PROCESS FOR THE PREPARATION OF CLAVULANIC ACID

Abstract: A process for preparation of clavulanic acid comprising 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.

Description: PROCESS FOR THE PREPARATION OF CLAvTJLANIC ACID 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. In addition to inhibition of beta lactamase, 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. Fed batch fermentation processes are well known for antibiotic production and have been preferred for production of clavulanic acid. Control and maintenance of desired levels of assimilable sources of nitrogen and carbon in the fermentation broth is well illustrated by Lee J S et al, Kor.Jour.Microbiol. 1978, Vol 15, No 1, p 21-29, which describes the improvement of the fermentation process for preparation of penicillin by control of addition of the assimilable nitrogen and carbon source according to the needs of microorganisms in the fermenter. Lilley G et al, J.Chem.Tech.Biotechnol. 1981, Vol 31, p 127-134 illustrates that the production of antibiotics by Streptomyces species can be controlled by changing of concentration of assimilable nitrogen and carbon source and source of phosphorus. For example, the production of thienamycin in the fermenter does not start until the concentration of phosphorus approaches zero. 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. According to the present invention 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. Alternatively or in addition 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. However we have found that use of ammonia both as the sole source of assimilable nitrogen and also for pH regulation is undesirable. A high concentration of ammonia can poison the microorganisms and a pH which is too low results in less effective clavulanic acid biosynthesis. Accordingly it is preferred that 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. According to a preferred aspect of the present invention 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 invention is further described by means of example but not in any limitative sense. EXAMPLE 1 A) CULTIVATION OF STREPTOMYCES SP.P 6621 FERM P 2804 Strain selection and maintenance 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. After 8 to 10 days the 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. After completion of the vegetative stage, 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. The entire procedure was carried out under aseptic conditions. 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 Media for slopes and Petri dishes Composition amount dextrin 10 g KH 2 P0 4 1 g MgS0 4 7H 2 0 1 g NaCl 1 g (NH « ) 2 S0 « 1 g CaC0 3 4 g Trace elements * l cm 3 agar 20 g demineralised water to 1000 cm 3 The composition was prepared in accordance with classical methods. Trace elements* Composition amounts CaCl 2 2H 2 0 10.0 g MgCl 2 6H 2 0 10.0 g NaCl 10.0 g FeCl 3 6H 2 0 3.0 g ZnCl 2 0.5 g CuCl 2 2H 2 0 0.5 g MnSO« H 2 0 0.5 g demineralised water to 1000 cm 3 Vegetative media for strain selection Composition amounts corn starch 10.0 g soybean flour 20.0 g KH 2 P0 4 0.6 g Estol (Priolube 1435) 5.0 g tap water to 1000 cm 3 Fermentation media for strain selection Composition amounts corn starch 9.6 g soybean flour 38.5 g KH 2 P0 4 1.2 g 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. VEGETATIVE PHASE IN PRE-SEED TANK Media for pre-seed tank Volume of pre-seed = 5001 Volume of medium = 3501 Composition amounts corn starch 7.0 kg soybean flour 7.0 g NaH 2 P0 4 0.185 kg Estol (Priolube 1435)* 0.7 kg synperonic 0.150 kg tap water to 350 1 ♦ soybean oil can be used instead of estol 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. Parameters of growth in pre-seed tank time/h pH PMV% decolourisation/min (h) 0 7.20 -- -- 4 7.25 10 > 5 10 7.35 8 > 5 16 7.30 10 > 5 22 7.20 16 4 28 7.02 17 2.5 34 6.85 18 0.5 39 6.66 20 0.3 45 6.60 21 0.5 51 6.52 22 1.0 56 6.39 22 1.0 61 6.45 20 1.3 Legend: pH = pH value of sample PMV% = volume % of culture in sample decolourisation = time necessary for decolourisation of methylene dye VEGETATIVE PHASE IN SEED FERMENTER Media for seed fermenter vol. of seed fermenter = 7500 1 vol. of media = 4500 1 Composition amount corn starch 90 kg soybean flour 90 kg NaH 2 P0 4 2.4 kg Estol (Priolube 1435)* 9 kg Synperonic 0.5 kg water to 4500 1 * Soybean oil can be used instead of estol . 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 μm. The vegetative phase lasted from 10 to 20 h at 28'±l'C, pressure 0.3 Bar, aeration by sterile air and constant mixing. Growth was monitoring by analysis of pH, PMV%, decolourisation of methylene and by microscopic examination of samples. Parameters of growth in pre-seed tank Time/h pH PMV% declourisation/min 0 7.20 6 7.10 15 > 5 12 6.87 20 1.5 16 6.65 22 0.3 Legend: pH = pH value of sample PMV% =- volume % of culture in sample decolourisation = time necessary for decolourisation of methylene dye B) BATCH FERMENTATION OF STREPTOMYCES SP. P 6621 FERM P 2804 IN FERMENTER Media for fermenters Vol. of fermenter = 90 000 1 Vol of media = 60 000 1 Composition amount corn starch 570 kg soybean flour 2300 kg NaCl 6 kg Estol ( Priolube 1435 ) * 1680 kg NaH 2 P0 4 5 kg MgCl 2 6H 2 0 7 kg FeCl 3 6H 2 0 1.6 kg ZnCl 2 0.5 kg MnS0 4 H 2 0 0.1 kg Synperonic 25 kg water to 60 m 3 Legend: - Estol is a generic name for glycerol trioleate; (Priolube 1435 registered trade mark of Unichem GmbH, Germany) Synperonic (registered trade mark of ICI GB) is a propylenglycol antifoam agent * Soybean oil can be used instead of Estol. 4700 1 of a culture of Streptomyces sp. PP 6621 FERM P 2804 in the vegetative phase of growth from the seed fermenter was inoculated by a sterile transfer into a sterile starting medium (60 0001) in a 90 000 1 stainless steel fermenter equipped for mixing and a delivery of sterile air through filters with a 0.2 μm pore size. The fermentation media and all inlet-pipes were sterilised and cooled by sterile air to 24 * C. The fermentation phase from seed fermenter was maintained at 24 * C - 25'C and 0.3 Bar. 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. During the course of the fermentation of Streptomyces sp. PP 6621 FERM P 2804 a source of phosphorus and an assimilable source of nitrogen (500 kg of soybean flour in 5000 1 of water and 25% aqueous solution of ammonium hydroxide) were added as follows : Concentration of assimilable sources of phosphorus and nitrogen in the fermentation broth time phosphorus nitrogen concentration (% w/v) concentrate (% w/v) 0 0,035 1, 73975 8 0,030625 1, 692286 16 0,0095 1, 331 24 0.005188 0, 9785 32 0,004638 0, 5527 40 0,003638 0, 69945 48 0,000863 0, 9128 56 0,000863 0, 8475 64 0 0, 709 72 0 0, 653625 80 0 0, 571 88 0 0, 47675 96 0 0,53825 104 0 0,7555 112 0 0, 77025 120 0 0, 673375 128 0 0, 78725 136 0 0, 734625 144 0 0, 8985 The concentration of phosphorus after the 51st hour of the fermentation was below the detection limit. The pH value reached in the first hours of the culture growth rose to almost 7.5. During this time phosphorus was consumed and clavulanic acid started to be produced, because of this the pH decreased and control of the pH of the media was necessary to maintain the level of pH at the optimum value. EXAMPLE 2 PROLONGATION OF THE VEGETATIVE PHASE OF FERMENTATION BY USE OF AMMONIUM SULPHATE AS ASSIMILABLE SOURCE OF NITROGEN AND SODIUM HYDROXIDE AS REGULATOR OF PH 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. Time Run 1, viscosity Run 2, viscosity (m Pa.s) (m Pa.s) 0 / / 8 / / 26 474 551 44 728 714 62 948 998 80 995 1076 98 936 1226 116 824 863 128 628 873

