JPH0124795B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a 7-aminocephalosporanic acid derivative represented by the following formula () (hereinafter, compound ()).
The present invention relates to a purification method for Compound () is derived from a cephalosporin compound having a D-α-aminoadipyryl group at the 7th position.
-Method using amino acid oxidase (Special Publication Act 1977-
No. 7158, JP-A-51-44695, JP-A-52-128295
(No. 86490/1986, 7986/1986) or a method of reacting with aldehyde derivatives (Japanese Patent Application Laid-open Nos. 51-86490 and 1982-7986). The above compound () is produced by using an enzyme produced by a bacterium belonging to the genus Comamonas or Pseudomonas to remove the glutaryl group at the 7-position, thereby producing 7-aminocephalosporanic acid, which is an important core of cephalosporin antibiotics. It is a useful intermediate substance that can produce compounds (Japanese Patent Application Laid-Open No. 101584-1984)
issue). In the above method for producing compound (),
In the solution after the reaction, the compound () coexists with unreacted starting materials, various reagents, reaction by-products, and various impurities including colored substances of unknown structure. Conventionally, compound () has been collected and purified by repeated organic solvent extraction with ethyl acetate and water dissolution. This solvent extraction method had to be repeated several times for a long time under strong acidity around pH 1.0, but during the operation, an emulsion was formed at the boundary between the solvent and water, making the separation operation difficult, and the The conditions were in the PH unstable region of compound (), and long-term operation increased deactivation, making it impossible to expect a high recovery rate. In addition, physical and chemical contaminants that are brought in from the culture of the cephalosporin compound, which is the reaction starting material for producing compound (), and that are produced as by-products during the reaction, often behave in the same way as compound (), and these impurities The separation was incomplete. Furthermore, the solution containing the purified and collected compound () will inevitably have a lower concentration than the liquid to be treated, and the usual method of heating and reducing pressure will be necessary for concentration, which will further improve the recovery rate. A decline was inevitable. From an industrial standpoint, the use of these organic solvents has been problematic in that it is expensive and highly dangerous. As a result of extensive research in order to overcome the above-mentioned problems, the present inventors have determined that the compound () can be selectively adsorbed using a non-polar large-mesh polymer synthetic adsorption resin and eluted with an eluent. , various reaction reagents and contaminant impurities such as reaction by-products are effectively separated in a short time, and the elution active fraction of the compound () is high in recovery rate and is higher than that in the liquid before resin adsorption. They found that it can be obtained at a high concentration and completed the present invention. That is, the present invention uses a nonpolar large-mesh polymer synthetic adsorption resin made of a cross-linked polymer of styrene-divinylbenzene or a cross-linked polymer of acrylic ester from an aqueous solution containing the compound (), and the pH is 2.0 to 2.0. Compound () was selectively adsorbed under the conditions of 3.0, and the compound () adsorbed on this adsorption resin
This is a method for purifying compound (2), which is characterized by elution using an eluent having a pH of 7.0 to 13.0. More specifically, an aqueous solution containing a compound (), especially an aqueous solution in which a large amount of impurities are dissolved, such as a reaction aqueous solution, and a most frequent pore size of 1000 Å or less,
Contact and adsorption with a non-polar large network polymer synthetic adsorption resin having a specific surface area of 100 to 800 m ² /g and a pore volume of 0.6 to 1.2 cm ³ /g at a pH of 2.0 to 3.0, and a pH of 7.0 to 3.0. 13.0
A purification process that can be eluted with a basic buffer solution or an alkali hydroxide or a hydrophilic organic solvent or an aqueous solution thereof, and collect an active eluate fraction containing the compound () alone and at a higher concentration than before adsorption to the resin. It's a method. The present invention will be explained in detail below. The non-polar large network polymer synthetic adsorption resin used in the present invention includes Diaion HP-10, a crosslinked polymer of styrene-divinylbenzene,
HP-20, HP-21, HP-30, HP-40,
HP-50 (registered trademark: manufactured by Mitsubishi Chemical Industries, Ltd.), Amberlite XAD-1, Amberlite XAD-2,
Amberlite XAD-4 (registered trademark: manufactured by Rohm and Haas) is mentioned, and examples of crosslinked polymers of acrylic acid ester include Amberlite XAD-7 and Amberlite XAD-4 (registered trademark: manufactured by Rohm and Haas).
