Title of Invention

"A METHOD TO PREPARE CRYSTALLINE 6-AMINO PENICILLANIC ACID (6-APA)"

Abstract The present invention discloses an improved process for the preparation of 6-amino penicillanic acid (6-APA) from a fermentatively produced N-aubstlfeuted penicillin, comprising the steps of extraction of the N-substituted penicillin compound as present in a fermentation broth or fluid to an organic solvent,dack exclaotiion of the N -suscstituted penicillin compound as water, treatment of the aqueoussg phpse with--a penicillin acylase and isolation of the 6-APA from the thus-obtained conversion solution by crystallization. Further impro-vements comprise extraction of the side chain to an organic solvesnt and isolation of 6-APA from the thus-obtaineel aqueous phase using a specific crystallization process.
Full Text The present invention relates to a method to prepare crystalline 6-amino penicillanic acid (6-APA). Further, the invention relates to a process for the preparation of penicillins, which are deacylateed at the 6~amino group, by the fermentation of a penicillin producing microorganism in the presence of a side chain precursor and by deacylation of fermentatively produced N-substitttted penicillin.
6-Amino penicillanic acid (6-APA) is an important intermediate in the pharmaceutical industry which is in general obtained through chemical or enzymatic cleavage ('deacylation') of penicillin G or penicillin V.
Penicillins themselves are obtained from fermentation using strains of Penicillium chrysogenum. In a typical penicillin G recovery process, the broth is filtered and washed, and penicillin G is extracted to butyl acetate or methyl isobutyl ketone after acidification of the filtrate. The solvent is then 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 sufficient butanol, the water content is reduced using evaporation of the butanol-water azeotrope, whereupon the penicillin G crystals formed are recovered by filtration and subsequent washing and drying.
In another process the crystalline material is directly obtained from the organic solution by addition of potassium acetate or other potassium salts and evaporation of the azeotrope, whereupon the crystals are filtered, washed with butanol, filtered and dried. For penicillin V the same process can 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.
6-Amino penicillanic acid is in general obtained from a solution of dissolved solid penicillin G or V, which is then
contacted with' immobilized penicillin G acylase or penicillin
V acylase, respectively. The conversion solution is subsequently
fed to a crystallizer where the solid product is formed by
precipitation at a pH of about 3.5 to 4.5. Generally, in this
crystallization process the pH is lowered stepwise to avoid
contamination of the final product 6-APA with high side chain
levels and coloured compounds. In addition, solvents like
methanol, ethanol, iso propyl alcohol, are added to avoid this
unacceptable contamination. It will be clear that these types
of production routes to 6-APA are lengthy and costly.
The present invention discloses an improved method for the production of 6-APA, wherein the isolation of penicillin G or
V as a solid intermediate is not required and wherein the colour
correction measures are made redundant.
Therefore, the present invention provides for a method to prepare crystalline 6-amino penicillanic acid (6-APA) which method comprises:
(a) obtaining a fermentation broth or fluid containing fermentatively produced N-substituted penicillin in a manner such as herein described;
(b) extracting the said broth or fluid of step (a) with an organic solvent such as herein described at a pH wherein said N-substituted penicillin is extracted into the said organic solvent to obtain an organic phase;
(c) extracting the organic phase obtained in step (b) with water in a manner such as herein described at a pH wherein an aqueous phase containing said N-substituted penicillin is obtained;
(d) treating said aqueous phase of step (c) in a manner such as herein described, without prior isolation of the N-substituted penicillin in a crystalline form, with a penicillin acylase to convert the N-substituted penicillin to 6-APA to obtain a treated aqueous phase containing 6-APA and a side chain; and
(f) crystallizing the 6-APA from the treated aqueous phase of step (d) in a manner such as herein described to prepare crystalline 6-amino penicillanic acid.
DESCRIPTION OF THE INVENTION
The present invention discloses an improved process for the production of 6-amino penicillanic acid (6-APA) from a fermentation broth of a penicillin producing microorganism.
In general the novel production and purification process comprises the following steps;
A penicillin-producing microorganism is fermented in the presence of a suitable side chain precursor. The fermentation broth is filtered, whereupon the filtrate is extracted with an organic solvent to obtain an organic phase containing the major part of N-substituted penicillanic acid. Subsequently, the N-substituted penicillanic acid is back extracted to water. The aqueous phase optionally ia stripped to remove traces of solvent. The remaining aqueous Solution phase is enzymatically treated to yield 6-APA and a side chain, as for instance phenoxy or phenyl acetic acid.
Subsequently, 6-APA is precipitated from the aqueous
conversion solution after acidification of said solution and
isolated as a crystalline product. However, a more preferred
embodiment of the invention comprises acidification of the
.conversion solution and subsequent extraction of the side chain


