WO1997039137A1 - Procede de preparation d'acide clavulanique - Google Patents

Procede de preparation d'acide clavulanique Download PDF

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Publication number
WO1997039137A1
WO1997039137A1 PCT/GB1997/001007 GB9701007W WO9739137A1 WO 1997039137 A1 WO1997039137 A1 WO 1997039137A1 GB 9701007 W GB9701007 W GB 9701007W WO 9739137 A1 WO9739137 A1 WO 9739137A1
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WO
WIPO (PCT)
Prior art keywords
assimilable
phosphorus
fermentation
source
concentration
Prior art date
Application number
PCT/GB1997/001007
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English (en)
Inventor
Sasa Kranjc
Artur Racman
Original Assignee
Lek Pharmaceutical & Chemical Co. Dd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lek Pharmaceutical & Chemical Co. Dd filed Critical Lek Pharmaceutical & Chemical Co. Dd
Priority to JP53684297A priority Critical patent/JP2001503244A/ja
Priority to AU25164/97A priority patent/AU2516497A/en
Priority to EP97916544A priority patent/EP0906446A1/fr
Priority to PL97329291A priority patent/PL185620B1/pl
Publication of WO1997039137A1 publication Critical patent/WO1997039137A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • This invention relates to a process for preparation of clavulanic acid.
  • Clavulanic acid is the common name for (2R, 5R, Z) - 30 ⁇ 2-hydroxyethylidene) -7-oxo-4-oxa-l-azabicyclo[3.2.0]heptane- 2-carboxylic acid.
  • Clavulanic acid and its alkali metal salts and esters are active as inhibitors of beta lactamase produced by some Gram positive as well as Gram negative micro-organisms.
  • clavulanic acid and alkali metal salts thereof also have a synergistic action with penicillin and cephalosporin antibiotics.
  • Clavulanic acid and its salts are used in pharmaceutical preparations to prevent the deactivation of beta lactam antibiotics.
  • Commercial preparations contain potassium clavulanate in combination with amoxycillin trihydrate. Potassium clavulanate is more stable than the free acid or other salts.
  • Clavulanic acid is prepared by fermentation of micro ⁇ organisms such as strains of Streptomyces for example S.clavuligerus NRRL 3585, S.jumonjinensis NRRL 5741 and S.katsurahamanus IFO 13716 and Streptomyces sp.P6621 FERM P2804.
  • the aqueous culture obtained after fermentation is purified and concentrated in accordance with conventional processes for example filtration and chromatographic purification as disclosed in GB 1508977, prior to extraction of the aqueous solution with an organic solvent to obtain a solution of impure clavulanic acid in the solvent.
  • WO95/23870 and W096/28452 disclose improved commercial processes for purification of clavulanic acid and preparation of potassium clavulanate.
  • Growth of an antibiotic producing micro-organism may include two phases; the growth phase during which biomass is produced and the subsequent stationary phase during which growth does not incur. Secondary metabolites such as antibiotics are usually produced during the stationary phase.
  • EP 82522 illustrates use of continuous or intermittent addition of the assimilable carbon source in fermentation of S.clavuligerus NRRL 3585. Regulation of the amount of ammonia is disclosed as W096/18743. Continuous fermentation processes have not been disclosed for clavulanic acid manufacture.
  • a process for production of clavulanic acid comprises fermentation of a clavulanic acid producing species of Streptomyces in a fermentation broth containing assimilable sources of carbon and nitrogen, wherein the concentration of phosphorus in the fermentation broth is less than 0.15% w/v.
  • the phosphorus concentration is preferably maintained below a limit of 0.15% w/v during the growth phase after which the phosphorus concentration may be allowed to decrease.
  • the growth phase for a typical clavulanic acid fermentation lasting for a total of 5 to 6 days may be complete by about 40 hours.
  • the source of assimilable phosphorus may be present as a phosphate salt for example sodium or potassium phosphate, sodium or potassium dihydrogen phosphate or disodium or dipotassium hydrogen phosphate or mixtures thereof.
