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Title:
PROCESS FOR THE RECOVERY OF A 'beta'-LACTAM ANTIBIOTIC
Document Type and Number:
WIPO Patent Application WO/1996/023797
Kind Code:
A1
Abstract:
Process for the recovery of a 'beta'-lactam antibiotic from a mixture containing the ammonium salt of the 'beta'-lactam antibiotic and the ammonium salt of the corresponding 'beta'-lactam core by subjecting the mixture to a physical treatment, for instance a treatment of stripping with steam or an inert gas or (steam) distillation at reduced pressure, in which ammonia is released from the ammonium salts that are present and is carried off as such and the precipitate of the 'beta'-lactam antibiotic released is recovered. The mixture for instance is the reaction mixture obtained after an enzymatic acylation reaction in which the 'beta'-lactam antibiotic is prepared from the corresponding 'beta'-lactam core and the corresponding acylation agent. Preferably, the mixture obtained after recovery of the precipitate of the 'beta'-lactam antibiotic is returned to the enzymatic acylation reaction. In such a process essentially no 'beta'-lactam core and 'beta'-lactam antibiotic are lost. Moreover, the enzyme can be re-used many times.

Inventors:
Boesten
Wilhelmus
Hubertus
Joseph
Application Number:
PCT/NL1996/000053
Publication Date:
August 08, 1996
Filing Date:
February 01, 1996
Export Citation:
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Assignee:
Chemferm V.
Boesten, Wilhelmus Hubertus Joseph
International Classes:
C07D499/00; C07D499/18; C07D501/00; C07D501/12; C07D501/22; C12P35/00; (IPC1-7): C07D501/12; C07D499/18; C12P35/04; C12P37/04
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Claims:
C L A I M S
1. Process for the recovery of a βlactam antibiotic from a mixture containing the ammonium salt of the β lactam antibiotic and the ammonium salt of the corresponding βlactam core, characterized in that the mixture is subjected to a physical treatment in which ammonia is released from the ammonium salts that are present and is carried off as such and the precipitate of the βlactam antibiotic released is recovered.
2. Process according to claim 1, characterized in that the mixture is subjected to a treatment of stripping with steam or an inert gas.
3. Process according to claim 1, characterized in that the mixture is subjected to (steam) distillation at reduced pressure.
4. Process according to any one of claims 13, characterized in that the treatment is carried out at a temperature of 030°C.
5. Process according to any one of claims 14, characterized in that the mixture is the reaction mixture obtained after an enzymatic acylation reaction in which the βlactam antibiotic is prepared from the corresponding βlactam core and the corresponding acylation agent.
6. Process according to claim 5, with a free amide being used as acylation agent, the molar ratio between the acylation agent and the βlactam core being between 0.5:1 and 2:1, the sum of the concentrations of the βlactam core and the βlactam antibiotic being between 200 and 800 mM and the mixture obtained after recovery of the precipitate of the βlactam antibiotic being returned to the enzymatic acylation reaction.
7. Process according to claim 5, with a free ester being used as acylation agent, the sum of the concentrations of the βlactam core and the βlactam antibiotic being between 200 and 800 mM and the mixture obtained after recovery of the precipitate of the βlactam antibiotic being returned to the enzymatic acylation reaction.
Description:
PROCESS FOR THE RECOVERY OF A β-LACTAM ANTIBIOTIC

The invention relates to a process for the recovery of a β-lactam antibiotic from a mixture containing the ammonium salt of the corresponding β-lactam core and the ammonium salt of the β-lactam antibiotic. In the process for preparation of a β-lactam antibiotic, involving the acylation of a corresponding β- lactam core by means of a suitable acylation agent, the recovery of the β-lactam antibiotic and the working up of the reaction mixture are difficult in general. Besides the β-lactam antibiotic the reaction mixture often still contains valuable components, such as for instance the β- lactam core. In order to get a commercially attractive process it is therefore necessary to recover in virtually pure form the β-lactam antibiotic formed, while at the same time minimizing the losses of β-lactam core and β- lactam antibiotic.

The object of the invention therefore is to provide a process which enables recovery of the β-lactam antibiotic in sufficiently pure form and working up or recirculating the remaining mixture simply and without significant losses of β-lactam core and β-lactam antibiotic. This is achieved according to the invention by subjecting the mixture to a physical treatment in which ammonia is released from the ammonium salts that are present and is carried off as such and the precipitate of the β-lactam antibiotic released is recovered. The fact is that it has been found that in the process according to the invention the free β-lactam

antibiotic is precipitated selectively, while the β-lactam core remains behind in solution. In this way it appeared to be possible to recover the β-lactam antibiotic with a purity greater than 90% on a dry weight basis, in particular greater than 98%.

