NL1007076C2 - Method for the recovery of a ß-lactam antibiotic. - Google Patents

Description

- 1 - PN 9314 METHOD FOR THE PRODUCTION OF A β-LACTAM ANTIBIOTIC 5

The invention relates to a method for recovering a β-lactam antibiotic from a mixture containing mainly β-lactam antibiotic and D-phenylglycine (FG) in solution, wherein the mixture is at a temperature between -5 and 20 ° C and at a concentration such that FG remains in solution, is brought to a pH between 3 and 8, the solid β-lactam antibiotic obtained is recovered and the residual liquid is subjected to a temperature increase to a temperature between 10 and 60 ° C, whereby solid FG is formed, FG is separated as a solid, and the mother liquor is recycled.

In the preparation of β-lactam antibiotics in which a β-lactam core is acylated with a D-20 phenylglycine derivative, recovery of the β-lactam antibiotic and work-up of the reaction mixture is generally difficult. Often the work-up is accompanied by significant losses of valuable components, in particular the β-lactam antibiotic, partly as loss of solubility and partly due to degradation due to the limited stability of β-lactam antibiotics.

The invention now provides a new concept for the recovery of β-lactam antibiotics, in which the losses of β-lactam antibiotic are greatly reduced in a simple, industrially applicable method, and valuable D-phenylglycine is also recovered. The invention is based on the fact that it has been found that at relatively low temperatures FG can be present in strong supersaturation in the solution containing FG and the antibiotic, and that it can also remain long without FG settling or crystallizing. Consequently, it is possible to selectively recover the β-lactam antibiotic at low temperature by separating it after crystallization using a pH shift where FG does not crystallize. Furthermore, it has been found that by increasing the temperature of the mother liquor FG crystallizes out quickly while the β-lactam antibiotic does not crystallize out. FG can be selectively recovered in this way. In the above manner, a complete separation between β-lactam antibiotic and FG can be achieved.

During the acylation reaction of the β-lactam core with a suitable acylating agent, in particular an enzymatic acylation reaction with, for example, an amide of D-phenylglycine, for example D-phenylglycine amide (FGA) or an ester of D-phenylglycine, for example the methyl ester of D- phenylglycine (FGM), hydrolysis of the acylating agent and the β-lactam antibiotic takes place, producing D-phenylglycine (FG).

In addition to the β-lactam antibiotic and FG, the mixtures obtained after an acylation reaction may also contain, for example, unreacted β-lactam core and / or an acylating agent, for example FGA or FGM. It has been found that the exact composition of the mixtures that can be used in the method according to the invention is not particularly critical. Mixtures suitable for use in the method according to the invention are preferably mixtures containing 10-1500 mM, in particular 50-1000 mM, β-25 lactam antibiotic; 0-1500 mM, especially 0-1000 mM FG; 0-1000 mM, especially 0-200 mM β-lactam nucleus and 0-1000 mM, especially 0-400 mM D-phenylglycine derivative.

The mixture of β-lactam antibiotic and FG is brought to such a concentration and pH that all components, in particular said components, are in solution. The pH is preferably chosen low for this purpose, for example between 0 and 3, in particular between 0.3 and 2. When the process is carried out on a large scale at 7 h 7 6: -3 -, preferably a more or less continuous work-up. By operating the dissolution continuously, the residence time at relatively high or low pH is shortened. If desired, any solid components 5 still present can be separated, for example by filtration or ultrafiltration.

According to the invention, the mixture, which may still contain a solid β-lactam antibiotic, is first brought to a pH between 3 and 8, preferably between 5.5 and 8.10, in particular between 6.5 and 7.5. , whereby it is ensured, for example by adding water, that the concentration of the reactants, in particular FG, is such that, optionally with the exception of the β-lactam antibiotic, they remain unsaturated in solution. The temperature is between -5 and 20 ° C, preferably between -3 and 15 ° C, in particular between 0 and 10 ° C. The temperature is kept relatively low because it has surprisingly been found that under these conditions it is possible to realize a strong supersaturation of FG without FG settling down.

