KR100479404B1 - Method for purification of cephalosporin C broth using absorbent - Google Patents

Abstract

The present invention relates to a method for purifying cephalosporin C (CPC) culture medium for the preparation of 7-aminocephalosporranic acid (7-ACA), more specifically, diacetylcephalo in CPC culture medium using an adsorbent. It relates to a method for separating and removing Sporin C (diacetyl CPC). In the case of the production method of the present invention, it is possible to produce 7-ACA having a very high purity compared to the prior art.

Description

Method for purification of cephalosporin C broth using adsorbents {Method for purification of cephalosporin C broth using absorbent}

The present invention relates to a method for purifying cephalosporin C (CPC) culture for the preparation of 7-aminocephalosporranic acid (7-ACA). More specifically, the present invention relates to a method for separating and removing diacetylcephalosporin C (diacetyl CPC) in a CPC culture medium using an adsorbent.

The method for preparing 7-ACA from CPC can be largely divided into chemical and enzymatic methods.

According to the chemical preparation method, crystals of the CPC salt form are obtained from the CPC culture, and then hydrolyzed to synthesize 7-ACA (Belgium Patent 615955). Chemical methods use toxic pollution reactants (e.g. chlorine solvents, chlorosilanes, dimethylaniline, etc.) and have the disadvantage of requiring strict process conditions (very low temperatures such as -50 ° C).

According to the enzymatic preparation, 7-ACA is prepared by purifying the CPC culture solution and the purified filtrate through the procedure of Scheme 1 below, ie, glutaryl-7 by reacting CPC with D-amino acid oxidase (DAOD). Aminocephalosporanic acid (Gl-7-ACA) was prepared and the resulting Gl-7-ACA was converted to glutaryl-7-aminocephalosporanic acid acylase (Gl-7-ACA acylase). React with to prepare 7-ACA.

The enzymatic method is advantageous in terms of yield compared to the chemical synthesis method because the process of crystallization in the form of CPC salt is omitted. However, since diacetyl CPC included in the purified filtrate reacts with CPC to produce by-product diacetyl 7-ACA as in Scheme 2, it is difficult to prepare 7-ACA with high purity.

The diacetyl CPC in the CPC culture medium is also present in the CPC salt obtained by crystallization, but in the chemical preparation method, the lactonation reaction occurs and is removed during the reaction process as shown in Scheme 3, which does not significantly affect the quality of the final product 7-ACA. Do not. However, in the enzymatic method, since diacetyl CPC is directly present in the form of diacetyl 7-ACA in 7-ACA, a process of removing diacetyl CPC in the CPC culture is essential.

The present invention was derived to solve the conventional problems as described above, to provide a method for producing 7-ACA with high yield by effectively removing the diacetyl CPC contained in the CPC culture medium.

The present invention relates to a method for purifying cephalosporin C (CPC) culture for the preparation of 7-aminocephalosporranic acid (7-ACA). More specifically, the present invention relates to a method for separating and removing diacetylcephalosporin C (diacetyl CPC) in a CPC culture medium using an adsorbent.

That is, (1) reacting cephalosporin C (CPC) culture with D-amino acid oxidase (DAOD) to prepare glutaryl-7-aminocephalosporonic acid (Gl-7-ACA), and (2 ) Gl-7-ACA was reacted with glutaryl-7-aminocephalosporranic acid acylase (Gl-7-ACA acylase) to prepare 7-aminocephalosporanic acid (7-ACA). In the manufacturing method of 7-aminocephalosporranic acid comprising the step, the step (1) after filtering the CPC culture medium, and remove the diacetyl CPC in the filtrate through the adsorption and elution process using an adsorbent, A method characterized by reacting with DAOD.

CPC culture medium is a fermentation product of Acremonium chrysogenium by the fermentation process, it is preferable to use a pH of 5.0 to 6.0.

The CPC culture broth removes impurities, for example, a medium, through a conventional filtering process. In addition, the filtration process may be performed after addition of an acid to the CPC culture medium, if necessary. On the other hand, the solution obtained after the filtration is called the filtrate of the CPC culture.

Purification of the filtrate is carried out using an adsorbent, and diacetyl CPC is removed by the difference in adsorption power of the CPC and the diacetyl CPC to the adsorbent. This can be seen by looking at the dissolution curve of FIG. When the filtrate of the CPC culture solution is passed through the adsorption resin, the CPC and diacetyl CPC molecules in the CPC filtrate are physically adsorbed to the adsorption resin by Van der Waals type relatively weak attraction force with the adsorption resin. At this time, the adsorption force of the molecule is different depending on the size and polarity of the molecule, the chemical structure of the adsorbent, the pore size and the like. If the adsorption is completely selective, the two materials can be separated during the adsorption process, but in general, complete selectivity is not shown for materials with similar molecular structures, such as CPC and diacetyl CPC. Therefore, a process called desorption is performed to separate these materials. Desorption from the adsorbent resin is carried out by using an eluent and reducing the van der Waals force of the molecules causing the adsorbing force by the pH, concentration, or organic solvent component of the eluent. [0035] As the adsorbent, a polystyrene resin is suitable. The polystyrene-based resin is not particularly limited, but for example, a high porosity polystyrene polymer, a copolymer of a high porosity styrene monomer and an ethylenic monomer, or a polymer crosslinked with a macronet thereof may be used. have. In addition, hydrogen of the benzene ring of the said styrene may be substituted by the other substituent. The substituent is not particularly limited, but for example, an alkyl group or an alkyl group substituted with halogen may be used. As the commercially available resin, for example, SEPABEADS of Mitsubishi Chemical Corporation of Japan SP series, in particular, SEPABEADS SP-700, SEPABEADS SP-825, SEPABEADS SP-850, also Amberlite from Rohm and Hass. XAD series, in particular, Amberlite XAD 16, Amberlite XAD 1600 can be used.

