HK1036794A - Novel process for the fermentative production of cephalosporin - Google Patents

Description

Novel method for producing cephalosporin by fermentation

The present invention relates to a process for the preparation of cephalosporins and cephalosporin derivatives. More particularly, the invention relates to the recovery of cephalosporins and their derivatives from complex mixtures of cephalosporins and other beta-lactam compounds. The invention also relates to the recovery of deacylated cephalosporins from a mixture of beta-lactam compounds and side chains, such as those obtainable by enzymatic side chain removal.

The semisynthetic routes for the preparation of cephalosporins mostly start with fermentation products (e.g. penicillin G, penicillin V and cephalosporin C) which are converted into the corresponding β -lactam rings, for example, according to the methods disclosed in the following documents: matsumoto, bioprocessing technology (bioprocess. technology.), 16 (1993), 67-88, J.G.Shewale & H.Sivaraman, Biochemical Advance (Process Biochemistry), 8.1989, 146-. The resulting betｃA-lactam ring is then converted into the desired antibiotic by coupling to ｃA suitable side chain, as described in particular in EP 0339751, JP-A-53005185 and CH-A-640240. By combining the side chains and the beta-lactam ring differently, various penicillin and cephalosporin antibiotics can be obtained.

7-aminodesacetoxycephalosporanic acid (7-ADCA) and 7-aminocephalosporanic acid (7-ACA) are known to be the most important intermediates for the production of antibiotics used in the pharmaceutical industry.

7-ADCA is obtained, for example, by chemical or enzymatic cleavage (deacylation) of phenylacetyledenoxycephalosporanic acid (to give 7-aminodesacetoxycephalosporanic acid and phenylacetic acid).

Phenylacetyledenoacetoxycephalosporanic acid is generally produced by chemical treatment of penicillin G sulfoxide, which is produced from penicillin G. In this production process, a large amount of chemical reagents are required to ensure the desired reaction. This is both expensive and constitutes a heavy burden on waste disposal. Furthermore, the overall yield of the process is not very high.

To overcome some of the disadvantages of the chemical processes, fermentation processes have been disclosed for the production of 7-ADCA, 7-aminodeacetylcephalosporanic acid (7-ADAC) and 7-ACA involving: n-substituted beta-lactams (e.g., adipoyl-7-ADCA, adipoyl-7-ADAC, or adipoyl-7-ACA) are produced by fermentation from a recombinant Penicillium chrysogenum strain which transgenically expresses a desacetoxycephalosporanic acid synthetase (DAOCS, also known as "expandase") (EP 0532341, EP 0540210, WO 93/08287, WO 95/04148). The expandase is responsible for the 5-membered ring expansion of certain N-acylated penicillanic acids, thus producing the corresponding N-acylated desacetoxycephalosporanic acid.

In order to produce the economically most important non-acylated cephalosporins (e.g.7-ADCA, 7-ADAC and 7-ACA), the acyl group is removed enzymatically using a suitable acyltransferase.

The known methods for recovering chemically or enzymatically produced penicillanic and cephalosporanic acids are not efficient for the recovery of N-substituted β -lactam intermediates and deamidated β -lactams. The main problem in the recovery of the fermentatively produced cephalosporin compounds mentioned above is the complexity of the fermentation broth or culture filtrate. Fermentation broths typically comprise various penicillanic acids [ e.g., α -aminoadipoyl-6-penicillanic acid, α -hydroxyadipoyl-6-penicillanic acid, 6-aminopenicillanic acid (6-APA) ], various cephalosporanic acids (including α -aminoadipoyl-and hydroxyadipoyl-7-ADCA) and a large number of proteinaceous materials. Known recovery methods do not give an acceptable grade of cephalosporanic acid product in purity. In deacylation, this leads to the following problems: reduced enzyme half-life, reduced bioconversion rates, and higher recovery costs and/or unacceptable levels of contaminants after bioconversion. Furthermore, after deacylation, such impurities hinder or at least hinder the recovery of the desired deacylated cephalosporin compound of the required specification.

The present invention provides a process for the recovery of a cephalosporanic acid compound of general formula (i) from a complex mixture containing, in addition to the compound of general formula (i), 6-aminopenicillanic acid (6-APA) and optionally one or more N-substituted beta-lactam compounds:wherein, ● R₀Is hydrogen or C_1-3An alkoxy group; ● Y is CH₂Oxygen, sulfur or oxidized forms of sulfur; ● R₁Is any one group selected from the group consisting of:

-hydrogen, and (C) hydrogen,

-a hydroxyl group,

-a halogen, in the form of a halogen,

-saturated or unsaturated, linear or branched alkyl (1 to 5 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy, halogen, aryl, alkoxy (1 to 3 carbon atoms) or acyl;

alkoxy (1 to 3 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy or halogen; or

-cycloalkyl (3 to 8 carbon atoms) optionally substituted by hydroxy, halogen, amino;

-an aryl group;

-a heteroaryl group; and ● R₂Selected from: adipoyl (1, 4-dicarboxybutane), succinyl, glutaryl, adipoyl, pimeliyl (pimelyl), suberyl (suberyl), 2- (carboxyethylthio) acetyl, 3- (carboxyethylthio) propionyl, higher alkyl saturated and higher alkyl unsaturated dicarboxylic acids, the process comprising the steps of: (a) acidifying the complex mixture to a pH below 6.5 and maintaining the mixture below the pH at a temperature between 10 ℃ and 150 ℃; and/or (b) contacting the complex mixture with a source of carbon dioxide; and (c) recovering the cephalosporanic acid compound of formula (I) from the mixture obtained after step (a) and/or (b). Preferably, the temperature is increased in step (a)The degree is maintained between about 50 ℃ and about 130 ℃ (preferably between 70 and 120 ℃) for 10 seconds to about 1 week, while the pH is maintained at or below pH 4.5. In a preferred method, the compound of formula (I) has been produced by fermentation of a competent microorganism, the complex mixture being a fermentation broth, a culture filtrate or any broth obtainable from a fermentation broth after fermentation.

Preferred compounds of formula (i) are selected from: adipoyl-7-ADCA, adipoyl-7-ADAC and adipoyl-7-ACA.

According to another aspect of the present invention, step (c) is carried out by: subjecting the mixture obtained after step (a) and/or (b) to chromatography, preferably adsorption chromatography, more preferably hydrophobic interaction chromatography.

According to a further aspect of the present invention there is provided the use of chromatography, preferably by adsorption chromatography, more preferably hydrophobic interaction chromatography, and even more preferably the use of Simulated Moving bed technology (Simulated Moving bed technology), in the recovery of a cephalosporin compound of formula (i).