8 (registered trademark: manufactured by Rohm and Haas). All of these resins have a wide pore size distribution of 50 to 1000 Å, and a large specific surface area of 100 to 800 m ² /g.
It has a pore volume of 0.6-1.2 m ³ /g. Individual resin brands can be identified by various combinations of the above-mentioned resin physical properties that are considered to have a large influence on adsorption capacity. The pore size affects whether or not the adsorbed substance is sufficiently able to migrate to the adsorption surface of the resin, and for resins with the same porosity, the larger the specific surface area, the larger the adsorption capacity. However, there is an inversely proportional relationship between pore diameter and specific surface area, and the smaller the pore diameter, the larger the surface area. From these points, regarding the adsorption of compounds (), the most frequent pore size is
Diaion HP-20, HP-21 and HP-40, which have a specific surface area of 250 Å to 460 Å, a specific surface area of 700 to 720 m ² /g, and a pore volume of 0.6 to 1.2 ^{cm 3} /g, have a high adsorption capacity. This can be said to be preferable in that it is large. Incidentally, for cephalosporin C, which is a related compound of compound (), a method of purifying it using a non-polar synthetic adsorption resin is known (U.S. Pat. No. 3,725,400, JP-A-51-32791); There are many aspects of the adsorption phenomenon to adsorption resins that are not yet understood.
It is not possible to predict with precision whether a given substance will be well adsorbed on a given synthetic adsorbent. In the case of compound () in the present invention, the non-polar polymer synthetic adsorbent has a very large adsorption capacity, and therefore it is possible to obtain a concentration effect that cannot be seen with cephalosporin C with a single adsorption/desorption operation. can. The method for bringing the aqueous solution containing the compound () into contact with the resin and adsorbing the compound () may be a batch method or a column method. Adsorption and elution are performed using the polarity fluctuation of the compound (), and in an aqueous solution, it is performed by manipulating the pH. The pore surfaces of these non-polar large network polymer synthetic adsorption resins are hydrophobic, and the higher the degree of hydrophobicity, the more
Since it tends to adsorb better, in order to increase the adsorption capacity, it is effective to shift the pH during adsorption to a lower side that suppresses the dissociation of the carboxyl group of the compound (). Considering the pH stability of the compound (2), a pH of 2.0 to 3.0 is preferable since it can greatly increase the adsorption capacity during adsorption. Moreover, the temperature during adsorption can be carried out in the range of 0° C. to 40° C., but a lower temperature side is preferable in order to increase the thermal stability of the compound () and the adsorption capacity. 3-acetoxymethyl-7β- of compound ()
(4-carboxybutanamide)3-cephem-
Regarding 4-carboxylic acid, as shown in Table 1,
The adsorption of compound () onto the resin is determined by Freundlich's isothermal adsorption formula q=KC 1/n (where q is the adsorption capacity,
C represents the adsorption stock concentration, and K and n represent constants determined by experiment. ), it was found that as the adsorption stock solution concentration increases, the adsorption capacity per unit amount of resin increases, so an increase in the total amount of adsorbed substances does not necessarily lead to an increase in the required amount of resin. This effect of the present invention is one of the major advantages brought about by the present invention, and greatly contributes to the concentration effect described below.