to an organic solvent prior to precipitation of 6-APA from the aqueous phase using a pH-shift,
In the process according to the invention, the following steps ,are described in more detail.
A fermentation broth containing the N-substituted penicillin is obtained from any suitable fermentation process, e.g from a fermentation of a strain of Penicillium chrysogenum in the presence of a suitable side chain precursor, such as phenyl acetic acid or phenoxy acetic acid.
The biomass is separated from the fermentation broth using any suitable technology, such as centrifugation or filtration, yielding a penicillin-containing fermentation fluid. Preferably, a filtration step is applied to obtain said separation. The residual solids optionally are washed.
The fermentation broth or fluid is acidified to a pH within a range of 2-4, preferably to a pH within a range of 2.5-3, by addition of an acid, such as sulfuric acid, hydrochloric acid or nitric acid or a combination thereof. The N-substituted penicillin is then separated from the acidified aqueous phase by extraction to an organic solvent. The organic solvent which is used preferably is amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, iso-butanol or n-butanol. The addition of a stiitable de-emulsif ier may improve the extraction significantly.
In one embodiment of the invention, part of the organic solvent may be added to the fermentation broth or fluid prior to acidification. This was found to be satisfactory in avoiding precipitation, said precipitation sometimes occurring as an undesired side effect of acidification of the fermentation broth or fluid. Precipitation is undesired since it will lead to loss of product and may cause subsequent operational problems.
In a next step, the N-substituted penicillin as present in the organic solvent is back extracted to water at a pH of 6-10, preferably at a pH of 7-9. The base used for pH adjustment for instance can be ammonia or an alkaline or earth alkaline hydroxide solution.
After phase separation, the aqueous phase optionally is stripped to remove the organic solvent.
Subsequently, the aqueous phase or solution is treated with a suitable penicillin acylase.
Organisms that have bein found to produce penicillin
acylase are, for example, Acetobacter, Aeromonas, Alcaligenes,
Aphanocladium, Bacillus sp., Cephalosporium, Escherichia,
Flavobacterium, Kluyvera, Mycoplana, Protaminobacter,
Providentia, Pseudomonas or Xanthomonas species . Enzymes derived from Acetojbacter pasteurioanim, Alcaligenes faecalis, Bacillus megaterium, Escherichia coli, Providentia rettgeri and Xanthomonas citrii have particularly proven to be succesful 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 immobilized form, for instance as has been described in EP 0 222 462 and WO 97/04086.
It is advantageously shovm by the present invention that is is feasible to subject a fementatively derived N-substituted penicillin compound, such as penicillin G, to an enzymatic conversion to 6-APA without the necessity for prior isolation of the N-substituted penicillin compound in a crystalline form.
The present invention also envisages the option to directly treat the fermentation fluid with penicillin acylase, i.e. without applying a prior extraction and back extraction step.
The conversion solution, resulting from the enzymatic reaction indicated above, cont ains 6-APA, a side chain such as phenyl or phenoxy acetic acid, coloured products and traces of unconverted product.
In one embodiment of the invention, the product 6-APA is isolated from the conversion solution by crystallization at acidic conditions. Typically, crystallization of 6-APA from an aqueous 6-APA solution is performed by adjusting the pH of the 6-APA solution to an acidic value by adding a titrant to the 6-APA solution until the pH has reached a value within a range of 2.5-4.5, preferably a value of 1+4.
In a preferred embodiment of the invention, crystallization of 6-APA from an aqueous solution is carried out by adding the 6-APA solution to a crystallization vessel which is kept at a fixed pH having a value within a range of 2.5-4.5, using a suitable titrant.
In an even more preferred embodiment of the invention, said crystallization is carried out by a stepwise adjustment of the pH of the 6-APA-solution to a final value within a range of 2.5-4.5 by adding the 6-APA solution to a series of interconnected crystallization vessels, i.e. adding the 6-APA solution to a first vessel, simultaneously adding the content of the first vessel to a second vessel, simultaneously adding the content of the second vessel to a third vessel, etc., wherein a pH range is applied in the interconnected vessels using a suitable titrant, starting at a pH in the first vessel which deviates about 0.5-2 pH units from the pH of the 6-APA solution and ending at a pH in the final vessel which has a value within a range of 2.5-4.5. Conveniently, the pH of the aqueous 6-APA solution is adjusted to the desired final value using a series of 2-6 interconnected vessels.
For instance, to obtain crystallization of 6-APA from the conversion solution, a decreasing pH range 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 crystallization vessels.
The two preferred embodiments described above are preferably performed in a continuous mode.
In a further preferred embodiment of the invention, the phenyl or phenoxy acetic acid side chain is removed prior to crystallization of 6-APA by extraction to an organic solvent. The conversion solution is acidified and then extracted with an organic solvent, for instance amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, iso-butanol or n-butanol. The acidification of the conversion solution is performed with an acid, such as sulfuric acid, hydrochloric acid or nitric acid or a combination thereof, preferably sulfuric acid, to a pH lower than 3, oreferably lower than 2. It was
unexpectedly found that also a high removal efficiency of the coloured impurities was obtained upon applying this extraction step.
In one embodiment of tku invention, the organic solvent extraction is performed in two to four stages of intensive contact extraction, for example a combination of an intense mixer, for instance a high shear mixer with a centrifugal separation, said stages preferably operated in counter current mode.
The organic solvent, containing phenyl or phenoxy acetic acid and coloured compounds, ttny contain a small quantity of 6-APA. Said 6-APA may optionally be back extracted to water, preferably in two to four extraction stages, in counter current mode. The water used for this back extraction is acidified to a pH lower than 6, preferably lower than 3. The preferred water:solvent ratio is 1:5, more preferably 1:20, most preferably 1:10. The thus-obtained aqueous phase can be fed to the 6-APA crystallization or recycled into the side chain extraction.
After organic solvent extraction of the side chain, the 6-APA product can be crystallized from the aqueous phase in several ways, such as the ways which are indicated hereinabove for crystallization of 6-APA trom an aqueous solution.
In a preferred mode of operation, the pH of the aqueous phase is increased to a pH having a value within a range of 2.5-5, preferably within a range of 3.5-4.5, by adding the 6-APA solution in one step to a crystallization vessel kept at the desired pH value or to a series of 2-6 interconnected crystallization vessels applying an increasing pH range. These processes can conveniently be carried out in continuous mode.
When applying this aspect of the process of the invention, the amount of side chain, e.g. phenyl or phenoxy acetic acid, and coloured compounds present in the 6-APA crystals is low as compared to conventional processes wherein the side chain is not extracted and batch mode crystallization from a neutral solution is applied.
All of the above mentioned steps, i.e. extraction, back extraction and crystallization, can be carried out in batch or fed batch mode, but because of stability reasons the preferred method is a continuous mode.
The obtained 6-APA crystals are isolated by filtration or centrifugation and dried in a conventional continuous or batch dryer.