According to a further aspect of the invention there is provided a process for the preparation of a compound of formula (ii):wherein, ● R₀Is hydrogen or C_1-3An alkoxy group; ● Y is CH₂Oxygen, sulfur or oxidized forms of sulfur; ● R₁Is any one group selected from the group consisting of:

-hydrogen, and (C) hydrogen,

-a hydroxyl group,

-a halogen, in the form of a halogen,

-saturated or unsaturated, linear or branched alkyl (1 to 5 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy, halogen, aryl, alkoxy (1 to 3 carbon atoms) or acyl;

alkoxy (1 to 3 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy or halogen; or

-cycloalkyl (3 to 8 carbon atoms) optionally substituted by hydroxy, halogen, amino;

-an aryl group;

-a heteroaryl group; the method comprises the following steps: preparation of a Compound of formula (I) (wherein R₀Y and R₁As defined above, and R₂Selected from: adipoyl (1, 4-dicarboxybutane), succinyl, glutaryl, adipoyl, pimeloyl, suberoyl, 2- (carboxyethylthio) acetyl, 3- (carboxyethylthio) propionyl, higher alkyl saturated and higher alkyl unsaturated dicarboxylic acids); deacylating the compound of formula (I) to obtain a conversion solution comprising the compound of formula (II). The transformation solution preferably also contains a cleaved side chain (denoted as R)₂)。

According to a preferred embodiment, the process further comprises a step of recovering the compound of formula (ii) from the solution by crystallization, preferably before and/or after (after solubilizing the crude crystals, i.e. by crystallization) treating the solution with a selected agent, such as activated carbon or an adsorption resin. According to a further aspect of the invention, a solvent, for example methanol, ethanol, (iso) propanol, isobutanol, n-butanol or acetone or a combination of any of the above, is added during or before the crystallization and/or recrystallization. Preferred adsorbent resins are selected from: XAD16(CAS No.102419-63-8), XAD1600(CAS No.153796-66-8), and HP20(CAS No. 55353-13-4). According to the invention, this method is preferred: wherein the 6-aminopenicillanic acid (6-APA) content is 10ppm or less with respect to the compound of formula (II). According to another aspect, a process is provided wherein, after deacylation, the solution is treated to at least partially remove R₂The cleaved side chain is shown. This step may be carried out or repeated after crystallization and solubilization (i.e. recrystallization) of the compound of formula (ii). The removal of cleaved side chains can also be carried out on the mother liquor obtained after crystallization or recrystallization.

Thus, a process is provided wherein the treatment to at least partially remove cleaved side chains is followed by solubilization of the crude crystals and recrystallization of the compound of formula (ii).

Preferably, the treatment to remove cleaved side chains comprises: the conversion liquor or mother liquor or both are subjected to membrane filtration at a pH below 5, preferably below 4, more preferably near or below 3. Thus, there is provided the use of membrane filtration for removing dicarboxylic acids from a mixture comprising dicarboxylic acids and a beta-lactam antibiotic. The mixture is preferably the mother liquor obtained after crystallization of the compound of formula (II) or the mixture obtained after deacylation of the compound of formula (I). The membrane filtration is preferably carried out at a pH of about 5 or less, preferably at a pH of 4 or less, more preferably at a pH of 3 or less, by ultrafiltration (nanofiltration). According to a further aspect of the invention, a process is provided in which the side chain R is at least partially removed from the transformation mixture by crystallization and/or recrystallization₂. According to a further aspect of the invention, a process is provided in which the side chain R₂Is at least partially removed from the conversion mixture by: the mixture is acidified to a pH below 3 and then contacted with an organic solvent (e.g., amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, isobutanol, or n-butanol).

The present invention relates to a process for the recovery of a cephalosporanic acid compound of general formula (I) from a complex mixture containing, in addition to the compound of general formula (I), 6-aminopenicillanic acid (6-APA) and optionally one or more N-substituted penicillanic acid compounds:wherein, ● R₀Is hydrogen or C_1-3An alkoxy group; ● Y is CH₂Oxygen, sulfur or oxidized forms of sulfur; ● R₁Is any one group selected from the group consisting of:

-hydrogen, and (C) hydrogen,

-a hydroxyl group,

-a halogen, in the form of a halogen,

-saturated or unsaturated, linear or branched alkyl (1 to 5 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy, halogen, aryl, alkoxy (1 to 3 carbon atoms) or acyl;

alkoxy (1 to 3 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy or halogen; or

-cycloalkyl (3 to 8 carbon atoms) optionally substituted by hydroxy, halogen, amino;

-an aryl group;

-a heteroaryl group; and ● R₂Selected from: adipoyl (1, 4-dicarboxybutane), succinyl, glutaryl, adipoyl, pimeloyl, suberoyl, 2- (carboxyethylthio) acetyl, 3- (carboxyethylthio) propionyl, higher alkyl saturated and higher alkyl unsaturated dicarboxylic acids, the process comprising the steps of: (a) acidifying the complex mixture to a pH below 6.5 and maintaining the mixture below the pH at a temperature between 10 ℃ and 150 ℃; and/or (b) contacting the complex mixture with a source of carbon dioxide; and (c) recovering the cephalosporanic acid compound of formula (I) from the mixture obtained after step (a) and/or (b). The invention further relates to a process for the preparation of a cephalosporin having the general formula (II):wherein, ● R₀Is hydrogen or C_1-3An alkoxy group; ● Y is CH₂Oxygen, sulfur or oxidized forms of sulfur; ● R₁Is any one group selected from the group consisting of:

-hydrogen, and (C) hydrogen,

-a hydroxyl group,

-a halogen, in the form of a halogen,

-saturated or unsaturated, linear or branched alkyl (1 to 5 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy, halogen, aryl, alkoxy (1 to 3 carbon atoms) or acyl;

alkoxy (1 to 3 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy or halogen; or

-cycloalkyl (3 to 8 carbon atoms) optionally substituted by hydroxy, halogen, amino;

-an aryl group;

-a heteroaryl group.

The compound of formula (i) may be produced by any series of steps which result in a complex mixture as defined herein from which recovery of the compound of formula (i) is effected. For the purposes of the specification and claims, a "complex mixture" is defined as a mixture comprising an N-substituted cephalosporin compound and a substituted or unsubstituted β -lactam compound.

The compound of formula (ii) is obtained by the following series of steps: (a) recovering, preferably purifying, the compound of formula (I); (b) deacylating the preferably purified compound of formula (I) to obtain a solution (conversion solution) comprising the compound of formula (II); and (c) recovering, preferably purifying, the compound of formula (II).