  • the phosphorus concentration referred to in this specification is determined as the percentage w/v of phosphorus equivalent to the amount of assimilable phosphorus compound present.
  • the phosphorus concentration is preferably 0.0015 to 0.15% w/v, more preferably 0.002 to 0.015% w/v.
  • the phosphorus concentration is preferably allowed to reduce to a low value, preferably zero by the 40th hour of fermentation.
  • Regulation of the amount of assimilable phosphorus in accordance with the present invention may afford unexpectedly high yields of clavulanic acid.
  • the concentration of assimilable carbon source may be selected by routine trials dependent on the characteristics of the Streptomyces strain employed.
  • the proportion of a carbon source such as glycerol, glycerol trioleate or corn starch in the starting medium may be higher than 5% w/v and further quantities of a carbon source may be added during the fermentation in accordance with usual fed batch procedures.
  • Assimilable nitrogen may be provided by proteinaceous matter in the starting media.
  • ammonia may be introduced into the fermenter.
  • Ammonia has also been used to regulate the pH of the fermentation broth during the course of the fermentation.
  • a high concentration of ammonia can poison the microorganisms and a pH which is too low results in less effective clavulanic acid biosynthesis.
  • an assimilable source of nitrogen for example soya bean flour, is added to the starting medium and that an ammonium salt such as ammonium sulphate is added during the course of the fermentation to provide further nitrogen as necessary.
  • a nitrogen free compound preferably ammonium hydroxide is used to control pH. This results in a later decrease in the level of biomass than is the case if ammonia is used as the sole nitrogen source and pH regulator.
  • the results of a comparison with a classic fermentation are shown in Figure 1.
  • the viscosity, which is proportional to the amount of the biomass is shown for a classical fermentation (broken line) and for a fermentation in accordance with the invention (solid line) .
  • the assimilable nitrogen source may be flour, for example soya flour or cotton seed flour.
  • the amount of assimilable nitrogen is preferably 0.5 to 15% w/v, more preferably l.5 to 7.5% w/v.
  • the amount of nitrogen is advantageously greater than 5%.
  • the invention relates particularly to fermentation of the Streptomyces species S.clavuligerus NRRL 3585, S.jumonjinensis NRRL 5741 and S.katsurahamanus IFO 13716 and particularly Streptomyces sp.P6621 FERM P2804.
  • the invention yield improved yields of clavulanic acid from S.clavuligerus
  • the invention finds particular application in relation to commercial scale fermentations particularly but not exceeding broth volumes of IO 4 1, preferably 5 x IO 4 1
  • the fermentation broth may be treated as disclosed in our WO95/23870 or by other known methods by which potassium clavulanate of high purity may be prepared.
  • the most productive clones of Streptomyces sp. PP 6621 FERM P 2804 were obtained by selection methods. The most productive cultures of this microorganism were stored and were further used as a source for new selection cycles.
  • a colony of Streptomyces sp. PP 6621 FERM P 2804 was aseptically transferred in to a sterile potter with sterile water (2cm 3 ) and homogenised. Fragments of the mycelium were transferred onto an agar slope and incubated to maturity (for 10 to 14 days) in a thermostat at 25'C.
  • agar surface was overgrown by a grey-green bacterial mycelium. Spores were scraped from the surface, aseptically inoculated into a seed vegetative medium and incubated on a shaker for 24 h at 250 rpm and at 25 * ⁇ 1 * C. Homogeneous suspension of spores from agar slopes may be stored in skimmed milk (which can be used as a protective medium) for more than two months.
