the concentration of vancomycin in the solution is concentrated to 150 to 200 g / l using a reverse osmosis membrane. The conductivity of the concentrate is adjusted, 1.5 to 2 volume times of ethanol is slowly added, and then left overnight at 4 ° C. to precipitate vancomycin into crystals, and then the crystals are recovered by a filter. The crystalline vancomycin precipitate is washed with ethanol and dried for one day while maintaining the temperature of 25 to 40 ° C. in a vacuum.

Powdered vancomycin hydrochloride containing less than 4% of water, more than 96% of vancomycin B when analyzed by HPLC (USP method) and having antibacterial effect of 1035mcg / mg (based on anhydride) with high purity Can be obtained.

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

A small amount of sulfuric acid was added to 100 L of an A. coli strain containing fermented vancomycin at a concentration of 8 g / L, and then adjusted to pH 2.0, followed by filtration using a diatomaceous earth filter. A column filled with cation exchange resin SK1B 5l, anion exchange resin PA408 15l and porous cation exchange resin PK208 25l was connected in series, and the filtrate was applied and adsorbed through a flow rate of 25l per hour, followed by 200l of water at the same flow rate. Washing with water. After washing was complete, vancomycin was eluted from PK208 resin with ammonia water at pH 13.0 and adjusted to pH 2.5 with hydrochloric acid immediately after elution. The volume of the eluate obtained was 60 L and the purity and yield of vantomycin were 87% and 96%, respectively. This eluate was passed through 15 L of alumina resin and concentrated using a reverse osmosis membrane to give 11 L of vancomycin concentrate having a concentration of 60 g / L.

Example 2: Comparison of C-8 Reverse Phase Columns and C-18 Reverse Phase Columns

The concentrate obtained in Example 1 was adjusted to a concentration of vancomycin at about 50 g / l, followed by a preparative high pressure liquid chromatography (Prep-HPLC) system using 15% methanol / 20 mM ammonium phosphate buffer (pH 2.7) as the mobile phase. It applied to the four columns shown in Table 1. The results using each column are shown in Table 1.

TABLE 1

Note) Sample concentration 51.2g / ℓ (Vn B purity 89.8%, highest impurity 2.8%)

From the results of Table 1, it can be seen that using a C-8 reverse phase column in the Prep-HPLC step can yield higher yields and shorten the working time compared with the case of using a C-18 reverse phase column. In particular, since the working time will be a very important variable when the industrial scale-up of the present invention in the future, it can be seen that the C-8 reverse phase column is better for industrial application in this respect.

Example 3: Effect of Salts on Mobile Phase

The concentrate obtained in Example 1 was adjusted to a vancomycin concentration of 60 g / l and then separated on a preparative high pressure liquid chromatography (prep-HPLC) system. In this case, vancomycin was separated by changing the column to C-8 reverse phase or C-18 reverse phase and changing the salt type in the mobile phase to ammonium phosphate or ammonium chloride, and the results are shown in Table 2.

TABLE 2

As can be seen from the results in Table 2, the C-8 reversed phase column shows a higher yield than the C-18 reversed phase column, which is the same as the result of Table 1 above. In addition, the use of AP (ammonium phosphate) as a salt in the mobile phase has been shown to significantly reduce the working time compared to the case of using AC (ammonium chloride).

Example 4 Impact of Overloading on a Column

The concentrate obtained in Example 1 was adjusted to a concentration of vancomycin at 50 g / l, followed by the use of a Kromasil 100-10-C8 column in a preparative high pressure liquid chromatography (prep-HPLC) system and 15% methanol / 20 mM phosphate buffer ( pH 2.7) was used as the mobile phase, and the conditions were changed as shown in Table 3 to investigate the appropriate loading amount for high purity vancomycin separation.

TABLE 3

As a result of the experiment, it was found that the larger the dropping amount, the larger the impurity was included in front of the Vn B peak.

Example 5 Production of High Purity Vancomycin Using Preparative High Pressure Liquid Chromatography

The concentrate obtained in Example 1 was concentrated to a vancomycin concentration of 80 g / l, and then Kromasil 100-10-C8 (250 mm x 20 mm) was used as a column, and 15% methanol / 25 mM ammonium phosphate buffer (pH2.7) was used as a mobile phase. The separation of vancomycin was performed 15 times in a preparative high pressure liquid chromatography (prep-HPLC) system, and only the vancomycin fractions were combined. Thereafter, the salt was removed from the separation solution and concentrated again, and then, the crystallization process was performed, recovered, and dried to analyze purity. The results are shown in Table 4 below. As can be seen from the results in Table 4, the purity of vancomycin was greatly improved by performing preparative high pressure liquid chromatography under specific conditions according to the present invention.

TABLE 4

1 is a result of concentrating the alumina pass-through and then analyzing by HPLC (USP method),

2 is a result of HPLC analysis of vancomycin fractionated from Prep-HPLC using a C-8 reversed phase column (USP method).

Claims (5)

The microbial fermentation broth containing vancomycin was adjusted to pH 2.0 to 4.0 to continuously pass strong acid cation exchange resin, strong base anion exchange resin, and porous strong acid cation exchange resin, and pass through activated alumina resin, and then Method for separating and purifying vancomycin by applying to preparative high pressure liquid chromatography. The method of claim 1, wherein 10-100 mM ammonium phosphate buffer or ammonium chloride buffer containing 10-20% methanol is used as the mobile phase in performing preparative high pressure liquid chromatography. The method of claim 2 wherein the pH of the buffer solution is 2.0 to 3.0. The method of claim 1 wherein the C-8 reverse phase column is Kromasil 100-5-C8. The method of claim 1 wherein the microorganism is Amycolatopsis orie-ntalis.