Furthermore, it has been found that upon increasing the temperature of the mother liquor remaining after the secretion of the β-lactam antibiotic, FG crystallizes rapidly in the form of large, easily filterable crystals. The temperature is thereby increased to a value between 10 and 60 ° C, preferably to a value between 12 and 50 ° C, in particular between 15 and 40 ° C, whereby the pH of the mixture may in principle vary in the range between 3 and 8. The pH is preferably between 5.5 and 8, in particular between 6.5 and 7.5. Because FG has crystallized, the concentration has been reduced to such an extent that the mother liquor remaining after FG crystallization and separation can be at least partially recycled after cooling, for example to the dissolving vessel to which also fresh mixture of β-lactam 70 070 7 6 - - 4 - antibiotic and FG is added. Preferably, this recirculation takes place at such a rate that FG remains in solution in both the dissolution vessel and oversaturated presence in the β-lactam antibiotic crystallization vessel.

Because the FG mother liquor can be reused at least partly for the β-lactam antibiotic crystallization, the solubility losses can be kept at a low level. Due to the favorable process conditions, the degradation losses are also relatively low.

Preferably, the process is carried out continuously, in which fresh mixture of β-lactam antibiotic and FG is always added and a small part of, for example, the FG crystallization mother liquor is always discharged. Preferably, the flow of the blowdown stream is selected such that the volume of the process stream at the various points in the process remains constant over time. In principle, a smaller purge flow can be realized in a continuous process. A possible process setup is shown in figure 1 as an illustration.

The method of the invention can be suitably used in the preparation of β-lactam antibiotics which have a phenylglycine side tail, for example, cephalexin, ampicillin, cefaclor, pivampicillin, bacampicillin, talampicillin and cepaloglycine.

In principle, any β-lactam core can be used; in particular a β-lactam core with the general formula (1) / \ 30 H2N --- 1 \

0 O ^ S) H

35

wherein R0 represents H or an alkoxy group with 1-3 C atoms; Y.

1 0 07076-5 - stands for CH2, O, S or an oxidized form of sulfur; and Z stands for V ”· Ί \

/ \ yX

/ CH3, Rx, R1, or CH2 wherein Rj. for example, represents H, OH, halogen, an alkoxy group with 1-5 C atoms, an alkyl group with 1-5 C atoms, a cycloalkyl group with 4-8 C atoms, an aryl or a heteroaryl group with 6-10 C- atoms, wherein the groups may be optionally substituted, for example, with an alkyl, an aryl, a carboxy or an alkoxy group having 1-8 C atoms; and wherein the carboxylic acid group may optionally be an ester group.

Suitable examples of β-lactam nuclei that can be used in the method according to the invention are penicillin derivatives, for example 6-aminopenicillanic acid (6-APA), and cephalosporin derivatives, for example a 7-aminocephalosporanoic acid with or without a substituent on the 3- site, for example, 7-aminocephalosporanic acid (7-ACA), 7-25 aminodesacetoxycephalosporanic acid (7-ADCA) and 7-amino-3-chloro-cef-3-em-4-carboxylic acid (7-ACCA).

In principle, any enzyme suitable as a catalyst in the coupling reaction can be used as the enzyme. Such enzymes are, for example, the enzymes known under the general term penicillin amidase or penicillin acylase. Such enzymes are described, for example, in J.G. Shewale et. al. Process Biochemistry, August 1989 p. 146-154 and in J.G. Shewale et. al. Process Biochemistry International, June 35 1990, p. 97-103. Examples of suitable enzymes are 1007076-6 enzymes derived from Acetobacter. in particular Acetobacter pasteurianum, Aeromonas. Alcaliaenes. in particular Alcaliaenes faecalis. Aphanocladium. Bacillus sp .. in particular Bacillus meaaterium. Cephalosoorium.

5 Escherichia. in particular Escherichia coli.

Flavobacterium. Fusarium, in particular Fusarium oyyspnrnin and Fusarium Solani. Kluvvera. Mvcoolana. Protaminobacter. Proteus. in particular Proteus rettoari. Pseudomonas and Xanthomonas, in particular Xanthomonas citrii.