  • part of the culture was aseptically transferred to a fermentation medium and was incubated on a rotary shaker for 96 h. After the finished fermentation state the content of clavulanic acid was analysed. Cultures which gave the highest yields were used as laboratory inoculum in the fermenter.
  • Strains may be stored on slope agar at 4'C maximum for 4 weeks, in skimmed milk in the same condition for 2 months and lyophilised strains may be stored at 4'C for a period of years.
  • composition of media for selection of strain for inoculation in the fermenter Composition of media for selection of strain for inoculation in the fermenter
  • composition was prepared in accordance with classical methods. Trace elements*
  • composition amounts of
  • MnSO H 2 0 0.5 g demineralised water to 1000 cm 3
  • composition amounts of
  • composition amounts of
  • Estol (Priolube 1435) 23.0 g glycerol 5.0 g morpholine propane sulphonic acid 12.0 g trace elements* 10.0 ml tap water to 1000 ml Preparation of the laboratory inoculum
  • the origin of culture for preparation of laboratory inocula was cultured from an agar slope.
  • the chosen slope agar was filled aseptically with sterile water (10cm 3 ) , spores were scraped off and homogenised in a sterile potter.
  • the solution of spores was used as a laboratory inoculum.
  • composition amounts of
  • the inoculum was transferred in a medium that had been sterilised in pre-seed tank and cooled by sterile air to 28 * C.
  • the vegetative phase lasted from 50 to 70 hours at a temperature 28" ⁇ 1 * C, pressure 0.3 Bar and with aeration using sterile air and with consistent mixtures.
  • the vegetative phase from the pre-seed tank was transferred under pressure into a medium that had been sterilised in the seed fermenter and cooled by sterile air to 28 * C. Air was sterilised by filters with a pore size of 0.2 ⁇ m.
  • the vegetative phase lasted from 10 to 20 h at 28' ⁇ l'C, pressure 0.3 Bar, aeration by sterile air and constant mixing.
  • Synperonic (registered trade mark of ICI GB) is a propylenglycol antifoam agent * Soybean oil can be used instead of Estol.
  • the broth was mixed and aerated during the course of whole fermentation and the pH of the media was maintained by addition of 25% aqueous solution of ammonium hydroxide at value 6.8 - 6.9.
  • the fermentation lasted for 96h and the resultant concentration of clavulanic acid was 3580 mg/1.
  • the concentration of phosphorus after the 51st hour of the fermentation was below the detection limit.
  • Example IB A medium used with the same proportions of ingredients as Example IB was placed in two stainless steel fermenters (5001 each) .
  • the fermentation in the first fermenter was run under the same conditions as were described in the Example IB.
  • the fermentation conditions in second fermenter differed from that described in Example IB only in that an 11% aqueous solution of ammonium sulphate at 9 cm 3 /l was added to the fermentation broth during the period between the 40th and 60th hours following inoculation.
  • the pH was maintained on the desired level by sodium hydroxide. After the 60th hour we stopped the addition of nitrogen source was stopped.
  • the viscosity of the fermentation broth which is proportional to the amount of biomass, was analysed during the course of fermentation.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention a pour objet un procédé de préparation d'acide clavulanique, qui comprend la fermentation d'une espèce de Streptomyces produisant de l'acide clavulanique dans un bouillon de fermentation contenant des sources assimilables de carbone et d'azote, la concentration de phosphore dans ledit bouillon de fermentation étant inférieure à 0,15 % en poids par volume.
PCT/GB1997/001007 1996-04-12 1997-04-11 Procede de preparation d'acide clavulanique WO1997039137A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP53684297A JP2001503244A (ja) 1996-04-12 1997-04-11 クラブラン酸の調製法
AU25164/97A AU2516497A (en) 1996-04-12 1997-04-11 Process for the preparation of clavulanic acid
EP97916544A EP0906446A1 (fr) 1996-04-12 1997-04-11 Procede de preparation d'acide clavulanique
PL97329291A PL185620B1 (pl) 1996-04-12 1997-04-11 Sposób wytwarzania kwasu klawulanowego