[Table] Elution can be performed with a hydrophilic organic solvent such as a basic buffer solution, alkali hydroxide, methanol, acetone, n-propanol, etc., and its aqueous solution, but the separation of the compound () from contaminant impurities and the degree of purification In order to obtain an appropriate elution pattern, it is more preferable to use an aqueous salt solution that has a strong buffering effect on the basic side and can rapidly cause pH fluctuations within the column. These salts include ammonium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium borate, and the like. The concentration of the aqueous salt solution used often depends on the solubility of the various salts, but generally the higher the concentration, the sharper the elution. However, in order to completely separate the target substance from impurities, an appropriate concentration must be determined for each salt, depending on the type of impurities, etc. The aqueous solution of these salts is adjusted to a pH range of 7.0 to 13.0 with ammonia water or an alkali hydroxide aqueous solution such as sodium hydroxide. Since the β-lactam, which is the characteristic skeleton of compound (), decomposes significantly at pH 13.0 or higher, it is necessary to consider its pH stability, and in order to obtain a high recovery rate, the pH at the time of elution must be PH8.0 as area
~9.0 can be said to be preferable. On the contrary, adsorption increased the adsorption capacity due to the increase in hydrophobicity.
During elution, in order to take advantage of the decrease in affinity with the resin due to increased hydrophilicity, the most preferable eluent is a buffer that can cause rapid pH fluctuations within the adsorption-elution system. It is desirable that the band of buffering action be more inclined toward the basic side. In this sense, determining an appropriate salt concentration is necessary to obtain a high recovery rate under an appropriate elution pattern. It is also possible to separate compounds () from each other by selecting the type and concentration of the eluent. The concentration effect of the purification method of the present invention varies depending on the adsorption capacity of the resin and the amount of the active elution fraction of the compound () obtained. Under an appropriate elution pattern that can effectively separate impurities, compound () can be obtained with a high recovery rate by collecting active elution fractions with a compound () concentration of 1000 μg/ml or more. The concentration of the active elution fraction collected in this way is concentrated between 2 and 5 times the concentration of the adsorption stock solution. Furthermore, fractions having a concentration of less than 1000 μg/ml can be effectively recovered by being recycled and reused as an eluent in the next batch of adsorption and elution operations. Compared to conventional methods such as solvent extraction, the purification method of the present invention has the following advantages:
A more pure compound () can be obtained with high recovery rate and high concentration. In other words, in the solvent extraction method, the compound () transferred to the solvent layer is concentrated by heating it under reduced pressure.
Extremely harsh conditions had to be used in terms of stability, and a portion of the separated and purified compound () often decomposed during the concentration process, lowering the purity and recovery rate. However, in the present invention, adsorption-
By simply passing liquid through the column for elution, an effective concentration effect can be easily achieved with high separation and purification in a short period of time. Furthermore, from an industrial standpoint, the method of the present invention can reduce the use of hazardous substances stipulated by the Fire Service Act to a very small amount, or even not use them at all, so it is safe and inexpensive. operation can be carried out and is very advantageous. EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 After adjusting the fermentation liquid of Cephalosporium acremonium to pH 3.0, it is sterilized by filtration and the residue is washed. The washing solution and the solution were combined to obtain a solution with a cephalosporin C concentration of 16,563 μg/ml. This solution 1 was reacted with glyoxylic acid in the presence of copper sulfate and pyridine at 0°C for 60 minutes, and the reaction was completed by adding aqueous hydrogen peroxide.
No. 51-86490, Japanese Patent Publication No. 52-7986). The reaction solution was adjusted to pH 2.0, and then insoluble matter was separately removed. The obtained liquid was 1927ml,
Contains 3-acetoxymethyl-7β-(4
-carboxybutanamide)3-cephem-4-
The concentration of carboxylic acid (hereinafter abbreviated as compound A) is
It was 7232μg/ml. So that the bed height is 117 mm in a column with a radius of 35 mm,
Filled with a non-polar large-mesh polymer synthetic adsorption resin (Diaion HP-20) with a most frequent pore diameter of 460 Å, a specific surface area of 718.0 m ² /g, and a pore volume of 1.077 cm ³ /g, and a pH of 2.0.
Thoroughly pre-wash the resin with acidic water. Pour the entire amount of the above liquid into this prepared column at SV = 1.0
The liquid was passed through. Thereafter, 2.0 CV of acidic water with a pH of 2.0 was passed through the column to wash the inside of the column. Then PH8.5
Elution was performed with 0.25M phosphate buffer. Most of the chemicals used in the reaction pass through the waste liquid during adsorption and acidic water washing. Reaction by-products were separated by 1.2CV after the start of elution, and 91.3% of the adsorbed compound A could be recovered from 1.2CV to 2.1CV. The active fraction obtained as the main fraction (hereinafter referred to as main stream) had a pH of 6.0 and a concentration of Compound A of 31448 μg/ml. This can be said to be about 4.3 times more concentrated than the adsorption stock solution. on the other hand,
Transfer of colored substances to the mainstream is 29.3% and 70.7%
has been removed. Example 2 In exactly ^the same manner as in Example 1, a non-polar large ^- mesh polymer synthetic adsorption resin (Diaion
Compound A adsorbed onto HP-20) was eluted using 0.2M borate buffer (PH9.5) instead of 0.25M phosphate buffer (PH8.5). 1.3CV〜
By 2.3 CV, 91.6% of Compound A was recovered with a high degree of purity. Compound A concentration in the main stream is 28376μg/ml
The concentration rate was 3.9 times. Example 3 As in Examples 1 and 2, the most frequent pore diameter was 460 Å,
Non-polar large mesh polymer synthetic adsorption resin (Diaion) with specific surface area 718.0m ² /g and pore volume 1.077cm ³ /g
Compound A adsorbed on HP-20) was eluted with a 70% methanol aqueous solution. 96.4% of compound A was recovered from 0.9 CV to 2.4 character CV. The concentration of Compound A in the main stream was 19908 μg/ml, and the concentration rate was 2.8 times. The removal rate of colored substances was 50.9%. Example 4 Cephalosporin C16563μg obtained in Example 1
The solution containing 24574 μg/ml was obtained by concentrating the solution containing 24574 μg/ml. This solution was subjected to the same reaction as in Example 1 to convert cephalosporin C to compound A. Next, the pH of the reaction-completed solution was adjusted to 2.0, insoluble impurities were separated, and the solution containing 9919 μg/ml of compound A was added.
Obtained 2080ml. So that the bed height is 153 mm in a column with a radius of 35 mm.
A synthetic adsorption resin (Diaion HP-20) slurried with acidic water of pH 2.0 was filled, and the entire amount of the above liquid was passed through at SV=1.0 to adsorb compound A onto the resin. Next, the inside of the column was thoroughly washed with acidic water of PH20 to wash away the conversion reaction agent, and then 0.25M
Elution was performed with phosphate buffer (PH8.5). 93.2% of compound A between 1.3CV and 2.2CV after the start of elution
was recovered, and the solution concentration was 36244 μg/ml.
The concentration ratio relative to the main adsorption stock solution was approximately 3.7 times. Example 5 A solution prepared in exactly the same manner as in Example 1 was
Packed in a 35 mm column with a bed height of 146 mm, the most frequent pore diameter is 250 Å, specific surface area is 704.7 m ² /g, and pore volume is 0.687.
cm ³ /g synthetic adsorption resin (Diaion HP-40)
was adsorbed to. After washing the inside of the column with 2.0CV PH2.0 acidic water, it was eluted with 0.2M phosphate buffer solution of PH8.5, and the concentration was 88.7% from 1.3CV to 2.5CV after the start of elution.
Compound A was recovered with a high degree of purity. The concentration of Compound A in the main stream was 18331 μg/ml, and the concentration ratio to the adsorption stock solution was 2.5 times. Example 6 53 g of 7-aminocephalosporanic acid with a purity of 95%
to 3.37 in pure water, adjust the pH to 3.37 with sodium bicarbonate.