WE CLAIM:
1. A method to prepare crystalline 6-amino penicillanic acid (6-APA)
which method comprises:
(a) obtaining a fermentation broth or fluid containing fermentatively produced N-substituted penicillin in a manner such as herein described;
(b) extracting the said broth or fluid of step (a) with an organic solvent such as herein described at a pH wherein said N-substituted penicillin is extracted into the said organic solvent to obtain an organic phases;
(c) extracting the organic phase obtained in step (b) with water in a manner such as herein described at a pH wherein an aqueous phase containing said N-substituted penicillin is obtained;
(d) treating said aqueous phase of step (c) in a manner such as herein described, without prior isolation of the N-substituted penicillin in a crystalline form, with a penicillin acylase to convert the N-substituted penicillin to 6-APA to obtain a treated aqueous phase containing 6-APA and a side chain; and
(e) crystallizing the 6-APA from the treated aqueous phase of step (d) in a manner such as herein described to prepare crystalline 6-amino penicillanic acid.
2. A method as claimed in claim 1, wherein the side chain obtained in
step (d) is extracted prior to the crystallization step (e).
3. A method as claimed in claim 1, wherein the crystallizing step (e) is performed as a pH lower than 3.
4. A method as claimed in claim 1, wherein the crystallization step (e) is performed by adding the treated aqueous phase obtained in step (d) containing 6-APA to a crystallization vessel which is kept at a fixed pH having a value within the range of 2.5-4.5 using a suitable titrant.
5. A method as claimed in claim 1, wherein the crystallization of step (e) is performed by adding the treated aqueous phase obtained in step (d) containing 6-APA to a series of interconnected crystallization vessels wherein the pH in the first vessel is maintained at a pH range within 0.5-2 pH units from the pH of the aqueous phase containing 6-APA and wherein the pH in the final vessel is maintained at a pH of 2.5-4.5.
6. A method as claimed in claim 1, wherein the pH in the extracting of step (b) is in the range of 2-4.
7. A method as claimed in claim 6, wherein the pH is in the range of 2.5-3.
8. A method as claimed in claim 1, wherein the organic solvent in step (b) is • amyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone, isobutanol or n-butanol.
9, A method to prepare crystalline 6-amino penicillanic acid (6-APA) substantially as herein described with reference to the foregoing examples.