One of the obstacles to the fermentative production of N-substituted cephalosporanic acids is the presence of undesirable contaminating β -lactam components (e.g., N-substituted 6-aminopenicillanic acid). According to one embodiment of the present invention, it has been found that these contaminants can be significantly reduced by: the fermentation broth, filtrate of the fermentation broth or liquid obtained from the fermentation broth by applying any biomass separation technique is incubated under acidifying conditions, preferably with elevated temperature. The fermentation broth is acidified to a pH below 6.5 (preferably below 4.5) using at least one known acid (e.g. sulfuric, hydrochloric or nitric acid or a combination thereof). The operation temperature is in the range of 20-150 ℃, preferably 70-120 ℃. The retention time under these conditions is in the range of a few seconds (at 150 ℃) or a few days (at 20 ℃), preferably from 10 seconds to 60 minutes. Preferably, the pH/temperature treatment is carried out for a period of time to provide a reduction of the N-substituted 6-APA by a factor of 100, preferably 1000, more preferably 1,000,000, relative to the compound of formula (II). This step can be carried out before or after the biomass separation and can be carried out batchwise or continuously.

According to another embodiment of the invention, the contaminating penicillin component (e.g. the N-substituted 6-APA) is prepared by reactingThe fermentation broth, filtrate of the fermentation broth, eluent, conversion solution or dissolved contaminating cephalosporin of formula (i) (usually at pH 5-7) is significantly reduced by contact with carbon dioxide. The carbon dioxide may be added to the solution in any suitable form, for example in solid or gaseous form or as a solution of carbonate ions. Mixing the solution with CO₂The source is contacted at a temperature of 10 to 60 ℃ (preferably 20 to 40 ℃), wherein the solution is contaminated with molecular CO₂Saturation is carried out for 4-10 hours. After reduction of the penicillin component, a purification of the cephalosporin of formula 1 as described above may be achieved.

The complex mixture as defined herein may be of any origin, but is preferably a culture broth or culture filtrate obtained after fermentation by a microorganism capable of producing the 7-N-acylated form of the compound of formula (I) under conditions leading to production, wherein the acyl group may be any acyl group supporting the cyclic extension enzyme (deacetoxycephalosporin synthase-DAOCS, or bifunctional expandase/hydroxylase (sometimes referred to as deacetylcephalosporin synthase DACS)) in the cephalosporin biosynthesis pathway. Biological processes for the in vivo production of 7-N-acyl substituted compounds of formula (i) are disclosed: WO 93/05158 (adipoyl-7-ADCA); WO 93/08287 (adipoyl-7-ADAC and adipoyl-7-ACA), WO 95/04148(2- (carboxyethylthio) acetyl-7-ADCA), WO95/04149(3- (carboxyethylthio) propionyl-7-ADCA) and higher alkyl saturated or unsaturated dicarboxylic acids. The relevant portions of these PCT applications are incorporated herein by reference. Preferred acyl groups are generally dicarboxylic acids such as adipoyl (1, 4-dicarboxybutane), 2- (carboxyethylthio) acetyl, 3- (carboxyethylthio) propionyl, muconic acid, and the like. Suitable host organisms include, but are not limited to, Penicillium chrysogenum and Acremonium chrysogenum. Suitable sources of expandases (including bifunctional expandases/hydroxylases) include, but are not limited to, Streptomyces clavuligerus (Streptomyces clavuligerus) and Acremonium chrysogenum. Methods of transformation, selection of transformed cells and regulatory factors indicative of filamentous fungi, which may be used to genetically modify the host cell, are well known in the art of recombinant DNA technology for β -lactam producing (filamentous) fungi.

Preferably, the fermentation broth is first subjected to biomass separation, for example by filtration by any suitable method, such as membrane filtration, vacuum filtration, ultrafiltration or a combination thereof, prior to acidification and optionally raising the temperature. Any other method of biomass separation is also suitable.

After the pH-lowering step and the optional temperature step, the recovered compound of formula (i) is preferably subjected to further purification in order to at least partially remove the undesired β -lactam component, especially the undesired N-substituted cephalosporins and penicillins. The further purification can be carried out by extraction with an organic solvent. In terms of extraction, it was found to be advantageous: washing the extract, back-extracting the N-substituted cephalosporin from the organic phase to an aqueous phase, and back-extracting the aqueous phase. The extraction organic solvent can be selected from amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, isobutyl alcohol, n-butyl alcohol, etc. In the present process, the preferred purification step is to purify the N-substituted cephalosporin by chromatography rather than by extraction with an organic solvent. The advantages of chromatography are the absence of solvents (solvents cause waste problems and contamination problems) and the improvement of the purity of the final product. Preferred is ion exchange chromatography or adsorption chromatography, more preferred is hydrophobic interaction chromatography. The filtrate is chromatographed using an adsorbent. The adsorbent comprises: activated carbon, such as Norit CG-1 or Cecarbon GAC 40; or an adsorption resin, for example, a styrene-divinylbenzene copolymer such as Dianion HP20 (CASNO.55353-13-4), Dianion HP 21(CAS No.92529-04-9), Dianion SP207(CAS No.98225-81-1) or Dianion SP 825 from Mitsubishi Kasei Corporation or Amberlite XAD1180(CAS No.97396-56-0), Amberlite XAD1600(CAS No.153796-66-8) or Amberlite XAD16(CAS No.102419-63-8) from Rohm and Haas or Amberchrom CG 161(CAS No.131688-63-6) from TosoHaas; preferably XAD16 or XAD 1600.

The complex mixture is adjusted to a pH of 1.0 to 5.0, preferably 2.5 to 3.5, by one or more known acids, such as sulfuric, hydrochloric or nitric acid or combinations thereof, prior to adsorption of the N-substituted cephalosporin. The operation temperature is in the range of 0 to 50 ℃ (preferably 5 to 25 ℃). The operation pressure is within the range of 0-1.0 MPa overpressure.

Undesirable beta-lactam components, especially undesirable N-substituted cephalosporins (e.g. alpha-aminocaproyl cephalosporanic acid) are also adsorbed on the adsorbent, but are replaced by the desired N-substituted cephalosporin.

After adsorption, a wash with water is employed to remove the undesirable β -lactam component in the interstitial volumes between the adsorbents and desorb the weakly bound, undesirable β -lactam component from the adsorbents. The water may be acidified to a pH of 1.0 by one or more known acids, such as sulfuric, hydrochloric, or nitric acids, or combinations thereof. To increase the osmotic pressure, salt may be added to the water. The operation temperature is in the range of 0 to 50 ℃ (preferably 20 to 40 ℃). The operation pressure is within the range of 0-1.0 MPa overpressure.

Elution can be carried out with a suitable buffer (e.g. acetate, phosphate, carbonate, bicarbonate or adipate), but also with rare organic solvents (e.g. acetone, isopropanol) or dilute bases (e.g. ammonium, caustic). The operation temperature is in the range of 0-80 ℃ (preferably 10-40 ℃). The operation pressure is within the range of 0-1.0 MPa overpressure.

