EP1957502A1

EP1957502A1 - Process for the preparation of a potassium salt of penicillin

Info

Publication number: EP1957502A1
Application number: EP06819870A
Authority: EP
Inventors: Van Der Thomas Does
Original assignee: DSM IP Assets BV
Current assignee: Centrient Pharmaceuticals Netherlands BV
Priority date: 2005-12-02
Filing date: 2006-11-30
Publication date: 2008-08-20
Also published as: EA017121B1; WO2007063107A1; BRPI0619157A2; HK1126476A1; EA200801491A1

Abstract

Process for the preparation of a potassium salt of penicillin G (Pen G K) or penicillin V (Pen V K) in crystal form from a suspension comprising Pen G K or penicillin V (Pen V K), an organic solvent and a C1 to C3 alcohol.

Description

PROCESS FOR THE PREPARATION OF A POTASSIUM SALT OF PENICILLIN

The present invention relates to a process for the preparation of a potassium salt of penicillin G (Pen G K) or penicillin V (Pen V K) in crystal form.

Penicillins are normally obtained from a fermentation process using microbial strains capable of producing penicillin in the presence of a suitable side chain precursor. Penicillin G for instance is produced by selected strains of Penicillium chrysogenum in the presence of the side chain precursor phenyl acetic acid. After fermentation, the penicillin is recovered from the fermentation broth according to known methods depending on the type of penicillin.

In a typical prior art penicillin G recovery process, the fermentation broth obtained is filtered and the filter cake washed after which the penicillin G present in the collected filtrate is extracted to an organic solvent such as n-butylacetate or methyl isobutyl ketone after acidification of the filtrate. The extract thus obtained may then be decolorized with aid of active carbon and the penicillin G acid is back extracted to water upon neutralization with an aqueous potassium salt solution such as potassium acetate. This solution is admixed with a sufficient amount of a second solvent such as n-butanol after which the water content is reduced using evaporation of the n-butanol-water azeotrope, whereupon the penicillin G K crystals formed are recovered by filtration and subsequent washing and drying. A disadvantage of this prior art recovery process is that during evaporation of the n-butanol- water azeotrope substantial losses of Pen G K occur due to degradation of Pen G K, resulting in a significantly reduced yield of Pen G K (e.g. <92%).

In another prior art process, crystalline Pen G K is directly obtained from the extract described above, by addition of potassium acetate or other suitable potassium sources followed by evaporation of the azeotrope, whereupon the Pen G K crystals that are formed are filtered, suspended in n-butanol, filtered and dried. A disadvantage of this prior art recovery process is that after isolation of the final product (Pen G K) the mother liquor comprises both n-butanol and n-butylacetate. Both solvents need to be recovered; however, since n-butanol and n-butylacetate are difficult to separate, this solvent recovery step requires expensive equipment. Moreover, in this process wherein Pen G K crystals are obtained by crystallisation in n-butylacetate, followed by suspending of the Pen G K crystals in n-butanol, two isolation steps of the Pen G K crystals are required instead of one.

For penicillin V in principle the same processes may be applied. However, due to better stability of penicillin V under acidic conditions, penicillin V can also be crystallized in the acid form instead of the potassium salt form, without economical unacceptable losses. The aim of the present invention to provide a simplified and therefore economically more attractive process for the preparation of a potassium salt of a penicillin selected from the group of Penicillin G and Penicillin V in crystal form in sufficiently high and preferably improved yield and of good preferably improved quality. "Yield" is defined herein as the %-yield of penicillin in the potassium salt of a penicillin selected from the group of Penicillin G and Penicillin V in crystal form relative to the penicillin present in the extract. Sufficiently high is defined herein as being preferably at least 90%, more preferably at least 92%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, most preferably at least 98%.

"Good quality" is defined herein as that the final potassium salt of a penicillin selected from the group of Penicillin G and Penicillin V in crystal form contains very little or no impurities, e.g. less than 5% (w/w), more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1 %, more preferably less than 0,5%, more preferably less than 0,25%, even more preferably less than 0,1%. Impurities may for instance be 6-amino-penicillanic acid (6-APA), phenylacetic acid (PA), parahydroxy-penicillin G, penicillic acid of penicillin G, penicilloic acids of penicillin G and penilloic acids of penicillin G and corresponding impurities for penicillin V.