When diacetyl CPC is separated and purified from the filtrate of the CPC culture by using an adsorbent, the flow rate of the filtrate of the CPC culture is 0.1 to 10 BVH (bed volume / hour), preferably 0.5 to 5 BVH. Thereafter, the CPC solution is eluted from the resin using a phosphate buffer, an acetate buffer, a carbonate buffer or a weak alkaline solution at pH 6-9. The flow rate of the eluate is 0.1 to 10 BVH, preferably 0.5 to 6 BVH. The temperature at the time of adsorption and elution is 2-25 degreeC, Preferably it is 5-15 degreeC .

After filtration of the fermented CPC culture medium, the elution curve as shown in FIG. 1 can be obtained by adsorbing and eluting the filtrate using a polystyrene-based resin. It can be seen from FIG. 1 that diacetyl CPC and CPC can be separated from each other. In Figure 1 to 3 BV is the adsorption process, after 3 BV corresponds to the elution process. As can be seen from the dissolution curve of FIG. 1, in the polystyrene-based resin, the adsorption force of CPC is stronger than that of diacetyl CPC. Thus, diacetyl CPC, which has a relatively weak adsorptive power during elution, elutes first and CPC elutes relatively late. In Figure 1 it can be seen that the diacetyl CPC is almost eluted at 7 BV, the main part is eluted after 6 BV. Therefore, a high purity CPC solution can be obtained in which diacetyl CPC has been removed when the eluate of 6 BV or more is taken. At this time, the permeability of 7-ACA produced by the color removal effect of the elution after adsorption is improved.

On the other hand, the manufacturing method of 7-ACA using the pre-treated CPC culture in more detail as follows. First, Gl-7-ACA is prepared by directly reacting the CPC solution after pretreatment with DAOD. The concentration of the substrate in the CPC solution is 25 to 50 g / L, preferably 30 to 40 g / L. The pH of the reaction is adjusted to 6.5 to 8.0, preferably 7 to 7.5, the temperature is 15 to 30 ℃, preferably 20 to 25 ℃. At this time, the pressure of the reactor is 0.5 to 1.5kgf / cm ² and oxygen is injected at 0.1 to 0.5vvm (volume / solution volume / min).

Next, the obtained Gl-7-ACA solution is pH 7.0-9.0, preferably 7.5-8.5, temperature 15-30 ° C, preferably 20-25 ° C, and the pressure is Gl-7-ACA acyl under conditions of atmospheric pressure. The 7-ACA can be prepared by reacting with lase.

Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited by the following Example.

Example 1 SEPABEADS Adsorption and Elution of Filtrate of Cephalosporin C Culture Using SP-700 Resin

SEPABEADS The filtered filtrate (diacetyl CPC content: 6%) was filtered and eluted at a rate of 3 BVH in a column containing SP-700 resin (diameter: 6 cm, height: 40 cm, bed volume: 3,000 mL). . The distribution of CPC and diacetyl CPC at adsorption and elution were as shown in FIG. 2.

When the eluate was taken after the separation line of FIG. 2, the content of diacetyl CPC in the CPC solution was 0.9%, and the content of diacetyl CPC was 3.5% on the separation line.

Example 2 SEPABEADS Adsorption and Elution of Filtrate of Cephalosporin C Culture Using SP-825 Resin

SEPABEADS SEPABEADS instead of SP-700 resin The filtrate of the CPC culture was adsorbed and eluted in the same manner as in Example 1 except that the SP-825 resin was used. The results are shown in FIG.

Example 3: SEPABEADS Adsorption and Elution of Filtrate of Cephalosporin C Culture Using SP-850 Resin

SEPABEADS SEPABEADS instead of SP-700 resin The filtrate of the CPC culture was adsorbed and eluted in the same manner as in Example 1 except that the SP-850 resin was used. The results are shown in FIG.

Example 4: Amberlite Adsorption and Elution of Filtrate of Cephalosporin C Culture Using XAD 1600 Resin

SEPABEADS Amberlite instead of SP-700 resin The filtrate of the CPC culture was adsorbed and eluted in the same manner as in Example 1 except that XAD 1600 resin was used. The results are shown in FIG.