Regeneration of the adsorbent can be carried out by any conventional method, for example using dilute base, dilute acid, or using a water miscible solvent (e.g., acetone, methanol, ethanol, or isopropanol), or a combination thereof. Optional heating to 100 ℃ may be carried out.

The regeneration liquid may be removed by washing with water. The water may be acidified to a pH of 1.0 by one or more known acids, such as sulfuric, hydrochloric, or nitric acids, or combinations thereof. The chromatographic process step can be carried out in several devices, for example in a single column, but also simulated moving bed technology can be applied. In the case of the simulated moving bed Technology, several devices are available, for example the ADSEP system of U.S. Filter, the ISEP/CSEP system of Advanced Separation Technology, the 'merry-go-around' system such as Aplexion or the SORBEX system of the Universal Oil Products Company (UOP).

The buffer in the eluate may also be removed by ultrafiltration. The membrane in the membrane filtration is characterized by a high retention of the desired N-substituted cephalosporin and a low retention of the buffer. Optionally, a concentration step is applied by any suitable concentration method, such as vacuum evaporation, reverse osmosis, microfiltration, or chromatography or nanofiltraction after extraction.

The recovered N-acylated compound is then deacylated using any suitable method known in the art. A preferred method is enzymatic deacylation using a suitable dicarboxylated acyltransferase. Many suitable acyltransferases (wild-type or mutant) are known in the art and include, but are not limited to, those from the following microorganisms: bacillus (Bacillus) (EP 0525861; EP 0405846), Pseudomonas (Pseudomonas) (EP 0482844; EP 0525861; EP 0475652; EP 0663445), Achromobacter (Achromobacter) (EP 0525861), Alcaligenes faecalis (EP 0638649), Acinetobacter (Acinetobacter) (EP 0469919), Arthrobacter (Arthrobacter) (EP 0283218), Escherichia coli (Escherichia coli) (US3, 945, 888), Kluyvera citrophila, providencia (Proteus rettgeri) (US3, 915, 798), and the like. Preferably, the dicarboxylated acyltransferase is derived from Pseudomonas SE83 or SY-77. The acyltransferase may optionally be in a mutated form (such as those disclosed in WO 91/16435, WO 97/20053, WO 97/40175) to increase or alter the affinity for the substrate. Another method for deacylating the N-acylated cephalosporin compounds of the present invention is by contacting the substrate with a microorganism capable of producing an acyltransferase (as disclosed in U.S. Pat. No.5,677,141).

The acyltransferase may be immobilised (US3, 930, 949) using techniques well known in the art on a membrane (EP 0243404) or a free-flowing carrier (e.g. glutaraldehyde-based carrier) or an azalide polymer (EP 0730035). Non-immobilized acyltransferases are also contemplated, using membranes to separate the reaction mixture (retentate) from the product (permeate), for example as disclosed in U.S. Pat. No.5,521,068. The process may be batch or (semi-) continuous, which is well known and not critical to the present invention. Enzymatic deacylation is typically carried out in a stirred tank reactor (with or without sieve trays, which are preferably inert, to facilitate separation of the immobilized enzyme from the reaction product). The pH is typically adjusted during the reaction to compensate for pH changes due to the removal of the (dicarboxy) side chains by any type of base (e.g. ammonium, caustic, carbonate, bicarbonate). The pH can be adjusted in the reactor and/or in a circulation loop (circulating loop) above the reactor. Other parameters (e.g., temperature, deacylated product or side chain concentration, etc.) may also be adjusted to account for the effect of these parameters on the reaction rate and/or equilibrium.

Additional stabilizers, such as sulfite (S), may be added prior to and/or during deacylation₂O₅ ^2-、HSO₃ ^-、SO₃ ^2-) EDTA, Dithiothreitol (DTT).

Generally, the deacylated cephalosporin compound of formula (I) is then recovered using any suitable combination of steps. Optionally, a concentration step may be taken, for example by vacuum evaporation, reverse osmosis, microfiltration or nanofiltraction prior to crystallization. Optionally, a water miscible solvent may be added. Optionally, the solution may be purified by treatment with activated carbon or an adsorption resin prior to crystallization. Optionally, the side chains may be removed prior to crystallization, characterized in that the aqueous phase is acidified, the side chains are extracted into the extraction organic solvent and the two phases are separated. The organic solvent can be selected from amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, isobutanol, n-butanol, etc.

The product can be crystallized from the aqueous phase formed in several ways. The most preferred method of operation is: neutralizing the aqueous solution, followed by the application of one or more known acids (e.g., H)₂SO₄、HCl、HNO₃Or a combination thereof) reducing the pH to 3-5 in 1-6 steps. This is preferably done in a continuous manner using a set of 1-6 interconnected continuously operating crystallizers (in sequence). Batch crystallization, semi-continuous crystallization or coordinated crystallization can also be adoptedAnd (4) crystallizing. The crystallization can be carried out directly without first neutralization in the same manner as described above. According to one embodiment of the present invention, it was found that the quality of the cephalosporin of formula (II) can be improved by the addition of water-miscible solvents such as methanol, ethanol, isopropanol, n-butanol, acetone, etc. Optionally, the quality of the compound of formula (ii) may be improved by treatment with activated carbon or with an adsorption resin prior to crystallization.

It has been found that the quality of the cephalosporin of formula (II) can be further improved by recrystallization, optionally after treatment with an adsorption resin, activated carbon and/or ethanol and/or acetate. The treatment is characterized by dissolving the cephalosporin of formula (II) at a pH in the range of 0.5 to 10.0, preferably between 7.5 and 8.5, and then crystallizing the product. The product can be crystallized in several ways. The most preferred method of operation is: using one or more known acids (e.g. H)₂SO₄、HCl、HNO₃Or a combination thereof) reducing the pH to 3-5 in 1-6 steps. This can be done in a continuous manner using a set of 1-6 interconnected continuously operating crystallizers (in sequence). Batch crystallization, semi-continuous crystallization or coordinated crystallization may also be employed. According to one embodiment of the present invention, it was found that the quality of the cephalosporin of formula (II) can be improved by the addition of water-miscible solvents such as methanol, ethanol, (iso) propanol, acetone, iso-butanol and n-butanol.

It has also been found that the quality of the cephalosporin of formula (II) can be improved by treating the conversion solution and/or the dissolved solution of the cephalosporin of formula (II) with an adsorbent. The adsorbent comprises: activated carbon, such as Norit Ultra SX; or an adsorption resin, for example, a styrene-divinylbenzene copolymer such as Dianion HP20 (CASNO.55353-13-4), Dianion HP 21(CAS No.92529-04-9) or Dianion SP207(CAS No.98225-81-1) from Mitsubishi Kasei Corporation or Amberlite XAD1180(CAS No.97396-56-0), Amberlite XAD1600(CAS No.153796-66-8) or Amberlite XAD16(CAS No.102419-63-8) from Rohm and Haas or Amberchrom CG 161(CAS No.131688-63-6) from TosoHaas; preferably XAD16, XAD1600 or HP 20.