It was dissolved while adjusting to 7.0. Add 90g of glutaric anhydride to this aqueous solution, and while stirring at room temperature.
PH was maintained at 7.0 using 4.0N NaOH. At the stage when no PH drop was observed during the reaction, 7NH ₂ SO ₄
When the pH is adjusted to 2.0 using
3.5 reaction solutions with a concentration of 19271 μg/ml were obtained. Average pore diameter is 90 Å, specific surface area 450 m ² /g, pore volume 1.144
cm ³ /g synthetic adsorbent (Amberlite XAD-7)
A column was filled with 1250 ml of the solution so that H/D≈3.0, and the column was washed with 100% methanol, and then thoroughly washed with acidic water having a pH of 2.0. After passing the entire amount of the above reaction solution through the prepared resin, the inside of the column was thoroughly washed with acidic water of PH2.0, and then elution was performed with 0.2M phosphate buffer. CV～
By 2.1 CV, 94.3% of Compound A was recovered.
The concentration of Compound A in the main stream was 42,403 μg/ml, which was 2.2 times more concentrated than the adsorption stock solution. Example 7 5.5 m ³ of a solution with a cephalosporin C concentration of 12,700 μg/ml, obtained by removing bacterial cells from the fermentation solution of Cephalosporium acremonium by suction, was reacted in the method shown in Example 1, and the reaction was completed. liquid
After adjusting the pH to 2.0, insoluble impurities were removed by suction again. The volume of the resulting solution was 9.4 ^m3 , and the compound A concentration was 6037 μg/ml. Pour the entire amount of liquid at SV = 1.0 through 2.3 m ³ of synthetic adsorption resin (Diaion HP-20) that has been thoroughly washed with PH2.0 acidic water, adsorb compound A, and then add PH2.0 acidic water.
It was thoroughly washed with water at 1.0 CV, and then eluted with 0.2 M phosphate buffer. 90.4% of the total amount of compound A is 1.2CV
It was recovered between ~2.4CV and the concentration of the main stream was
It was 18834μg/ml. In addition, 7.6% of the total amount was recovered as a downstream up to 4.0 CV, and the concentration of compound A was 1106 μg/ml. A similar operation was carried out with a new cephalosporin C-containing disinfectant solution, and the above-mentioned wake was used as a circulating eluent at the start of elution fractionation from a synthetic adsorption resin (Diaion HP-20).
The transfer rate of Compound A into the main stream in the second elution was 96.4%, indicating that Compound A contained in the wake of the previous elution was added to the main stream in the second elution and was effectively recovered. In this way, by continuous batch type resin operation,
It was also possible to recover most of the low concentration Compound A that migrated to the downstream stream.

Claims (1)

[Claims] 1 Formula () From an aqueous solution containing a 7-aminocephalosporanic acid derivative represented by Expression () under the condition that is between 2.0 and 3.0
The 7-aminocephalosporanic acid derivative represented by the formula () is selectively adsorbed, and the 7-aminocephalosporanic acid derivative represented by the formula () adsorbed on this adsorption resin is eluted using an eluent with a pH of 7.0 to 13.0. A method for purifying a 7-aminocephalosporanic acid derivative, the method comprising: 2 The most frequent pore diameter of non-polar large network polymer synthetic adsorption resin is 50 Å to 1000 Å, and the specific surface area is 100 to 800 m ² /
The method according to claim 1, wherein the pore volume is 0.6 to 1.2 cm ³ /g. 3 The most frequent pore diameter of non-polar large network polymer synthetic adsorption resin is 250 Å to 460 Å, and the specific surface area is 700 to 720 m ² /
3. The method according to claim 2, wherein the pore volume is 0.6 to 1.2 cm ³ /g. 4. The method according to claim 1, wherein the eluent is a basic buffer solution with a pH of 8.0 to 9.0. 5. The method according to claim 1, wherein a fraction having a concentration of 7-aminocephalosporanic acid derivative of 1000 μg/ml or more is collected in the eluate.