Claims: CLAIMS 1. A process for production of clavulanic acid comprising fermentation of a clavulanic acid producing species of Streptomyces in a fermentation broth containing assimilable sources of carbon and nitrogen, wherein the concentration of assimilable phosphorus in the fermentation broth is less than 0.15% w/v. 2. A process as claimed in claim 1, wherein the phosphorus concentration is maintained below a limit of 0.15% w/v during the growth phase. 3. A process as claimed in claim 2 wherein the phosphorus concentration is allowed to decrease after cessation of the growth phase. 4. A process as claimed in any preceding claim, wherein the phosphorus concentration is allowed to decrease after a fermentation time of 40 hours. 5. A process as claimed in any preceding claim, wherein the phosphorus concentration up to a fermentation time of 40 hours is 0.0015 to 0.15% w/v. 6. A process as claimed in claim 5, wherein the phosphorus concentration is 0.002 to 0.05% w/v. 7. A process as claimed in any preceding claim, wherein no assimilable phosphorus is added after a fermentation time of 40 hours. 8. A process a claimed in any preceding claim, wherein the source of assimilable nitrogen does not include ammonia. 9. A process as claimed in claim 8, wherein the sole source of assimilable nitrogen is not ammonia. 10. A process as claimed in any preceding claim, wherein the source of assimilable nitrogen is flour. 11. A process as claimed in any preceding claim, wherein the assimilable nitrogen source is ammonium sulphate. 12. A process as claimed in any preceding claim, wherein the concentration of the assimilable nitrogen source is 0.5 to 15% w/v. 13. A process as claimed in any preceding claim, wherein the starting concentration of the assimilable nitrogen source is greater than 5% w/v. 14. A process as claimed in any preceding claim, wherein the source of assimilable phosphorus is sodium or potassium phosphate, sodium or potassium dihydrogen phosphate or disodium or dipotassium hydrogen phosphate or a mixture thereof. 15. A process as claimed in any preceding claim, wherein the microorganism is Streptomyces clavuligerus, Streptomyces jumonjinensis, Streptomyces katsurahamanus or Streptomyces sp. P6621. 16. A process as claimed in any preceding claim, wherein the process is fed batch or continuous with intermittent or continuous addition of an assimilable source of phosphorus. 17. A process as claimed in any preceding claim, wherein the volume of the fermentation broth is greater than 10 4 1. 18. A process as claimed in claim 17, wherein the volume is 5 x IO 4 1.

Inventors: KRANJC SASA, RACMAN ARTUR, KRANJC, SASA
Applicants: BROWNE ROBIN FORSYTHE, KRANJC SASA, LEK TOVARNA FARMACEVTSKIH, RACMAN ARTUR, BROWNE, ROBIN, FORSYTHE, KRANJC, SASA, LEK PHARMACEUTICAL & CHEMICAL CO. DD
Application year: 1997
Main classification: C12P17
IPC codes: C12P17, C12R1