The crystals are separated by filtration or centrifugation and dried in a conventional continuous or batch dryer. The crystals may be milled by any type of mill (e.g., ball mill, jet mill, etc.).

Optionally, water miscible solvent may be added during the crystallization process. After dissolution, the solution may be treated with activated carbon or an adsorbent resin.

This operation will give better overall yield and product quality than the previously described currently known methods.

According to another aspect of the present invention, there is provided a process for removing and recovering adipic acid from the conversion liquor or mother liquor (the liquor obtained after crystallization of the compound of formula (II)). It was found that adipic acid can be advantageously isolated at low pH (e.g. below pH5, preferably below pH4, more preferably below pH3, at or near pH3, 3) using membrane filtration. Preferred according to the invention is this embodiment: wherein the filtration is performed by reverse osmosis.

In addition to the saving of raw materials, this has the advantage of the purity and/or yield of the solution thus treated when crystallized.

The invention is illustrated by the following non-limiting examples. Experiment of

A fermentation broth comprising adipoyl-7-ADCA, in particular 6-APA, adipoyl-6-APA and alpha-amino-adipoyl-7-cephalosporanic acid as undesired contaminants, was obtained as a complex mixture by fermentation of a strain of Penicillium chrysogenum transformed with an expansase derived from Streptomyces clavuligerus (deacetoxycephalosporin C synthetase), as described in International patent application WO 93/05158 published 3/18 1993.

The transformed Penicillium strain was cultured as described in example 1 of WO 93/05158 (incorporated herein by reference).

And after fermenting for 5-7 days, using the fermentation liquor for a recovery experiment.

This complex mixture can also be simulated by formulating an aqueous mixture of the following compounds: 6-aminopenicillanic acid, adipoyl-6-aminopenicillanic acid, alpha-aminoadipoyl-6-aminopenicillanic acid, adipoyl-7-aminodesacetoxycephalosporanic acid and alpha-aminoadipoyl-7-cephalosporanic acid.

Example 1

pH/heat-treatment this example shows the advantage of pH treatment (preferably combined with a temperature-raising treatment) for removing the undesired β -lactam component from complex mixtures.

The fermentation broth from the Penicillium chrysogenum fermentation (see experimental section) comprising a complex mixture of adipoyl-7-ADCA and penicillanic and cephalosporanic acids contaminants was filtered. The concentrate was washed with process water until the total combined filtrate volume was about 2 times the volume of the initial broth. The following experiments were performed: A. acidifying a portion of the filtrate to pH = 3.5; heating to 70 deg.C, cooling to 40 deg.C after 30 min; B. acidifying a portion of the permeate to pH = 2.7; heating to 110 deg.C, cooling to 25 deg.C after 4 min; acidifying a portion of the permeate to pH =3.0 and without further treatment.

Subjecting the pretreated solution to the following treatment to obtain a compound of formula (II); 7-ADCA. Adsorption chromatography

Three solutions (A-C) were filtered on a Seitz K100 filter, and the solution at pH 3.0 was then pumped onto a column packed with 1.6 liters of XAD-1600 resin; the resin was then washed with 4.8 liters of water and eluted with 0.2M bicarbonate solution. The first eluate fraction (1.1 l) was removed and discarded. A second fraction (3.2 liters) was collected and analyzed. The resin was purified by washing with caustic soda and acetone, again with acidified water. Concentrating

The eluate is concentrated under vacuum (5-10 mm Hg) at 20-30 ℃ until a concentration of 40 g/l adipoyl-7-ADCA is obtained. Enzymatic deacylation

Next, adipoyl-7-ADCA was treated with acyltransferase as follows. To 1 liter of the eluate were added 1 gram of sodium metabisulfite, 20mM EDTA and 100g of immobilized acyltransferase (including Pseudomonas SE83 dicarboxylated acyltransferase). The solution was stirred at 30 ℃ for two hours. The pH was maintained at 8.5 with 4N sodium hydroxide. Separating the immobilized acyltransferase from the liquid using a glass-sintered filter. Crystallization of 7-ADCA

Precipitating 7-aminodesacetoxycephalosporanic acid (7-ADCA) by lowering the pH to 3.6 at a temperature of 30 ℃ with stirring; the pH of the solution was lowered to 3.6 with 6N sulfuric acid in 45 minutes. After cooling to 20 ℃ the crystals were separated on a glass-sintered filter, washed with water and dried at 35 ℃. Dissolving 7-ADCA crystals

The 7-ADCA is dissolved by means of ammonia. For this purpose, 15 g of 7-ADCA were suspended in 255ml of water. Dissolving 7-ADCA by means of 4N ammonium hydroxide at a pH of 7.5-8.5. After filtration on a glass sintered filter, water was added to give 300ml of solution. Treatment with adsorbent resins

The solution was treated with an adsorbent resin. This solution was pumped over 45 minutes onto 15ml XAD 1600. Then, 75ml of water was pumped over the resin to obtain 375ml of solution. Recrystallization

Precipitating 7-ADCA by lowering the pH to 3.6 at a temperature of 30 ℃ with stirring; the pH was lowered to 3.6 with 6N sulfuric acid in 45 minutes. After cooling to 20 ℃ the crystals were separated on a glass-sintered filter, washed with water and dried at 35 ℃.

The 7-ADCA thus produced showed good results in terms of 6-APA reduction. (6-APA ratio was calculated relative to 7-ADCA).

TABLE 1A results of experiments 1A, 1B and 1C

Experiment of	6-Aminopenicillanic acid content (ppm)
		1A	＜10
1B	＜10
		1C	950

It is evident that the pH/temperature treatment reduces the 6-aminopenicillanic acid content of contaminating adipoyl-7-ADCA formulations.

Is constantly decreased by 10^-66-aminopenicillanic acid the correlation between pH, temperature and time of the treatment was determined (Table 1 b). TABLE 1b

6-APA reduction	pH	Temperature (C)	Time(s)	Time (min)	Time (h)
						10-6	3	25	35050	584	9.74
10-6	3	50	3057	50.9	0.85
						10-6	3	75	378	6.3	0.11
10-6	3	100	62	1.0	0.02
						10-6	4	25	148857	2481	41.35
10-6	4	50	12982	216.4	3.61
						10-6	4	75	1607	26.8	0.45
10-6	4	100	263	4.4	0.07

Example 2 adsorption chromatography

This example shows: (a) when adsorption chromatography is used, the degree of loading of the column (2A-2D), (b) washing of the column (2E-2G) with different amounts of water before elution, (c) the effect of the pH of the material on the purification of adipoyl-7-ADCA (2H-2J). This embodiment of the adsorption chromatography process in simulated moving bed mode is given as "experiment 2K".

The fermentation broth was pretreated as described in example 1A. Next, adipoyl-7-amino-desacetoxycephalosporanic acid was purified by adsorption chromatography:the solution was pumped onto a column packed with 1.6 liters of XAD-1600 resin, washed with varying amounts of water (2A-2D and 2H-2K: 4.8 liters; 2E-2F: see Table 2b), and eluted with 0.2M bicarbonate solution. The first eluate fraction (1.1 l) was removed and discarded. A second fraction (3.2 liters) was collected and analyzed. The resin was purified by washing with caustic soda and acetone, again with acidified water. Several variables of the process conditions were applied (see table 2). The decrement is calculated as: (Compound-i)_Material(s)Compound 1_Material(s)) /(Compound-i)_EluateCompound 1_Eluate)。

Table 2 a. results of experiment 2
										Experiment of	Material(s)			Eluate (g)			Decrease (-)		Compound 4(ppm)
	Compound 1(g)	Compound 2(g)	Compound 3(g)	Compound 1(g)	Compound 2(g)	Compound 3(g)	Compound 2	Compound 3
									2A	34	3．3	9．3	30	3．1	5．9	1	1
2B	80	5．9	18．5	70	0．5	0．09	10	173											＜6
									2C	127	10．5	32．8	76	0．3	0．07	24	301	19
2D	255	18．2	59．4	67	0．1	0．03	38	501											30
									Compound 1: adipoyl-7-ADCA Compound 2: α -hydroxyadipoyl-7-ADCA Compound 3: α -aminoadipoyl-7-ADCA compound 4: content of 6-APA relative to Compound 1

These results clearly show the positive effect of overloading the column on the reduction of compounds 2 and 3 in the eluate.

Table 2 b. results of experiment 2
										Experiment of	Material(s)			Washing machine	Eluate			Decrease (-)
	Compound 1(g)	Compound 2(g)	Compound 3(g)	(1)	Compound 1(g)	Compound 2(g)	Compound 3(g)	Compound 2	Compound 3
										2E	85	6．6	14．0	1．6	81	2．1	1．2	3	11
2F	74	6．4	12．6	4．8	71	1．1	0．13	6	94
										2G	75	5．8	13．0	7．3	68	0．5	0．1	12	122
Compound 1: adipoyl-7-ADCA Compound 2: α -hydroxyadipoyl-7-ADCA Compound 3: alpha-aminoadipoyl-7-ADCA

Example 2b shows a continuous wash couple before elution with sodium bicarbonateReducing the undesirable positive effects of cephalosporin compounds. TABLE 2c

Experiment of	Material(s)				Eluate			Decrease (-)
											Compound 1(g)	Compound 2(g)	Compound 3(g)	pH(-)	Compound 1(g)	Compound 2(g)	Compound 3(g)	Compound 2	Compound 3
2H	79	8．4	22．2	2．5	83	0．6	0．09	15	248
										2I	80	5．9	18．5	2．9	70	0．5	0．09	10	183
2J	83	8．2	21．7	3．5	62	0．5	0．14	12	118
										Compound 1: adipoyl-7-ADCACompound 2: α -hydroxyadipoyl-7-ADCA Compound 3: alpha-aminoadipoyl-7-ADCA

The above examples show the effect of pH on the reduction of the undesirable 7-N acylated cephalosporin compound when crystallization is carried out. H₂SO₄Is used as the acid.

Table 2 d. results of experiment 2 (30 l resin in SMB system)
												Experiment of	Material(s)				Eluate				Decrease (-)
	Compound 1(kg)	Compound 2(kg)	Compound 3(kg)	Compound 4(kg)	Compound 1(kg)	Compound 2(kg)	Compound 3(kg)	Compound 4(kg)	Compound 2	Compound 3	Compound 4
												2K	1．46	0．08	0．20	0．15	1．38	0．01	0．01	0．02	7	18	7
Compound 1: adipoyl-7-ADCA Compound 2: α -hydroxyadipoyl-7-ADCA Compound 3: α -aminoadipoyl-7-ADCA compound 4: adipic acid

This example illustrates the use of adsorption chromatography on the kilogram scale according to the so-called simulated moving bed technique. This technique can easily be further scaled up.

Fractions 2A to 2K thus treated were treated with acyltransferase as described in example 1 to produce 7-ADCA. Excellent conversion results were obtained as described in example 3.

Example 3 enzymatic transformation

This example illustrates the results of an enzymatic conversion of adipoyl-7-ADCA to 7-ADCA. adipoyl-7-ADCA was recovered as disclosed in example 2K (pH-treatment as in example 1A, optimization of the adsorption chromatography process by overloading and washing). The conversion was carried out as described in example 1 at the pH indicated in table 3. Examples a to E represent different batches.

TABLE 3
						Experiment of	Substrate Compound 1(mmol)	Substrate Compound 2(mmol)	pH(-)	Product flow Compound 1(mmol)	Product flow Compound 2(mmol)
A	69．7	2．2	8．5	1．1	68．6
						B	144．2	2．4	8．5	5．9	143．3
C	181．4	2．3	8．5	13．5	174．5
						D	113．1	1．7	8	5．4	108．7
E	113．4	3．1	9	1．2	112．2
						Compound 1: adipoyl-7-ADCA Compound 2: 7-ADCA

When adipoyl-7-ADCA is pretreated using a pH/temperature step, both conversion and yield are superior compared to untreated. Further purification using chromatography resulted in further improvement of purity (not shown in the table).

Example 4 crude crystallization

The fermentation broth was subjected to pH/heat-treatment (example 1) and enriched for adipoyl-7-ADCA by adsorption chromatography as described in example 2. Next, the transformation was performed as described in example 1.

The concentration of the converted solution (the solution obtained after the deacylation) was increased by concentrating it by reverse osmosis.

A portion of the solution was removed and 7-ADCA was crystallized by lowering the pH to pH 3.6, 4 or 5 (see Table 4 a). TABLE 4a. crude crystals

Experiment of	Compound 1 in solution	pH(-)	Product (g) after separation, washing and drying	Compound 1(%)
A	49.5	3.6	48.9	97.5
B	49.5	4	48.7	97.4
C	49.5	5	48.2	98
Compound 1: 7-ADCA

Crystallization at all tested pH was satisfactory. In the following experiment, the pH was 3.6. Illustrating the effect of concentrating the solution.

TABLE 4b crude crystallization
				Experiment of	Compound 1(g) in solution	Product (g) after separation, washing and drying	Compound 1(%)
D	15.5	14.8	94.3
E	36.1	35.7	95.3
F	49.5	48.9	97.5
Compound 1: 7-ADCA

It is clear that the concentration of 7-ADCA in the conversion broth has an influence on both purity and yield after crystallization.

The following examples illustrate the effect of different adsorbents on product quality (color and clarity in solution).

TABLE 4c crude crystallization
						Experiment of	Compound 1(g) in solution	Treatment of	Compound 1(%)	Colour in solution at 425nm (-)	Transparency in HCl (EBC)
G	25	HP20	97．2	0．16	3．6
						H	25	HP20(2 times)	97．8	0．11	2．4
I	25	IRA67	97．2	0．18	6
						J	25	IRA67+HP20	97．7	0．1	0．8
K	25	Is free of	96．3	0．39	7．3
						Compound 1: 7-ADCA

Example 5 treatment of solubilized 7-aminodesacetoxycephalosporanic acid this example shows the treatment of 7-AD after treatment of 7-ADCA solution with different adsorption resins before crystallizationTransparency and color of the CA. A solution containing 7-ADCA (the adsorption column used was XAD-1600 resin) was prepared as disclosed in example 2K.

TABLE 5
					Experiment of	Dissolved Compound 1(g)	Treatment of	Colour in solution at 425nm (-)	Transparency in HCl (EBC)
A	40	--	0．18	4．2
					B	40	XAD16	0．05	1．8
C	40	HP20	Not measured out	0．5
					D	40	XAD1600	0．09	0．5
E	25		Not measured out	3．6
					F	25	+1％ EtOH	0．11	0．6
G	50	+3％ EtOH	0．18	0．9
					H	50	+ 2% of charcoal	0．04	Not measured out
I	50		0．17	1．4
					J	50	+ 5% of charcoal	0．03	0．8
Compound 1: 7-aminodesacetoxycephalosporanic acid

EXAMPLE 6 recovery of adipoyl-7-ADCA by extraction with n-butanol

The fermentation broth containing adipoyl-7-ADCA was treated as described in example 1.

After acidification, a portion of adipoyl-ADCA was purified by adsorption chromatography. This solution was pumped onto a column packed with XAD-16 resin, washed with water and eluted with 0.2M acetate solution. The first eluate fraction containing a low content of cephamycephalic acid adipamoate is removed and discarded. The second fraction was collected. The resin was purified by washing with caustic soda and acetone, again with acidified water.

A portion of adipoyl-7-ADCA was thus purified: extracting, washing the extract, back-extracting the N-substituted cephalosporin from the organic phase to an aqueous phase, and back-extracting the aqueous phase; the organic solvent for extraction is n-butanol.

7-aminodesacetoxycephalosporanic acid (7-ADCA) is produced by treating adipoyl-7-ADCA with an immobilized acyltransferase. A portion of 7-ADCA was isolated by lowering the pH. Adipoyl deacetoxycephalosporanic acid was dissolved with the aid of caustic soda. Isolating 7-aminodesacetoxycephalosporanic acid by lowering the pH.

Acidifying a part of 7-aminodesacetoxycephalosporanic acid aqueous solution, extracting the side chain into an extraction organic solvent, and then carrying out phase separation; the organic solvent for extraction is n-butanol.

Finally, the crystal mass was filtered, washed and dried.

TABLE 6
					Experiment of	Description of the invention	Compound 1(%)	Colour in solution at 425nm (-)	Transparency in HCl (EBC)
A	Extraction/crystallization	98．3	0．13	2．4
					B	Chromatography/extraction/crystallization	98．9	0．09	1．5
C	Chromatography/crystallization	97．6	0．12	-
					D	Chromatography/crystallization/recrystallization	98．7	0．05	1．1
Compound 1: 7-aminodesacetoxycephalosporanic acid

The results with separate extraction are not given in the table, but the purity is much poorer than when chromatographic treatment is used (even without recrystallization). Combining chromatography with recrystallization or chromatography with extraction gives the best results.

EXAMPLE 7 recovery of adipic acid from 7-ADCA crystallization mother liquor

The 7-ADCA crystallization mother liquor was obtained as described in example 4. Adipic acid was determined using HPLC: aminex HPX-87H column, 300 mm. times.7.8 mm, packed with 9um cationic gel (Biorad) and treated with 0.2M H at 65 deg.C₂SO₄The aqueous solution was eluted and measured using an RI Waters 410 refractometer. In the examples given below, optimization is directed to purity, not yield.

Example 7A

Recovery of adipic acid by acidification

The pH of the 7-ADCA crystallization mother liquor (250ml, 13.6g/l adipic acid) was lowered to 0.7 with an aqueous 12M H2SO4 solution at 20 ℃. After 16h at 0 ℃ no crystals could be detected. The pH was raised to 3.4 using 6M aqueous KOH. The crystals formed were recovered by filtration to give 7.7g of material (which was a mixture of salt and adipic acid, which was not further analyzed).

Example 7B

Recovery of adipic acid by acidification and concentration

At 20 ℃ with 12M H₂SO₄The aqueous solution reduced the pH of the 7-ADCA crystallization mother liquor (500ml, 9.4g/l adipic acid) to 1.5, followed by concentration at 40 ℃ under reduced pressure to give a viscous mixture which was isolated by filtration to give 6.9g of adipic acid after drying, 53% pure (78% yield).

Example 7C

Recovery of adipic acid by reverse osmosis using a Nanomax 50 membrane

At 20 ℃ with 6M aqueous KOH or 12M H₂SO₄The aqueous solution adjusted the pH of the 7-ADCA crystallization mother liquor (500ml, 9.4-18.2 g/l adipic acid, Yuan.) to the values mentioned in the table. The resulting solution was subjected to reverse osmosis using a Nanomax 50 membrane from Millipore. A pressure of 30 bar was applied with the aid of nitrogen to give a filtrate and a retentate, wherein the amount of adipic acid was determined by HPLC. In most cases, a handler is applied, which comprises: concentrate under reduced pressure until crystalline, then filter the product and dry.

	TABLE 7
								pH	Adipic acid (g/l)			Retention (%)	Volume of permeate (ml)	Yield after treatment (g)	Purity after treatment (%)
Initiation of	Penetrating fluid	Retentate
			1．5	13．6	11．0	14．8	26		400	4．4	96
2．0	18．2	13．1						20．8				37	260	2．4	99
			3．0	9．4	6．7	8．7	23		360	1．9	97
7．2	13．6	5．3						31．9				83	400	Untreated	Untreated

Example 7D recovery of adipic acid using reverse osmosis of DK U19F membranes at pH 2.0

With 12M H of 2.91₂SO₄The aqueous solution reduced the pH of the 7-ADCA crystallization mother liquor (100 l, containing 25.0g/l adipic acid) to 2.0. With 57 l of waterThe solution formed was subjected to reverse osmosis in a membrane filtration unit P2-B200 from Hydro Air Research at 2.5m2 using DK U19F membrane. At a pressure of 30 bar, 8.8 l/m are achieved²Average flow/h, the results summarized in the table are given.