Documents:

1045-del-1998-abstract.pdf

1045-del-1998-assignment.pdf

1045-del-1998-claims.pdf

1045-del-1998-complete specification (granted).pdf

1045-del-1998-correspondence-others.pdf

1045-del-1998-correspondence-po.pdf

1045-del-1998-description (complete).pdf

1045-del-1998-form-1.pdf

1045-del-1998-form-2.pdf

1045-del-1998-form-3.pdf

1045-del-1998-form-4.pdf

1045-del-1998-form-6.pdf

1045-del-1998-form-9.pdf

1045-del-1998-gpa.pdf

1045-del-1998-pct-308.pdf

1045-del-1998-pct-409.pdf

1045-del-1998-pct-416.pdf

1045-del-1998-petition-others.pdf


Patent Number 190382
Indian Patent Application Number 1045/DEL/1998
PG Journal Number 30/2003
Publication Date 26-Jul-2003
Grant Date 08-Feb-2005
Date of Filing 23-Apr-1998
Name of Patentee DSM N.V..,
Applicant Address HET OVERLOON 1, 6411, TE HEERLEN, THE NETHERLANDS
Inventors:
# Inventor's Name Inventor's Address
1 RIENK HENDRIK KUIPERS WATERLUSTLAAN 18,2804 KZ GOUDA,NETHERLANDS
2 HUBERTUS GERARDUS MARIA WALRAVEN VAN VREDENBURGHWEG 787,2284 TM RIJSWIJK NETHERLANDS
3 PIETER THEODORUS KERKHOF WESTERERF 2,3181 JD ROZENBURG,NETHERLANDS
PCT International Classification Number A61K 31/43
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA