MXPA98010768A - Improved process for the fermentative production of cephalosporin - Google Patents

Abstract

The present invention relates toa novel process for the preparation of cephalosporins having been deacylated at the 7-amino group, by fermentation of a cephalosporin producing microorganism in the presence of a side chain precursor, extraction of the N-substituted cephalosporin compound as present in the fermentation broth or fluid to an organic solvent, back extraction of the N-substituted cephalosporin compound to water, treatment of the aqueous phase with a dicarboxylate acylase and isolation of the crystalline cephalosporin compound according to formula (1) from the conversion solution, characterized in that the fermentation broth or fluid is incubated at acidic conditions and an elevated temperature prior to extraction of the N-substituted cephalosporin compound to an organic solvent. Further improvements of the process are obtained by washing the first organic solvent extract with acidified water and/or by extraction of the side chain to an organic solvent and/or by treating an aqueous cephalosporin solution produced at one or more stages in the process of the invention with carbon dioxide.

Description

IMPROVED PROCESS FOR THE FERMENTAL PRODUCTION OF CEPHALOSPORINE FIELD AND BACKGROUND OF THE INVENTION The present invention relates to recovery of the fermentatively produced cephalosporin compounds. The semi-int routes to prepare cefporporins start mainly from fermentation products such as penicillin.

G, penicillin V and cephalosporin C, which are converted to the corresponding β-lactam nuclei, for example in a manner as described in K. Matsumoto, Bioprocess. Techn., 16, (1993), 67-88, J.G. Shewale & Siv raman, Process Bicchemistry, August 1989, 146-154, T.A. Savidge, Biotechnology of Industrial Antibiotics (Ed. E.J.

Vandamme) Marcel Dekker, New York, 1984, or J. G.

Shewale et. ai., Process Biochemistry International, June 1990, 97-103. The obtained ß-lactam nuclei are subsequently converted to the desired antibiotic by coupling to a suitable side chain, as has been described among others in European Patent EP 0 339 751, in REF: 28991 Japanese Patent JP-A-53! and in the Chinese Patent n-f * - or • 0. By performing the different combinations of side chains and -b-lactam nuclei, a variety of penicillin and cephalosporin antibiotics can be obtained. The 7-amino-deacetoxycephalosporanic acid (7-ADCA) and 7-inocephalosporanic acid (7-ACA) are known to be the most important intermediates for the production of antibiotics used in the pharmaceutical industry. 7-ADCA is, for example, obtained by the chemical or enzymatic cleavage (deacylation) of phenylacetyl-7-ADCA, producing 7-ADCA and acid phenylacetic. Phenylacetil til-7-ADCA is usually produced by clinical treatment of penicillin G sulfoxide, which is formed from penicillin G. In this production process large amounts of chemicals are required to ensure that the desired reaction has place. This is clear and places a heavy burden on waste management. In addition, the total throughput of the process is not as high as could be desired. To overcome some of the disadvantages ? > of the chemical process, a fermentative process has been described for the production of 7-ADCA and 7-ACA, involving the fermentative production of ß-lactam, N-their ituides, such as adi-il-7-ADCA or adipil-7 ACA, by means of a strain of recombinant Peni cl ll um chrysogen um, capable of expressing an acid synthase of pharosporic toxin toxice (DAOCS) also known as "expandase" from a transgene (European Patent Nos. 0 532, 341, 0 540 210, Inter-national Patent Applications No. WO 93/08287, WO 95/04148, WO 95/04149). The expandase is responsible for the expansion of the 5-membered ring of certain N-acylated penicilic acid acids, thereby producing N-acylated sporical deacetylated acids, known processes for the recovery of penicillanic and cephalosporinic acids. chemically or enzymatically produced, they are not effective for the recovery of the N-substituted β-lactam intermediates and deacylated amino-β-lactams. The main problem with the recovery of the fermentatively produced cephalosporin N-substituted compounds, mentioned hereinabove, is the complexity of the culture broth or filtrate. The broth usually comprises various penicillanic acids, such as α-hydroxyadipyl-6-penicillanic acid, α-hydroxyadipyl-6-penicillanic acid, 6-ami openc 11 ani co (6-APA) acid, various cephalosporinic acids including -aminoadipyl - and α-hydroxyadipyl-7-ADCA and too much proteinaceous material. The known recovery processes do give an acceptable quality of the cephalosporanic acid product in terms of purity. In. Enzymatic deacylation this leads to problems in terms of the reduced half-life of medicine, slower bioconversion rate and more expenses in recovery after bioconversion and / or levels of unacceptable contaminants. further, after deacylation, such impurities prevent or at least prevent the recovery of the desired deacylated cephalosporin compound, with the desired specifications. Therefore, in the known processes, penicillins and cephalosporins do not give an acceptable quality of the final product: the final product, for example, 7-ADCA or 7-ACA, contains an unacceptable amount of penicillin components as impurities.

DESCRIPTION OF THE INVENTION The present invention relates to an improved process for the production of deacylated β-lactam, for example 7-ADCA or 7-ACA, from a fermentation broth of a cephalosporin-producing microorganism. In particular, the present invention describes an improved process for the preparation of cephalosporins which have been deacylated in the group 7 -ami or and which has a general formula (I) (i) e do of: R is hydrogen or alkoxy of 1 to 3 atoms of CSGDO? O } And it is CH;, oxygen, sulfur, or an oxidized form of sulfur; and R; is any of the selected groups of hydrogen, hydroxyl, halogen, alkoxy of 1 to 3 carbon atoms, alkyl of 1 to 5 carbon atoms straight or branched, saturated or unsaturated, optionally substituted, or optionally containing one or more heteroatoms, preferably methyl, optionally substituted C 5 -C 8 cycloalkyl, optionally containing one or more heteroatoms, optionally substituted aryl or heteroaryl, or optionally substituted benzyl, and monitoring of a cephalosporin producing microorganism in the presence of a side chain precursor , the extraction of the N-substituted cephalosporin compound as present in the broth or in the fermentation fluid, to an organic solvent, the retro-extraction of the N-substituted cephalosporin compound to the water, the treatment of the aqueous phase with an dicarboxy lato-acylase and the isolation of the crystalline cephalosporin compound according to formula I from of the aqueous phase, characterized in that the fermentation broth or fluid or the back-extract is incubated under acidic conditions and at an elevated temperature before the extraction of the broth or the fermentation fluid with an organic solvent or before further processing of the back-off. abstract. Additional improvements of the process are obtained by washing the first extract with organic solvent containing the N-substituted cephalospic kidney compound, with acidified water and / or by trimming the aqueous cephalosporin solutions produced in one or more stages in the process of the invention, with carbon dioxide or by extraction of the enzymatically released side chain towards an organic solvent, prior to the crystallization of the deacylated cephalosporin. The process according to the invention will give a better total yield and a better product quality than the currently known processes. In the novel processes of recovery, gone; Note: omitted cecid deacylated from its N-acylated counterpart, for example, 7-ADCA of adipi 1-7 -ADCA or 7-ACA from adi? Il-7-ACA, are described in more detail broth is obtained from any suitable fermentation process, for example, from a fermentation using a Peni ci um chrys ogen um strain in the presence of an appropriate side chain precursor, as mentioned above in the I presented. The biomass is separated from the fermentation broth using any suitable technology, such as centrifugation or filtration, producing a fermentation fluid containing cephalosporin. Preferably, a filtration step is applied to obtain said separation. The residual solids are optionally washed. One of the obstacles to the production of N-substituted cephalosporanic acid is the presence of the unwanted contaminant ß-lactam components, especially 6-aminopencilnic acid (6-APA), 6-APA N-substituted or α-aminoadipyl-7-ADCA. In a preferred embodiment of the invention, the contaminants are substantially reduced by incubating an aqueous solution containing the N-substituted cephalosporin compound produced at any stage in the process of the invention, under acidic conditions and at an elevated temperature. The aqueous solution containing N-substituted cephalosphorin compound is acidified to a pH that is less than 4, preferably less than 3, using one or more known acids, for example sulfuric acid, hydrochloric acid or nitric acid or a combination thereof. The operating temperature is in the range of 20 to 140 ° C, preferably 60 to 80 ° C. The residence time to these conditions is in the range of several days to several minutes, preferably less than 60 minutes, more preferably 1 to 30 minutes. The above incubation step according to the invention can be applied to the fermentation broth or fluid or to the aqueous back-extract containing the N-substituted cephalosporin. Preferably, the incubation step is applied to the fermentation broth or fluid. The incubation step can also be carried out either before or after the separation of the biomass. Preferably, the incubation is carried out before filtration, to have an advantage in the filtration. The N-substituted cephalosporin compound is separated from the aqueous phase, for example, the fermentation broth or fluid, by acidification of the broth to fermentation fluid and the subsequent extraction of the N-substituted cephalosporin compound into an organic solvent. . Acidification typically occurs at a pH of less than 4, preferably less than 3, and only in the case where the fermentation broth or fluid is no longer subject to the aforementioned incubation under acidic conditions and at an elevated temperature. A 1 ^? Suitable indicator can be added to the broth or fermentation fluid to significantly improve extraction. Preferably, the organic solvent is selected from the group of amyl acetate, butyl acetate, ethyl acetate, met il-isobutyl-t-tone, cyclohexaxione, is? -butanol or n-but-nol. Extraction with an organic solvent as described above has no satisfactory selectivity towards the unwanted β-lactam products such as α-aminoadipyl-7-ADCA and 6-APA. Therefore, in a preferred embodiment of the invention, a washing process is carried out for the specific elimination of these compounds. The washing process is characterized by the mixing of the organic solvent extract with a small amount of acidified water, followed by phase separation. Acidified water typically has a pH that is less than 4, preferably less than 3, more preferably less than 2. In addition, the proportion of the phases is typically between 1: 1 to 1:20 of water: solvent, preferably 1: 2 water: solvent. The N-substituted cephalosporin compound is back-extracted into water in conventional manners by extracting the organic phase with an alkaline solution, to produce an aqueous back-extract, with a pH in the range of 6 to 9. Typically, a phase ratio of 1:10 (water / solid sol) is applied. The alkaline solution is an aqueous solution that contains a toxic mineral base, such as sodium hydroxide or ammonia. The extraction, washing and back-extraction are preferably carried out in a series of continuous intensive contact extractors, for example a combination of an intensive mixer, for example a high-shear mixer, with a centrifugal separation, preferably 2 to 3, more preferably 3 to 6, and more preferably 4 to After phase separation, the aqueous phase is optionally purified to remove the Subsequently, the aqueous solution is contacted with a suitable dicarboxylate acylase enzyme, to deacylate the N-substituted cephalosporin compounds. For example, to form 7-ADCA or 7-ACA from the corresponding N-adipyl derivatives. The organisms that have been found to produce dicarboxylate acylase are species of Al cal i gen es, Ar th r cb a c t e r, Achromobacter, Aspergillus, Acinetobacter, Bacillus and Pseudomonas More specifically, the following species produce highly suitable dicarb-xylate-acyiasa: Achromobacter xylosocxidans, Arthrobacter viscosis, AL throbact CA128, Baciilus CA78, Bacillus magaterium ATCC53667, Bacillus cereus, Bacillus iatercsporus Jl, Paecilomyces C2106, Pseudomonas diminutive sp N176, Pseudomonas diminuta sp V22, Pseudomonas paucimobilis Pseudomonas diminuta BL072 Pseudomonas strain C427, Pseudomonas sp SE83, Pseudomonas sp 3? 49, Pseudomonas ovalis ATCC950, Comamonas sp SY77, Pseudomonas GK 16, Pseudomonas SY- 7-l, Pseudomonas sp Al 4, Pseudomonas vesiculari s B955, Pseudconas syringae, Ps putida ATCC17390, Ps aeroginosa NCTC 10701, Proteus vulgaris ATCC9634, Ps fragi DSM3881, and B. subtilus IFO3025. The dicarboxylate acylase can be obtained from the microorganism by which it is produced in any suitable manner, for example as described for the SE83 strain of Pseudomonas sp in US Patent No. 4,774,179. Also, genes for example for the dicarboxylate acylases of SE83 or SY77, can be expressed in a different suitable host, such as E. coli as shown in FIG. reported by Matsuda et al., in J. Bacteriology, 169, (1987), 5818-5820, for strain SE83, and in US Patent No. 5,457,032 for strain SY77. Enzymes isolated from previous sox sources are often referred to as glutaryl acylases. However, the specificity of the side chain of the enzymes is not limited to the glutaryl side chain, but also comprises smaller and larger dicarboxylate side chains. Some of the dicarboxylate acylases also express gamma-glutami 1-transpept idase activity and are therefore sometimes classified as gamma-glutamyl transpeptidase. The dicarboxy lato-acylase can be used as the free enzyme, but in any immobilized, appropriate form, for example as described in European Patent EP 0 222 462. In one embodiment of the invention, the deacylated cephalosporin compound, by Example 7-ADCA or 7-ACA is isolated from the conversion solution by crystallization under acidic conditions. Typically, the crystallization of a deacylated cephalosporin compound from a solution is performed by measuring the pH of the aqueous solution to an acidic value by adding a titrant to the aqueous solution until the pH has changed. reached a value within a range of 2.5 to 4.5, preferably a value of 3 to 4. In a preferred embodiment of the invention, the crystallization of a deacylated cephalosporin compound from an aqueous solution is carried out by the addition from the aqueous solution to a crystallization vessel which is maintained at a fixed pH having a value within a range of 2.5-4.5, using a suitable titrant. In a still even more preferred embodiment of the invention, the crystallization is carried out by a gradual adjustment of the pH of the aqueous solution to a final value within one hour. by adi; aqueous solution to a series of interconnected crystallization vessels, for example, the addition of the aqueous solution to a first container, simultaneously adding the contents of the first container to a second container, simultaneously adding the contents of the second container to a third container , etc., where the pH range is applied in the interconnected vessels using a suitable titrator, starting at a pH in the first vessel which deviates approximately 0.5-2 pH units from the pH of the aqueous solution containing the deacylated cephalosporin, and ending at a pH in the final vessel which has a value within a interval of 2.5-4.5. Conveniently, the pH of the aqueous solution containing the deacylated cephalosporin is adjusted to the desired final value using a series of 2-6 interconnected vessels. For example, to obtain the crystallization of a deacylated cephalosporin from the conversion solution, a pH range that decreases from 8 to 3 can be applied using a titrant which is an acid, such as sulfuric acid, hydrochloric acid and / or nitric acid, applying a series of 3-4 interconnected containers. The two preferred embodiments described above are preferably performed in a continuous mode. In a further preferred embodiment of the invention, the side chain, colorful products and traces of the unconverted compound are removed from the conversion solution prior to crystallization by following the steps as set forth hereinafter. The conversion solution is acidified and contacted with an organic solvent, for example, amyl acetate, vinyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, isobutanol or n-butanol, to remove the chain lateral before crystallization. The acidification is carried out with acid, such as sulfuric acid, hydrochloric acid or nitric acid or a combination thereof, preferably sulfuric acid, at a pH of less than 3, preferably less than 2. Unexpectedly, a high efficiency of elimination of the colored impurities besides that of the side chain. According to another preferred embodiment of the invention, the contamination of the penicillin components, for example amphoteric 6-APA, as is present in aqueous solutions containing cephalosporin, produced in one or more stages of the process of the invention, such as the fermentation broth or fluid, the retro-extract, the conversion solution, or the solution containing the deacylated cephalosporin dissolved according to formula (I), are markedly reduced by contacting the contaminated fluid with penicillin, typically at a pH from 5 to 7, with carbon dioxide. The carbon dioxide can be added to the solution in any suitable manner, such as in a solid or gaseous form or as a carbonate ion solution. The aqueous solution containing cephalosporin is contacted with the C02 source, at a temperature of 10 to 60 ° C, preferably 20 to 40 ° C, where the solution is saturated with molecular CO2 for 4 to 10 hours. After the reduction of the penicillin components, the purification of the cephalosporin compounds according to the formula (I) can be obtained. After extraction of the side chain to an organic solvent, the deacylated cephalosporin compound can be crystallized from the aqueous phase of several persons, such as the forms that are indicated hereinabove for the crystallization of a cephalosporin compounds. deacylated, from an aqueous solution. In a preferred embodiment, the pH of the aqueous phase is increased to a pH having a value within the range of 2.5-5, preferably within a range of 3.5-4.5, by the addition of the solution containing the compound of deacylated cephalosporin in a passage to a crystallization vessel maintained at a desired pH value or to a series of 2-6 interconnected crystallization vessels applying an increasing pH range. These processes can be conveniently carried out in a continuous mode. The crystals are isolated by filtration or centrifugation and dried in a conventional continuous or batch dryer. All the steps mentioned above, for example, extraction, washing, back-extraction and crystallization, can be carried out in a continuous or batch mode, but due to stability reasons, the preferred method is a continuous mode. The following example is for exemplification only and should not be considered as a limitation of any kind.EXAMPLE A 1 1 sample of adipil-7-ADCA broth was filtered to remove the biomass. The mycelium was washed with tap water to obtain a final volume of the filtrate of approximately 2 1. Approximately 2 1 of the filtrate was acidified at 40 ° C with 250 ml of 6N H2SO4, at pH 1.5. N-butanol was added to 2/3 of the volume of the acidified filtrate and after vigorous mixing, it was separated. The aqueous phase was subjected to 2 or more of these t treatments with n-butanol. Subsequently, the combined organic phases were washed with 0.25 1 portions of acidified water having a pH of 2. The resulting organic phase was back-extracted with 245 ml of 2N sodium hydroxide solution at 20 ° C and after separation of the phase the traces of n-butanol in the aqueous phase were removed by vacuum purification. 135 g of the aqueous phase were diluted with demineralised water to a total of 650 ml at 30 ° C and mixed with 4N sodium hydroxide until a pH of 8.5 was reached. 50 g of the immobilized deacylation enzyme were added, and after 2 h at 30 ° C, pH 8.5, under addition of 13.5 ml of 4N sodium hydroxide, the aqueous phase was collected. The filtrate was extracted with 3 portions of 125 ml of n-butanol saturated with water at a pH of 0.4. During the extraction a total of 50.6 ml of 37% hydrochloric acid was added. The remaining aqueous phase was neutralized with 56.5 ml of 8N sodium hydroxide and the product was crystallized from the aqueous phase, to free it from the droplets of n-butanol, by lowering the pH to 5.3 with 6N sulfuric acid. After 5 minutes the pH was further decreased to the final value of 3.5. In total 15 ml of the acid was used. The suspension was filtered and the crystalline cake was washed with 50 ml of water, the cake was dried and 4.1 g of 96% pure 7-ADCA were obtained.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the one that is clear from the present description of the ixivexició. Having described the invention as above, property is claimed as contained in the following:

Claims (11)

1. The process for the preparation of a cephalosporin according to the formula (I) (I) which has been deacylated in the 7-amino group, by fermentation of a cephalosporin producing microorganism in the presence of a side chain precursor, extraction of the N-substituted cephalosporin compound as present in the broth or in the fermentation fluid, towards an organic solvent, the retro-extraction of the N-substituted cephalosporin compound into water, the treatment of the aqueous phase with a dicarboxylate acylase and the isolation of the cephalosporin compound according to formula (I) from the conversion solution obtained in this way, by crystallization, characterized in that the fermentation broth or fluid or the back-extract is incubated under acidic conditions and at an elevated temperature before the extraction of the broth or the fermentation fluid with an organic solvent or before further processing of the retro-extract.

2. The process according to claim 1, characterized in that the fermentation broth or fluid or the challenge-extract is incubated at a pH of less than 4, preferably less than 3.

3. The process according to claim 1 or 2, characterized in that the fermentation broth or fluid or the back-extract is incubated at a temperature within a range of 20 to 140 ° C, preferably 60 to 80 ° C.

4. The process according to claim 2 or 3, characterized in that the incubation time under acidic conditions and at an elevated temperature is less than 60 minutes.

5. The process according to any of claims 1 to 4, further characterized in that the organic solvent extract containing the N-substituted cephalosporin compound is washed with acidified water having a pH that is less than 4, preferably lower. of 3, more preferably less than 2, before retro-extraction.

6. The process according to any of claims 1 to 5, further characterized in that the side chain as present in the conversion solution is extracted into an organic solvent prior to crystallization of the cephalosporin compound according to formula (I) .

7. The process according to any one of claims 1 to 6, further characterized in that an aqueous solution containing cephalosporin in one or more stages such as the fermentation broth or fluid, the retro-extract, the conversion solution, or the solution containing the dissolved cephalosporin compound according to formula (I), is contacted with carbon dioxide.

8. The process according to claim 7, characterized in that the carbon dioxide is in the solid or gaseous form, or as a solution of carbonate ions. •

9. The process according to any of claims 1 to 8, characterized in that the organic solvent is amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, isobutanol or n- butanol.

10. The process according to any of claims 1-9, characterized in that the crystallization of the ketalosporin compound according to the formula (I) from the conversion solution or the aqueous phase is carried out by the addition of the aqueous solution. or from the aqueous phase to a crystallization vessel which is maintained at a fixed pH having a value within a range of 2.5-4.5, using a suitable titrant.

11. The process according to any of claims 1 to 9, characterized in that the crystallization of the cephalosporin compound according to the formula (I) from the conversion solution or the aqueous phase is carried out by the addition of the. aqueous solution or from the aqueous phase to a series of interconnected crystallization vessels while applying a pH range using a suitable titrant, starting at a pH in the first vessel and which deviates approximately 0.5-2 pH units from the pH of the 6-APA solution and ending at a pH in the final container which has a value within a range of 2.5-4.5.