Preferably, an immobilized enzyme is used, since the enzyme can then easily be separated and reused. A suitable immobilization technology is described, for example, in EP-A-222462. Another suitable technology is to immobilize the Penicillin G acylase on a support containing a gelling agent, for example, gelatin, and a polymer with free amino groups, for example, alginate amine, chitosan or polyethyleneimine. In addition, enzymes can also be used as a crystalline substance (Clecs).

For example, among the immobilized enzymes commercially available, the Escherichia coli enzyme from Boehringer Mannheim GmbH, commercially available under the name Enzygel®, the immobilized Penicillin-G acylase from Recordati and the immobilized Penicillin-G acylase from Pharma Biotechnology Hanover.

In the (enzymatic) acylation reaction, as acylating agent, for example, D-phenylglycine in activated form, preferably a (primary, secondary or tertiary) amide or salt thereof, or a lower alkyl (1-4C) ester, for example a methyl ester, can be used.

The temperature at which the enzymatic acylation reaction is carried out is usually below 40 ° C, preferably between -5 and 35 ° C. The pH at which the 1007076-7 enzymatic acylation reaction is carried out is usually between 5.5 and 9.5, preferably between 6.0 and 9.0.

Preferably the reaction is stopped almost completely when the maximum conversion has been reached.

A suitable embodiment to stop the reaction is to lower the pH, preferably to a value between 4.0 and 6.3, especially between 4.5 and 5.7. Another suitable embodiment is to lower the temperature of the reaction mixture once the maximum conversion has been reached. A combination of both embodiments is also possible.

After the reaction has virtually stopped upon reaching maximum conversion, the reaction mixture is usually in the form of a suspension containing multiple solids, for example, the antibiotic, D-phenylglycine, and optionally immobilized enzyme. Due to the process economy, the immobilized enzyme is preferably recovered. This can conveniently be done, for example, by filtering the reaction mixture over a screen while stirring, preferably choosing the direction of rotation of the stirrer such that the suspension is pumped up in the center of the stirrer. Valuable components, for example the antibiotic and FG, can then be recovered by the method according to the invention in which the solid components, optionally apart from solid antibiotic, are first dissolved, for instance by means of a pH shift.

For the purposes of the invention, a pH reduction can be effected, for example, by adding an acid to the mixture. Suitable acids are, for example, mineral acids, in particular sulfuric, hydrochloric or nitric acid. Hydrochloric acid is preferably used. For example, a pH increase can be accomplished by adding a base to the mixture. Suitable bases are, for example, inorganic bases, in particular ammonia, potassium hydroxide or caustic soda. Ammonia is preferably used.

The enzymatic acylation reaction and the work-up of the reaction mixture are in practice usually carried out in water. If desired, the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30% by volume. Examples of organic solvents that can be used are alcohols with 1-7 C atoms, for example a monoalcohol, in particular methanol or ethanol; a diol, especially ethylene glycol or a triol, especially glycerol.

The process of the invention is particularly suitable for use in the work-up of the reaction mixture obtained after the enzymatic acylation reaction in which 6-APA is acylated with an amide of D-phenylglycine, for example FGA, or an ester of D -phenylglycine, for example FGM.

In a preferred embodiment it is hereby ensured that the concentration of 6-APA in dissolved form present in the reaction mixture is kept relatively low, so that a higher conversion can be achieved than when the concentration of dissolved 6-APA was chosen as high as possible. In addition, it has been found that the stirability of the reaction mixture is considerably better when the concentration of dissolved 6-APA is kept low.

In this context, conversion is understood to mean the molar ratio of ampicillin formed to the amount of 6-APA used. The concentration of dissolved 6-APA is expressed as the amount of 6-APA in moles per kg of reaction mixture; the total concentration, dissolved and undissolved, 6-APA and ampicillin is expressed as the amount of 6-APA plus ampicillin in moles per kg of total reaction mixture; the total reaction mixture may contain a number of solids in addition to the solution, for example, 6-1UU7J 7 6-9 APA, ampicillin, phenylglycine and immobilized enzyme.

The molar ratio of acylating agent to 6-APA, i.e. the total amount of phenylglycine derivative added divided by the total amount of 6-APA added, expressed in moles, is preferably less than 2.5. The molar ratio is preferably between 1.0 and 2.0, in particular between 1.2 and 1.8.