The term 'physical treatment' in the framework of the present invention is opposed to chemical treatment, for instance the chemical reactions in which inorganic ammonium salts are formed by means of a strong acid. Examples of suitable physical treatments to which the mixture can be subjected are stripping with steam or an inert gas such as described for instance in Perry's Chemical Engineers' Handbook, R.H. Perry, D. Green, 2nd edition (1985), (steam) distillation at reduced pressure, in particular thin-film evaporation, such as described for instance by Mutzenberg et al. in Chem. Eng. 12_, 175-190 (1965), evaporation in a spray tower, such as described for instance by Mehta and Shorma in Br. Chem. Eng. 15 1440, 1465 (1970), gas membrane separation, such as described for instance by D.J. Brose and P. v. Eikeren in Appl. Biochem. and Biotech., 24/25 (1990), 457-468, and electrodialysis, such as described for instance in the above-mentioned reference work by R.H. Perry et al. The process according to the invention is suitable for application in the preparation of known β- lactam antibiotics, for instance cephalexin, amoxicillin, ampicillin, cephaclor, cephadrin, cephadroxyl and cephotaxim.

The treatment is mostly carried out at a temperature between -5 and 40°C, preferably between 0 and 40°C, more preferably between 0 en 30°C particularly between 5 and 30°C.

The pH of the mixture to be treated depends on the nature and the concentration of the individual components. In view of the stability of the β-lactam core and the β-lactam antibiotic, the treatment is mostly carried out at a pH lower than 8.5, in particular at a pH

between 5.5 and 8.5, preferably at a pH between 6 and 8. During the treatment ammonia is removed, so that the pH is lowered. By adjusting the intensity and the duration of the treatment, the pH resulting at the end of the treatment can be varied. The optimum final pH depends on the composition of the mixture and is chosen such that an optimum separation of β-lactam core and β-lactam antibiotic is obtained. The optimum final pH in practice is a compromise between on the one hand high purity of the recovered antibiotic, which is achieved if the pH is lowered relatively little, so that the β-lactam antibiotic is still partially and the β-lactam core still fully in solution, and on the other hand a high yield, which is achieved if the pH is lowered so far that the β-lactam antibiotic is precipitated virtually completely, but at the same time part of the β-lactam core is precipitated. For the person skilled in the art it is easy to determine the optimum pH in a given situation.

The process according to the invention is in particular suitable for application to the reaction mixture obtained after an enzymatic acylation reaction in which a β-lactam antibiotic is prepared by enzymatic acylation of the corresponding β-lactam core with a suitable acylation agent. Suitable examples of β-lactam cores that can be used in the process according to the invention are penicillic acid derivatives, for instance 6- aminopenicillic acid (6-APA), and cephalosporanic acid derivatives, for instance 7-aminocephalosporanic acid (7- ACA), 7-aminodesacetoxy-cephalosporanic acid (7-ADCA) and 7-amino-3-chlorocephalosporanic (7-ACCA) .

Suitable acylation agents that can be used in the process according to the invention are for instance α- amino acid derivatives, in particular amides and esters of phenyl glycine, p-hydroxyphenyl glycine and dihydrophenyl glycine.

In principle any enzyme can be used that is

suitable as catalyst in the coupling reaction. Such enzymes are for instance the enzymes that are known under the general designations 'penicillin amidase' and 'penicillin acylase'. Examples of suitable enzymes are enzymes derived from Acetobacter, Aeromonas. Alcaliαenes, Aphanocladium, Bacillus SP. , Cephalosporium. Escherichia, Flavobacterium, Kluvvera, Mvcoplana, Protaminobacter, Pseudomonas and Xanthomonas, in particular Acetobacter pasteurianum, Acaliαenes faecalis, Bacillus meσaterium, Escherichia coli and Xanthomonas citrii.

Preferably an immobilized enzyme is used, since the enzyme can be easily re-used then. Immobilized enzymes are known as such and are commercially available. Highly suitable enzymes have appeared to be the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the name 'Enzygel®', the immobilized Penicillin-G acylase from Recordati, the immobilized Penicilline-G acylase from Pharma Biotechnology Hannover, and an Escherichia coli penicilline acylase isolated as described in WO-A-92/12782 and immobilized as described in EP-A-222462.

The enzymatic acylation reaction is mostly carried out at a temperature between -5 and 35°C, particularly between 5 and 35°C, preferably between 0 and 28°C, particularly between 20 and 28°C.

The pH at which the acylation reaction is carried out is mostly between 6 and 8.5. The optimum pH depends on, among other things, the antibiotic, since the stability and the solubility of the β-lactam antibiotic as well as the β-lactam core depend on the pH. If a phenyl glycine derivative is used as acylation agent the pH is preferably between 6.2 and 8.5, particularly between 7 and 8; if a p-hydroxyphenyl glycine derivative is used as acylation agent the pH is preferably between 6 and 7.5, particularly bewteen 6 and 7. Besides, the enzyme activity is also pH-related.

In practice the acylation reaction is mostly

carried out in water. Optionally, the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol.%. Examples of organic solvents that can be used are alcohols with 1-7 carbon atoms, for instance a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol or a triol, in particular glycerol.

In the known enzymatic acylation processes in which the β-lactam core is acylated by means of amide as acylation agent, a mixture of the ammonium salts of the β- lactam antibiotic and the β-lactam core is obtained. In these acylation processes the pH is generally kept at a constant level by titration with a strong acid. But this results in the formation of inorganic salts which hamper the working up of the reaction mixture and which moreover, it has appeared to the applicant, affect the stability of the enzyme, so that the relatively costly enzyme can be re-used less often.

The process according to the invention is preferably applied to a mixture that has been obtained by enzymatic acylation of a β-lactam core by means of an amide as acylation agent, the concentration of inorganic salts in the reaction mixture being lower than 1000:n mM, n representing the valency of the anion. In such a process preferably a free amide is used as acylation agent and the molar ratio between the acylation agent and the β-lactam core is chosen between 0.5:1 and 2:1. Such a process can be carried out at relatively high concentrations; the sum of the concentrations of the β-lactam antibiotic and the β-lactam core is for instance between 200 and 800 mM, preferably between 300 and 700 mM, particularly 300 and 600 mM. It has appeared that under such conditions the pH remains sufficiently low during the acylation reaction, so that the pH need not be controlled through titration during the acylation reaction. This preferred process offers the advantage that during the entire process essentially no inorganic salts are formed, so that the

reaction mixture obtained after the acylation reaction can as such be returned to the acylation reaction after the physical treatment, in which ammonia is released and carried off, and after separation of the precipitated β- lactam antibiotic. In such a process therefore essentially no β-lactam core and β-lactam antibiotic are lost.

In the known enzymatic acylation processes in which the β-lactam core is acylated by means of an ester, the pH is generally kept at a constant level by titration with ammonia, so that also a mixture of the ammonium salts of the β-lactam antibiotic and the β-lactam core is obtained. In such a process a free ester is preferably used as acylation agent. Such a process can be carried out at relatively high concentrations; the sum of the concentrations of the β-lactam antibiotic and the β-lactam core is for instance between 200 and 800 mM, preferably between 300 and 700 mM, particularly between 300 and 600 mM. This preferred process offers the advantage that during the entire process essentially no inorganic salts are formed, so that the reaction mixture obtained after the acylation reaction can as such be returned to the acylation reaction after the physical treatment, in which ammonia is released and carried off, and after separation of the precipitated β-lactam antibiotic. In such a process as well, essentially no β-lactam core and β-lactam antibiotic are lost.

In addition it has been found that in such a process, in which the β-lactam antibiotic is recovered from the reaction mixture obtained after the enzymatic acylation reaction as described above, the enzyme can be re-used many times without much loss of activity.

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

Abbreviations: CEX = cephalexin

CEX.H 2 0 = cephalexin monohydrate 7-ADCA = 7-aminodesacetoxy-cephalosporanic acid D-PGA = D-phenyl glycine amide D-PG = D-phenyl glycine

Example I

At 21°C 6 ml of 25 mass% ammonia (78 mmol) were added to a mixture of 50 mmol (10.7 g) of 7-ADCA, 50 mmol (18.2 g) of CEX.H 2 0 and 25 mmol of D-PGA in 215 ml of water. The pH of the clear solution was 7.92 at 21°C.

Successively, at a pressure of 1.6 kPa (12 mm Hg), 125 ml were distilled off, 125 ml of water were added, 130 ml were distilled off and 130 ml of water were added. The pH of the resulting suspension was 7.55 at 21°C. The CEX was filtered off, rewashed with 2x10 ml of methanol and 2x10 ml of acetone and dried at 20 mm Hg/20°C.

Yield: 7.8 g of CEX.H 2 0 in solid form. The content of CEX, calculated on a water-free basis, was 99.2 mass% (HPLC).

Example II

The starting mixture used was a mixture obtained after the enzymatic acylation reaction of 7-ADCA and D-PGA containing 50 mmol (10.7 g) of 7-ADCA, 50 mmol (18.2 g) of CEX.HjO a-nd 25 mmol of D-PGA (3.8 g), 26 mmol of D-PG (3.9 g), 220 ml of water and ammonia (77 mmol), mainly present as ammonium salt of CEX.H 2 0, 7-ADCA and D-PG. The pH of the clear solution at 21°C was 7.90. Successively, at a pressure of 1.6 kPa (12 mm Hg), 140 ml were distilled off and 140 ml of water were added. The pH of the resulting suspension at 21°C was 7.38. The CEX was filtered off, rewashed with 2x10 ml of methanol and 2x10 ml of acetone and dried at 2.7 kPa (20 mm Hg)/20°C. Yield: 8.7 g of CEX.H 2 0 in solid form. HPLC analysis: CEX 92.8 mass%

7-ADCA 0.08 mass% D-PGA 0.19 mass% and

D-PG 0.1 mass%.

Water content: 6.0 mass% (Karl Fischer titration). The content of CEX, calculated on a water-free basis, was 98.7 mass%.

Example III

218.6 g (purity: 99%) of CEX.H 2 0, 128.7 g (purity: 98.3%) of 7-ADCA, 63.8 25 wt.% ΝH 4 OH and 2640 g of demineralized water - a total of 3051 g - were added to a vacuum crystallizer. The pH was 8.06. The temperature was 20.5°C. A clear liguid was obtained. The concentrations of CEX.H 2 0 and 7-ADCA were 195 and 191 mM, respectively. Evaporation was started by evacuation to 2.4 kPa (24 mbar) (approx. 28°C). By suppletion of demineralized water the level was kept constant. The temperature of the heating element was set to 45°C. In this way 3056 g of condensate was collected in 65 minutes (average rate of evaporation: 2820 g/h). In this situation the superficial vapour velocity was approx. 2 m/s. During the evaporation crystallization occurred.

When the evaporation had been stopped the crystallization of the slurry was continued for approx. 1 hour.

The final pH was 7.42 (T = 20.5°C). The slurry was filtered through a glass filter, without any problem. The weight of the wet cake was 197.1 g.

After washing with 3x50 ml of acetone and drying for about 16 hours at 30°C and a relative humidity of 30%, 110.1 g of CEX.H 2 0 was obtained. This substance consisted of needle-shaped crystals with an average length of 150 μ and an average diameter of 10-15 μm.

The content of CEX, calculated on a water-free basis, amounted to 98.4 mass%.

Example IV 218.6 g (purity: 99%) of CEX.H 2 0, 130.8 g

(purity: 98.3%) of 7-ADCA, 64.9 g of 25 wt.% agueous NH 4 OH and 2641 g of demineralized water - a total of 3055 g -

were added to a vacuum crystallizer. The pH was 8.06. The temperature was 20°C. A clear liguid was obtained. The concentrations of CEX.H 2 0 and 7-ADCA were 194 and 194 mM, respectively. Evaporation was started by evacuation to 2 .2 kPa ( 22 mbar) (approx. 25°C). The temperature of the heating element was set to 45°C. In this way 1602 g of condensate was collected in 34 minutes (average rate of evaporation: 2830 g/h). In this situation the superficial vapour velocity was approx. 2 m/s. During the evaporation crystallization occurred.

Subseguently, 1500 g of demineralized water was supplied and the evaporation was continued. In 21 minutes 1437 g of condensate was collected (average rate of evaporation: 4106 g/h, vapour velocity approx. 3 m/s). The final pH was 7.19 (T = 23.4°C). The slurry was filtered through a glass filter, without any problem. The weight of the wet cake was 197.9 g.

After washing with 1x100 ml of demineralized water, 1x100 ml of 80 vol.% acetone and drying for about 16 hours at 30°C and a relative humidity of 30%, 110.5 g of CEX.H 2 0 was obtained. This substance consisted of needle-shaped crystals with an average length of 150 μm and an average diameter of 10-15 μm.

The content of CEX, calculated on a water-free basis, amounted to 98.1 mass%.