Example 5 Preparation of Gl-7-ACA

The CPC solution from which diacetyl CPC was removed in Example 1 was concentrated to a concentration of 75 mM by reverse osmosis, and 1,000 mL of this was put in a reactor. 2 kU of D-amino acid oxidase (DAOD) was added and 5% ammonia was injected while maintaining 20 ° C. to maintain a pH of 7.2. At this time, the oxygen was injected at 0.1 vvm (air volume / working volume / min) so that the pressure inside the reactor was 1 kgf / cm ² . The conversion was 99% in 45 minutes and the composition of the product after complete conversion is shown in Table 1.

compound Composition ratio (%) CPC 0.0 GL-7-ACA 90.8 Diacetyl Gl-7-ACA 1.2

Example 6: Preparation of 7-ACA

5kU of Gl-7-ACA acylase was added to the solution in which the conversion to Gl-7-ACA was carried out as in Example 5, and the reaction was performed at 20 ° C. under atmospheric pressure. At this time, the pH was maintained at 8.0 using 5% ammonia. The maximum conversion yield to 7-ACA was obtained after 95 minutes as 95%. 10% HCl was added to the reaction solution to adjust the pH to 3.5, and crystals were recovered by filtration. Crystallization yield was 88%. Composition ratios of the 7-ACA crystals were as shown in Table 2.

compound Composition ratio (%) 7-ACA 97.8 Diacetyl 7-ACA 1.00

Comparative Example 1: Preparation of Gl-7-ACA

After filtering the cephalosporin C culture medium, 1000 mM of cephalosporin C filtrate was added to a reactor without any separate separation process as in Example 1, and reacted in the same manner as in Example 5. The composition ratio after completion of the reaction is shown in Table 3.

compound Composition ratio (%) CPC 0.0 GL-7-ACA 85.6 Diacetyl Gl-7-ACA 4.7

Comparative Example 2: Preparation of 7-ACA

The reaction solution converted to Gl-7-ACA in Comparative Example 1 was reacted in the same manner as in Example 6. After the reaction was completed, 7-ACA crystals were recovered, and the composition ratio of 7-ACA crystals is shown in Table 4.

compound Composition ratio (%) 7-ACA 95.7 Diacetyl 7-ACA 2.5

 According to the present invention, by separating the diacetyl CPC contained in the CPC culture medium using the adsorbent, it is possible to prepare 7-ACA having a very high purity compared to the prior art.

Figure 1 shows a typical elution curve form when the fermentation-completed cephalosporin C (CPC) culture solution is filtered and then the filtrate is adsorbed using a polystyrene-based resin to elute (diacetyl CPC ( ), CPC (◇)).

2 shows SEPABEADS as a result of Example 1 Elution curve showing the result of adsorption and elution of filtrate of cephalosporin C culture solution using SP-700 resin (diacetyl CPC (), CPC (◇)).

3 shows SEPABEADS as a result of Example 2. FIG. Elution curve showing the result of adsorption and elution of filtrate of cephalosporin C culture solution using SP-825 resin (diacetyl CPC ( ), CPC (◇)).

4 shows SEPABEADS as a result of Example 3. FIG. Elution curve showing the result of adsorption and elution of filtrate of cephalosporin C culture solution using SP-850 resin (diacetyl CPC ( ), CPC (◇)).

5 is Amberlite as a result of Example 4 This is an elution curve showing the result of adsorption and elution of the filtrate of cephalosporin C culture using XAD 1600 resin (diacetyl CPC ( ), CPC (◇)).

Claims (10)

(1) reacting cephalosporin C (CPC) culture with D-amino acid oxidase (DAOD) to produce glutaryl-7-aminocephalosporanic acid (Gl-7-ACA) and (2) production Reacting the prepared Gl-7-ACA with glutaryl-7-aminocephalosporanic acid acylase (Gl-7-ACA acylase) to produce 7-aminocephalosporanic acid (7-ACA) In the manufacturing method of 7-aminocephalosporonic acid comprising a, The step (1) is a polystyrene-based filter comprising a CPC culture medium, and comprises a highly porous polystyrene-based polymer, a copolymer of a highly porous styrene monomer and an ethylenic monomer, or a polymer obtained by crosslinking them in a macronet shape. Resin was used as an adsorbent to elute the CPC solution from the adsorbent using an adsorption process for adsorbing the filtrate at a flow rate of 0.1 to 10 BVH and a phosphate buffer, acetate buffer, carbonate buffer or weak alkaline solution at pH 6-9. After elution to remove the diacetyl CPC in the filtrate, characterized in that the reaction with DAOD. The method of claim 1, wherein the pH of the CPC culture is characterized in that 5.0 to 6.0. delete delete delete delete The method of claim 1, wherein the flow rate of the eluate is 0.1 to 10 BVH. The method according to any one of claims 1, 2 or 7, wherein the pH at the reaction of step (1) is 6.5 to 8.0. 8. Process according to any one of claims 1, 2 or 7, wherein the pH in the reaction of step (2) is 7.0 to 9.0. The method according to claim 1, wherein the content of diacetyl 7-ACA contained in the obtained 7-ACA crystal is 1.0% or less.