In one aspect, the invention provides a process for the preparation of a potassium salt of a penicillin selected from the group of Penicillin G and Penicillin V in crystal form from a suspension comprising the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V, an organic solvent and an alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol. Preferably the penicillin is Penicillin G. Surprisingly it was found that the yield of the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V in crystal form in the process according to the present invention was sufficiently high as defined hereinbefore and as high as, or even higher than in the processes known in the art, while at the same time the quality of the crystals obtained was good as defined hereinbefore. Another advantage of the process of the present invention is that only one isolation step of the crystals is required which not only contributes to the sufficiently high yields but also to a substantial economic advantage.

The organic solvent in the process according to the present invention may be any suitable organic solvent known by the man skilled in the art. Examples of such suitable organic solvents are amylacetate, n-butylacetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, iso-butanol or n-butanol. Preferably the organic solvent is n-butylacetate. The alcohol in the process according to the present invention is selected from the group consisting of a Ci, C₂ and C₃ and is preferably methanol, ethanol, n-propanol or iso-propanol. Most preferably, the alcohol is methanol.

The penicillin in the process for the preparation of the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V in crystal form according to the present invention may be obtained from any suitable production process known in the art. Penicillin G may for instance be produced by fermenting a penicillin G-producing microorganism e.g. a strain of Penicillium chrysogenum preferably in the presence of the side chain precursor phenylacetic acid according to methods known in the art. The biomass may be separated from the fermentation broth using any suitable technology, such as centrifugation or filtration, thus yielding a penicillin containing fermentation fluid. Preferably, a filtration step is applied to separate the biomass from the broth. Optionally, the residual solids are washed.

The fermentation broth or fluid is acidified to a pH within a range of 2 to 4, preferably to a pH within a range of 2.5 to 3, by addition of at least one acid, such as sulphuric acid, hydrochloric acid or nitric acid or any combination of any of them. The penicillin is then separated from the acidified aqueous phase by extraction to an organic solvent. Any suitable organic solvent may be used, for instance, amylacetate, n-butylacetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, iso-butanol or n-butanol. Preferably the organic solvent is n-butylacetate. The addition of a suitable de-emulsifier may improve the extraction significantly.

"Extract" is defined herein as the organic solvent to which the penicillin selected from the group of Penicillin G and Penicillin V has been extracted. The extract comprising the penicillin may optionally be treated with activated carbon to remove impurities according to methods known in the art.

In one embodiment of the invention, the extract comprising the penicillin selected from the group of Penicillin G and Penicillin V as defined herein before, optionally treated with activated carbon, may subsequently be mixed with a suitable potassium source to convert the penicillin selected from the group of Penicillin G and Penicillin V into the corresponding potassium salt of the penicillin thereby obtaining a mixture comprising the organic solvent and potassium salt of the penicillin.

The amount of the suitable potassium source mixed with the extract comprising the penicillin selected from the group of Penicillin G and Penicillin V, is preferably such that between 0.4 and 3 mole-equivalents of potassium are added with regard to the penicillin. Preferably, between 0.6 and 2 mole-equivalents, more preferably between 0.7 and 1.6 mole-equivalents, more preferably between 0.8 and 1.4 mole-equivalents, more preferably between 0.9 and 1.2 mole-equivalents of potassium are added with regard to the penicillin. Most preferably 1 mole-equivalent of potassium is added with regard to the penicillin. A suitable potassium source is any potassium salt of a preferably weak acid. Examples of such suitable potassium sources are potassium acetate or potassium carbonate. The potassium source may be used in solid form or may first be dissolved in water to form an aqueous solution of the potassium source. A suitable concentration of the potassium source in aqueous solution is for instance between 10 to 60% w/v, for instance between 15 to 55% w/v, for instance between 20 to 50%.

Subsequently, part of the water or part of an azeotrope comprising water and the organic solvent, e.g. an azeotrope of water and n-butylacetate, may be removed from the mixture comprising the organic solvent and the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V. The water which is present in the mixture may origin from the fermentation broth or from the organic solvent or may have been introduced with the aqueous solution of potassium source. Preferably the water content of the mixture comprising the organic solvent and the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V after removal of part of the water or the azeotrope is <10% (v/v), more preferably <5% (v/v), more preferably <2.5% (v/v), more preferably <1 % (v/v), more preferably <0.5% (v/v) and most preferably <0.2% (v/v).