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SIP-9600120 1995-04-12
SI9600120A SI9600120A (en) 1996-04-12 1996-04-12 New and improved fermentative procedure for the production of clavulanic acid and its salts

Publications (1)

Publication Number Publication Date
WO1997039137A1 true WO1997039137A1 (fr) 1997-10-23

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/001007 WO1997039137A1 (fr) 1996-04-12 1997-04-11 Procede de preparation d'acide clavulanique

Country Status (9)

Country Link
EP (1) EP0906446A1 (fr)
JP (1) JP2001503244A (fr)
AU (1) AU2516497A (fr)
CA (1) CA2251596A1 (fr)
PL (1) PL185620B1 (fr)
RU (1) RU2188868C2 (fr)
SI (1) SI9600120A (fr)
WO (1) WO1997039137A1 (fr)
ZA (1) ZA973139B (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998054352A1 (fr) * 1997-05-28 1998-12-03 Dsm N.V. Production par fermentation d'acide clavulanique dans des conditions de regulation de phosphate
WO2000018947A1 (fr) * 1998-09-29 2000-04-06 Dsm N.V. Fermentation d'acide clavulanique a un niveau regule d'ammoniac
EP0811689B1 (fr) * 1995-11-23 2002-06-12 Antibioticos, S.A. Procede de production d'acide clavulanique et/ou de sels de celui-ci
US6440708B1 (en) * 1998-09-29 2002-08-27 Dsm N.V. Fermentation of clavulanic acid at a controlled level of ammonia
EP2589663A1 (fr) 2011-11-04 2013-05-08 LEK Pharmaceuticals d.d. Procédé de production d'acide clavulanique

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2328045A1 (fr) * 1975-10-13 1977-05-13 Beecham Group Ltd Procede de production d'acide clavulanique
US4110165A (en) * 1974-04-20 1978-08-29 Beecham Group Limited Process for the production of clavulanic acid
US4144242A (en) * 1975-02-07 1979-03-13 Glaxo Laboratories Limited Process for the purification of clavulanic acid
GB1571888A (en) * 1977-02-04 1980-07-23 Glaxo Lab Ltd Clavulanic acid production
EP0182522A1 (fr) * 1984-10-27 1986-05-28 Beecham Group p.l.c. Préparation de l'acide clavulanique et de ses sels et esters

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4110165A (en) * 1974-04-20 1978-08-29 Beecham Group Limited Process for the production of clavulanic acid
US4144242A (en) * 1975-02-07 1979-03-13 Glaxo Laboratories Limited Process for the purification of clavulanic acid
FR2328045A1 (fr) * 1975-10-13 1977-05-13 Beecham Group Ltd Procede de production d'acide clavulanique
GB1571888A (en) * 1977-02-04 1980-07-23 Glaxo Lab Ltd Clavulanic acid production
EP0182522A1 (fr) * 1984-10-27 1986-05-28 Beecham Group p.l.c. Préparation de l'acide clavulanique et de ses sels et esters

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 95, no. 3, 20 July 1981, Columbus, Ohio, US; abstract no. 22886g, LILLEY, G ET AL.: "Control of the production of cephamycin C and thienamycin by Streptomyces cattleya NRRL 8057" page 516; XP002035709 *
DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; DEMAIN A L: "PRODUCTION OF BETA LACTAM ANTIBIOTICS AND ITS REGULATION.", XP002035710 *
J.CHEM.TECHNOL.BIOTECHNOL., vol. 31, no. 2, 1981, pages 127 - 134 *
PROC NATL SCI COUNC REPUB CHINA PART B LIFE SCI 15 (4). 1991. 251-265. CODEN: PNBSEF ISSN: 0255-6596 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0811689B1 (fr) * 1995-11-23 2002-06-12 Antibioticos, S.A. Procede de production d'acide clavulanique et/ou de sels de celui-ci
WO1998054352A1 (fr) * 1997-05-28 1998-12-03 Dsm N.V. Production par fermentation d'acide clavulanique dans des conditions de regulation de phosphate
WO2000018947A1 (fr) * 1998-09-29 2000-04-06 Dsm N.V. Fermentation d'acide clavulanique a un niveau regule d'ammoniac
US6440708B1 (en) * 1998-09-29 2002-08-27 Dsm N.V. Fermentation of clavulanic acid at a controlled level of ammonia
US6991925B2 (en) 1998-09-29 2006-01-31 Dsm Ip Assets B.V. Fermentation of clavulanic acid at a controlled level of ammonia
EP2589663A1 (fr) 2011-11-04 2013-05-08 LEK Pharmaceuticals d.d. Procédé de production d'acide clavulanique
WO2013064687A2 (fr) 2011-11-04 2013-05-10 Lek Pharmaceuticals D.D. Procédé de production d'acide clavulanique

Also Published As

Publication number Publication date
JP2001503244A (ja) 2001-03-13
CA2251596A1 (fr) 1997-10-23
PL185620B1 (pl) 2003-06-30
AU2516497A (en) 1997-11-07
EP0906446A1 (fr) 1999-04-07
RU2188868C2 (ru) 2002-09-10
SI9600120A (en) 1997-12-31
PL329291A1 (en) 1999-03-15
ZA973139B (en) 1998-08-05

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