TABLE 8
					Components	Concentration (g/l)			Retention (%)
Initiation of	Penetrating fluid	Retentate
			Adipic acid	25.0		11.6	17.8	35
7-ADCA	0.72	<0.01	0.72	＞99
Adipic acid-7-ADCA	1.51	<0.03	1.54	＞98

Claims (25)

1. A process for the recovery of an N-substituted cephalosporanic acid compound of general formula (i) from a complex mixture containing, in addition to the compound of general formula (i), 6-aminopenicillanic acid (6-APA) and optionally one or more N-substituted β -lactam compounds:wherein, ● R₀Is hydrogen or C_1-3An alkoxy group; ● Y is CH₂Oxygen, sulfur or oxidized forms of sulfur; ● R₁Is any one group selected from the group consisting of:

-hydrogen, and (C) hydrogen,

-a hydroxyl group,

-a halogen, in the form of a halogen,

-saturated or unsaturated, linear or branched alkyl (1 to 5 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy, halogen, aryl, alkoxy (1 to 3 carbon atoms) or acyl;

alkoxy (1 to 3 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy or halogen; or

-cycloalkyl (3 to 8 carbon atoms) optionally substituted by hydroxy, halogen, amino;

-an aryl group;

-a heteroaryl group; and ● R₂Selected from: adipoyl (1, 4-dicarboxybutane), succinyl, glutaryl, adipoyl, pimeloyl, suberoyl, 2- (carboxyethylthio) acetyl, 3- (carboxyethylthio) propionyl, higher alkyl saturated and higher alkyl unsaturated dicarboxylic acids, the process comprising the steps of: (a) acidifying the complex mixture to a pH below 6.5 and maintaining the mixture below the pH at a temperature between 10 ℃ and 150 ℃; and/or (b) contacting the complex mixture with a source of carbon dioxide; and (c) recovering the cephalosporanic acid compound of formula (I) from the mixture obtained after step (a) and/or (b).

2. The method of claim 1, wherein in step (a) the temperature is maintained between about 50 ℃ and about 130 ℃, preferably between 70 and 120 ℃ for 10 seconds to about 1 day, while the pH is maintained at pH4.5 or below pH 4.5.

3. The method of claim 1 or 2, wherein the compound has been produced by fermentation of a microorganism capable of producing the compound, and wherein the complex mixture is a fermentation broth, a culture filtrate, or any culture broth obtainable from a fermentation broth after fermentation.

4. A process according to any one of claims 1 to 3 wherein the compound of formula (la) is selected from: adipoyl-7-ADCA, adipoyl-7-ADAC and adipoyl-7-ACA.

5. The process of any one of the preceding claims, wherein step (c) is carried out: subjecting the mixture obtained after step (a) and/or (b) to chromatography.

6. The method of claim 5, wherein the chromatography is adsorption chromatography, more preferably hydrophobic interaction chromatography.

7. Use of chromatography in the recovery of an N-substituted cephalosporin compound of formula (i) in claim 1.

8. Use according to claim 7, wherein the chromatography is adsorption chromatography, preferably hydrophobic interaction chromatography.

9. Use according to claim 8, wherein the chromatography is carried out using simulated moving bed technology.

10. A process for preparing a compound of formula (ii):wherein, ● R₀Is hydrogen or C_1-3An alkoxy group; ● Y is CH₂Oxygen, sulfur or oxidized forms of sulfur; ● R₁Is any one group selected from the group consisting of:

-hydrogen, and (C) hydrogen,

-a hydroxyl group,

-a halogen, in the form of a halogen,

-saturated or unsaturated, linear or branched alkyl (1 to 5 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy, halogen, aryl, alkoxy (1 to 3 carbon atoms) or acyl;

alkoxy (1 to 3 carbon atoms; optionally substituted by one or more heteroatoms), optionally substituted by hydroxy or halogen; or

-cycloalkyl (3 to 8 carbon atoms) optionally substituted by hydroxy, halogen, amino;

-an aryl group;

-heteroaryl, the process comprising the steps of: use of a process according to any one of claims 1 to 6 for the preparation of a compound of formula (i); deacylating the compound of formula (I) to obtain a conversion solution comprising the compound of formula (II).

11. The method of claim 10, wherein said conversion solution further comprises R₂Shown are cleaved side chains.

12. The method of claim 10, wherein said deacylation is enzymatically performed using a dicarboxy acyltransferase.

13. The process of any of claims 10 to 12, comprising the further step of: recovering the compound of formula (ii) from the solution by crystallization.

14. The method of claim 13, wherein the solution is treated prior to said crystallizing with an agent selected from the group consisting of: adsorbent resins, activated carbon, methanol, ethanol, (iso) propanol, isobutanol, n-butanol, acetone, or combinations of any of the foregoing agents.

15. The process of claim 14, wherein at least one adsorbent resin selected from the group consisting of: XAD16, XAD1600, and HP 20.

16. The process according to claim 10 or 11, wherein the 6-aminopenicillanic acid (6-APA) content is 10ppm or less with respect to the compound of formula (ii).

17. The method of claim 11 or 12, wherein the solution is treated after deacylation to at least partially remove the cleaved side chain represented by R2.

18. The process of claim 17, wherein the treatment for at least partially removing cleaved side chains is performed on a mother liquor obtained after crystallization.

19. The process of claim 18, wherein the treatment to at least partially remove cleaved side chains is followed by solubilization of the crude crystals and recrystallization of the compound of formula (ii).

20. The method of claim 18, wherein the solution is treated with an agent selected from the group consisting of: adsorbent resins, activated carbon, methanol, ethanol, (iso) propanol, isobutanol, n-butanol and acetone or any combination of these agents.

21. The method of any one of claims 17 to 20, wherein the treating comprises: the conversion or mother liquor is subjected to membrane filtration at a pH below 5, preferably below 4, more preferably near or below 3.

22. Use of membrane filtration for removing dicarboxylic acids from a mixture comprising dicarboxylic acids and a beta-lactam antibiotic.

23. Use according to claim 22, wherein the mixture is the mother liquor obtained after crystallization of the compound of formula (ii).

24. Use according to claim 22 or 23, wherein the filtration is carried out at a pH of about 5 or lower, preferably at a pH of 4 or lower.

25. The use of claims 22 to 24 wherein the filtration is by nanofiltraction at pH3 or below pH 3.