After evaporation of part of the water or of an azeotrope of water/organic solvent originally as described above, an alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol is added to the mixture to obtain a suspension comprising a potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V, an organic solvent and the alcohol. The alcohol may be methanol, ethanol, n-propanol or iso- propanol. Preferably, the alcohol is methanol.

Preferably, the alcohol selected from the group consisting of a C₁, C₂ and C₃ alcohol is dry. As used herein, dry means that the alcohol contains less than 10% (v/v) of water, preferably less than 5% (v/v) of water, preferably less than 2% (v/v). Most preferably, the dry alcohol contains 0% (v/v) of water.

The suspension comprising the potassium salt of a penicillin selected from the group of Penicillin G and Penicillin V, the organic solvent and the alcohol may be agitated during a certain period of time after having been brought into contact with each other. Preferably, the suspension is agitated during a period of time of between 5 min to 24 hours, preferably of between 10 min to 12 h, more preferably of between 20 min to 8 h, more preferably of between 0.5 h and 6h, more preferably of between 45 min and 3 h. Preferably, the suspension is agitated until white crystals of the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V are formed. More preferably, the suspension is agitated until white crystals of the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V are formed without lumps. The suspension may be agitated at any suitable temperature, preferably between 0 and 4CO, more preferably between 5 and 30 °C, most preferably between 10 and 25 °C. In the suspension of the process according to the present invention the ratio of the volume of the alcohol to the volume of the mixture comprising an organic solvent and the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V, may be any suitable ratio. Preferably this ratio is between 1 :20 to 1 :3, preferably between 1 :15 to 1 :4, preferably between 1 :10 to 1 :6. The potassium salt of penicillin selected from the group of Penicillin G and

Penicillin V may crystallise partly or wholly during the process of the present invention. For instance, the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V may start to crystallise upon addition of the suitable potassium source to the extract comprising the penicillin and/or during evaporation of part of the water or part of the azeotrope of water/organic solvent from the mixture comprising the organic solvent and the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V. The potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V may also crystallise further or recrystallise when the mixture comprising the organic solvent and the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V, optionally partly or wholly crystallized, is brought into contact with the alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol and/or during agitation of the suspension comprising the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V, the organic solvent and the alcohol.

In another embodiment, crystals of the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V obtained after addition of a suitable potassium source to an extract containing a penicillin selected from the group of Penicillin G and Penicillin V obtained and defined as described hereinbefore and evaporation of the azeotrope comprising the organic solvent and water, are collected, e.g. by filtration. The crystals thus obtained are resuspended in a mixture comprising the organic solvent as described hereinbefore and the alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol under the same conditions (e.g. with respect to the ratio of alcohol/organic solvent, time, temperature) as described in the previous embodiment.

The potassium salt of penicillin selected from the group of Penicillin G and Penicillin V in crystal form may be isolated from the suspension in any embodiment of the invention by any suitable method known in the art, for instance by centrifugation or filtration. A wet cake of the potassium salt of the penicillin in crystal form may then be obtained. Optionally the wet cake is washed with organic solvent or a mixture of organic solvent and the alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol. If a washing step is applied, the organic solvent is preferably the same organic solvent as used in the extract and the alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol is preferably the same as used in the suspension. The wet cake of the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V in crystal form may be dried using any suitable technology known in the art. In this embodiment of the invention, the penicillin is preferably penicillin G.

Optionally the wet cake comprising the crystals of the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V is mixed with water to obtain an aqueous solution of the potassium salt of penicillin selected from the group of Penicillin G and Penicillin V. The aqueous solution may then be stripped to remove traces of organic solvent and/or the alcohol selected from the group consisting of a C₁ , C₂ and C₃ alcohol. Stripping comprises evaporation of water, the organic solvent and/or the alcohol. The aqueous solution of the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V may be used for enzymatic conversion to 6-amino penicillanic acid (6-APA) by known methods described in the art, for instance as described in EP 0977 883. The solution comprising the dissolved the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin V may be treated with a suitable penicillin acylase, e.g. a suitable penicillin G acylase to deacylate penicillin G and a suitable penicillin V acylase to deacylate penicillin V. Organisms that have been found to produce suitable penicillin acylases are, for example, Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus sp., Cephalosporium, Escherichia, Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Providentia, Pseudomonas or Xanthomonas species. Enzymes derived from Acetobacter pasteurioanum, Alcaligenes faecalis, Bacillus megaterium, Escherichia coli, Providentia rettgeri and Xanthomonas citrii have particularly proven to be successful in a method according to the invention. In the literature, penicillin acylases have also been referred to as penicillin amidases. The penicillin acylase may be used as a free enzyme, but also in any suitable immobilised form, for instance as has been described in EP 0 222 462 and WO 97/04086.The enzymatic conversion of dissolved Pen G K by a penicillin acylase results in the formation of 6-amino penicillanic acid (6-APA) and phenyl acetic acid.

Example 1

Comparative Example To 1000 ml of an activated-carbon-treated extract containing approximately 100,000 Oxford Units Penicillin G per ml (1 Oxford Unit equals circa 0.6 microgram of penicillin G) in n-butylacetate, a 30% (w/w) solution of K₂CO₃ in water was added such that the mixture contained approximately 1.0 mole-equivalent of potassium with regard to penicillin G. This back extract was mixed with 200 ml n-butanol and the resulting mixture was heated under vacuum using a rotary film evaporator (bath at 50-6CO). During evaporation n-butanol was added in portions whereby the total amount of n-butanol added was 200 ml. In total 250 ml n-butanol/water was evaporated and the residue was cooled to room temperature. The Pen G K crystals that were formed were isolated by filtration, washed with n-butanol and dried. The yield (as defined herein before) was 91%.

Example 2

A series of experiments was carried out in which to 1000 ml of an activated- carbon-treated extract containing approximately 100,000 Oxford Units Penicillin G per ml in n-butylacetate, a 50% (w/w) solution of K₂CO₃ in water was added such that the mixture contained approximately 1.0 mole-equivalent of potassium with regard to penicillin G. The resulting mixture was heated under vacuum using a rotary film evaporator (bath 45-48⁹C) whereby a certain quantity of the mixture was evaporated - see Table 1 for the amount for each experiment. Subsequently, methanol was added to the residue and the mixture was agitated during 1 h at 25^. The suspension was filtered and the wet cake was washed with 70 ml n-butylacetate after which the wet cake was dried.

Table 1 : cr stallization of Pen G K in n-but lacetate + addition of methanol to the residue

The volume evaporated is the total volume evaporated and the value in brackets represents the volume of the water layer in the distillate. The column with the water content represents the water content of the residue after evaporation. All experiments yielded white Pen G K crystals in high yield (as defined hereinbefore) with regard to the penicillin content in the carbon treated extract (=100%) and only limited penicillin losses to the mother liquor. The crystals from experiment 1 contained 99.3% Pen G K (i.e. good quality).

Claims

1. Process for the preparation of a potassium salt of a penicillin selected from the group of Penicillin G and Penicillin V, in crystal form from a suspension comprising the potassium salt of the penicillin selected from the group of Penicillin G and Penicillin

V, an organic solvent and an alcohol selected from the group consisting of a Ci, C₂ and C₃ alcohol.

2. The process according to claim 1 characterized in that the penicillin is Penicillin G.

3. Process according to claim 1 or 2, characterized in that the organic solvent is selected from the group consisting of amylacetate, n-butylacetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, iso-butanol or n-butanol, preferably is n- butylacetate.

4. Process according to anyone of claims 1 -3, characterized in that the alcohol is selected from the group consisting of methanol, ethanol, n-propanol or iso-propanol, preferably methanol.

5. Process according to anyone of the claims 1 to 4, further characterised in that the potassium salt of the penicillin in crystal form is isolated from the suspension to obtain a wet cake of the potassium salt of the penicillin in crystal form and optionally washed and/or dried.

6. Process according to claim 5, characterized in that the wet cake is mixed with water to obtain an aqueous solution of the potassium salt of the penicillin and subsequently used in the enzymatic conversion to 6-amino penicillanic acid (6-APA).