The enzymatic acylation reaction is preferably carried out as a batch process. If desired, it is also possible to run the reaction continuously, controlling the concentration of dissolved 6-APA in line.

The total concentration of 6-APA plus ampicillin (in dissolved and undissolved form) in the reaction mixture is preferably chosen to be above 250 mM, more preferably above 300 mM, especially above 350 mM.

The concentration of dissolved 6-APA in this preferred form during the preparation of ampicillin is kept essentially below 300 mM, preferably below 250 mM. At a higher concentration of the acylating agent 20, a higher concentration of dissolved 6-APA can optionally be chosen than at a lower concentration. Namely, at a higher concentration of the acylating agent, the reaction speed is higher, as a result of which 6-APA is only present in high concentration in dissolved form for a relatively short time.

The concentration of 6-APA in dissolved form present in the reaction mixture can be kept low in various ways. One possibility to keep the concentration of dissolved 6-APA low is to present only part of the total amount of 6-APA and dose the rest during the reaction. A drawback of this, however, is that 6-APA must then be dosed as a solid, which presents practical drawbacks. Preferably, therefore, the total amount of 6-APA in a batch process is presented at the start of the reaction, after which during the enzymatic acylation reaction the concentration of 107076 '- 10 - 6-APA in the reaction mixture will decrease and the concentration of ampicillin will increase. A suitable method to still achieve a low concentration of dissolved 6-APA is, for example, by keeping the pH at a lower value compared to the 5 pH value, whereby a maximum solubility of the reactants is achieved. A particularly suitable method of keeping the 6-APA concentration in dissolved form low is, for example, by ensuring that the concentration of the phenyl glycine derivative is kept low, for example by partially dosing the phenyl glycine derivative in the course of the reaction.

Namely, it has been found that when the phenylglycine derivative concentration is kept low, little 6-APA goes into solution, so that the concentration of 6-APA in solution can be controlled by dosing the phenylglycine derivative.

A particularly suitable embodiment is obtained when FGA is added in the form of a salt thereof, preferably the salt of FGA and a mineral acid, for example FGA.HCl, FGA.1 / 2H2SO4 and FGA.HN03. In this way it is in fact easily possible to achieve an optimum dosage of the FGA by keeping the pH constant. Preferably FGA.1 / 2H2SO4 is used because this salt has a very high solubility.

Within the scope of this invention, the various components may be present in the reaction mixture in the free form or as salts. The said pH value always means the pH value measured at room temperature.

The invention will be further elucidated by means of the examples without, however, being limited thereto.

Abbreviations; 1007076 '- 11 - AMPI = ampicillin AMPI.3H20 = ampicillin trihydrate 6-APA = 6-amino-penicillanic acid FGA = D-phenylglycine amide 5 FG = D-phenylglycine FGHM = D-p-hydroxyphenylglycine methyl ester

Assemblase ™ is an immobilized Escherichia coli penicillin acylase from E. coli ATCC 1105 as described in WO-A-97/04086. The immobilization was performed as described in EP-A-222462, using gelatin and chitosan as gelling agent and glutaraldehyde as crosslinker.

The final activity of the Escherichia coli penicillin acylase is determined by the amount of enzyme 15 added to the activated beads and amounted to 3 ASU / g dry weight with 1 ASU (Amoxicillin Synhesis Unit) defined as the amount of enzyme containing 1 g Amoxicillin per hour .3H20 generates from 6-APA and FGHM (at 20 ° C; 6.5% 6-APA and 6.5% FGHM).

20

Example I

Preparation of FGA.1 / 2H2SO4 solution.

301.6 g of FGA (2.00 mol) was suspended in 650 g of water at T = 5 ° C. 102.1 g of 96% H 2 SO 4 (1.00 mol) was added dropwise with stirring over 1 hour, the temperature being kept at T <25 ° C by cooling.

Example II

Enzymatic synthesis of ampicillin.

30 An enzyme reactor (1.5 l, diameter 11 cm), equipped with a sieve bottom with 175 μτα gauze, was filled with 300 g net-wet assemblase ™ (the term net-wet refers to the mass of the enzyme obtained after the enzyme has been separated from an enzyme slurry with a glass filter).

1 0 0 70 7 6 * - 12 -

A production reactor (1.2 L) was charged with 131.6 g of 6-APA (0.600 mol), 30.2 g of FGA (0.200 mol) and 400 ml of water (T = 10 ° C). This mixture was stirred at T = 10 ° C for 15 minutes and then transferred to the enzyme reactor at t = 0 using 100 ml of water (T - 10 ° C).

The stirrer was started in the enzyme reactor at t = 0. The temperature was always kept at 10 ° C. For 233 minutes, 423.7 g (0.800 mol) of FGA. H 2 SO 4 solution was added at a constant rate. The pH was approx.

10 6.3. From t = 295 minutes, the pH was kept at 6.3 by titration with 6N H 2 SO 4. At t = 570 minutes, the amount of AMPI was maximal and the pH was lowered to 4.7 by adding 6N H 2 SO 4. The enzyme reactor now contained:

15 575 mmol AMPI

15 mmol 6-APA 50 mmol FGA 365 mmol FG

Example III

Separate the AMPI / FG slurry from the enzyme reactor.

Using stirred filtration, the AMPI / FG slurry, prepared as described in Example II, was removed from the enzyme reactor via the sieve bottom. An angled-blade agitator was used, which was placed 0.5 cm above the sieve. Stirred at about 500 rpm. The AMPI / FG slurry separated from the reactor was filtered through a G3 glass filter. The AMPI / FG wet cake was set aside and the mother liquor was returned to the enzyme reactor, after which the stirred filtration, followed by the G3 filtration of the next AMPI / FG slurry, was resumed. In this way, the enzyme reactor was washed with the AMPI / FG mother liquor until no more solid was rinsed from the reactor. The last, at the G3 filtration, '1 i V.

Mother liquor collected was combined with the AMPI / FG wetcake into an AMPI / FG slurry.

The AMPI / FG slurry thus obtained contained> 99.0% of the total AMPI produced in the enzyme reactor and> 95.5% of the total FG produced in the enzyme reactor. It

Assemblase ™ was> 99.5% in the enzyme reactor after this stirred filtration.

Example IV

10 Ampicillin recrystallization.

The recrystallization was carried out in an arrangement (see figure 1) which consisted successively of: a storage vessel (2 1), a pump, a dissolving vessel (0.05 1), a filter fitted with a Seitz filter plate, a pump, an AMPI 15 crystallization vessel (0.5 1), two parallel-connected glass filters IA and 1B, a pump, a heat exchanger, an FG crystallization vessel (0.5 1), two parallel-connected glass filters 2A and 2B, a pump and finally a heat exchanger, which was reconnected to the dissolution vessel.

The line between filters 2A and 2B and the dissolution vessel contained a three-way valve through which part of the flow could be vented. All vessels were equipped with a stirrer, a thermometer and a pH electrode.

The AMPI / FG slurry, which was isolated as described in Example III, was quantitatively transferred to the storage vessel, and cooled to 2 ° C with stirring.

The recrystallization loop (solvent vessel up to and including heat exchanger) was filled with a total of about 1350 grams of starting solution, which consisted of an aqueous solution of 0.6% AMPI and 0.6% FG. The recrystallization run (except the FG

crystallization vessel and glass filters 2A and 2B) were cooled to 1-2 ° C. The FG crystallization vessel and the glass filters 2A and 2B were kept at 20 ° C.

The circulation in the recrystallization loop was started 1007076-14 (flow = 1.2 liters per hour). The pH in the dissolution vessel was adjusted to pH = 1.25 by titration with 8N HCl solution. The pH in the AMPI crystallization vessel was adjusted to pH = 6.5 by titration with 25 wt% NH 3 solution. In the AMPI crystallization vessel, 13.0 grams of AMPI.3H20 were placed as a seed. 10.0 grams of FG graft was placed in the FG crystallization vessel.

At t = 0, recrystallization was started by starting the dosing of the AMPI / FG slurry from the storage vessel to the dissolution vessel (flow = 0.14 liters per hour). The contents of the storage vessel were added to the dissolving vessel in about 8 hours. The levels in the dissolution vessel and the AMPI and FG crystallization vessels always remained constant. This was accomplished by not recirculating a portion of the FG mother liquor to the dissolution vessel, but by spitting it out.

The AMPI slurry from the AMPI crystallization vessel was continuously filtered on glass filter IA while the mother liquor was pumped to the FG crystallization vessel. The FG slurry from the FG crystallization vessel was filtered on glass filter 2A and the mother liquor was pumped back to the dissolution vessel. The AMPI / FG slurry from the storage vessel and the FG mother liquor were mixed in the dissolution vessel in a ratio of 1 to 8.6 throughout the test. After about 8 hours, the storage vessel was empty and a total of 330 ml of 8 N HCl solution had been dosed to the dissolution vessel.

At t = 8 hours, the storage vessel was filled with a new AMPI / FG slurry, which was again isolated as described in Example III. The AMPI separation in the recrystallization loop was switched from glass filter IA to 1B, and likewise the FG separation was switched from glass filter 2A to 2B.

The AMPI wet cake on glass filter IA was washed with 2 x 175 ml water (T = 5 ° C) and dried. The FG wet cake on glass filter 2A was washed with 2 x 60 ml water and dried.

1007076 '- 15 -

The recrystallization was continuously operated by filling the storage vessel every 8 hours with AMPI / FG slurry and the glass filters IA and 1B, and 2A and 2B, alternately switching and emptying. The mean flow of the FG 5 mother liquor purge was approximately 0.18 liters per hour.

The average yield per storage vessel fill (600 mmol 6-APA dosed to the enzyme reactor in Example II) was: 220 grams of AMPI.3H20 (excluding AMPI graft; 91% over 600 mM 6-APA) and 30 grams of FG ( excluding FG graft).

10 070 76

Claims (16)

A method for recovering a β-lactam antibiotic from a mixture containing the β-lactam antibiotic and D-phenylglycine (FG) in solution, wherein the mixture is at a temperature between -5 and 20 ° C and at a concentration such that FG remains in solution, is brought to a pH between 3 and 8, the solid β-lactam antibiotic obtained is recovered and the remaining liquid is subjected to a temperature increase to a temperature between 10 and 60 ° C at which solid FG FG is separated as a solid and the mother liquor is at least partly recycled. 2. A method according to claim 1, wherein the temperature of the mixture is between -3 and 15 ° C. The method of claim 2, wherein the temperature of the mixture is between 0 and 10 ° C. 4. A method according to any one of claims 1-3, wherein the temperature increase to a value between 12 and 50 ° C is carried out. The method of claim 4 wherein the temperature elevation is performed to a value between 15 and 40 ° C. 6. A method according to any one of claims 1-5, wherein the mixture is brought to a pH between 4 and 7. A method according to any one of claims 1-6, wherein the mother liquor is recycled to the mixture containing the β-lactam antibiotic and FG in solution. 8. A method according to any one of claims 1-7, characterized in that the method is carried out continuously. A method according to any one of claims 1-8, wherein the starting mixture mainly containing β-lactam antibiotic and FG comes from an enzymatic acylation reaction in which the corresponding β-1007076 -17-lactam core is acylated with a D-phenylglycine of ivate. 10. A method according to claim 9, wherein the mixture obtained after the acylation reaction is first subjected to a pH reduction to a pH between 0 and 3. The method of claim 10, wherein the mixture is subjected to a pH drop to a pH between 0.3 and 2. The method according to any one of claims 1-11, wherein the mixture contains 10-1500 mM β-lactam antibiotic, 0-1500 mM FG, 0-1000 mM D-phenylglycine derivative and 0-1000 mM β-lactam core. The method of any one of claims 1-12, wherein the β-lactam core is 6-aminopenicillanic acid (6-APA) and the β-lactam antibiotic is ampicillin. The method of any one of claims 1-12, wherein the β-lactam nucleus is 7-aminodesacetoxycephalosporanic acid (7-ADCA) and the β-lactam antibiotic is cephalexin. The method of any one of claims 1-12, wherein the β-lactam core is 7-amino-3-chloro-cef-3-em-4-carboxylic acid (7-ACCA) and the β-lactam antibiotic is cefaclor. 16. Method for the recovery of a β-lactam antibiotic as described and illustrated by the examples. 1007076