EP2115152A1 - Procédé de fermentation pour la préparation de pravastatine - Google Patents

Procédé de fermentation pour la préparation de pravastatine

Info

Publication number
EP2115152A1
EP2115152A1 EP08708571A EP08708571A EP2115152A1 EP 2115152 A1 EP2115152 A1 EP 2115152A1 EP 08708571 A EP08708571 A EP 08708571A EP 08708571 A EP08708571 A EP 08708571A EP 2115152 A1 EP2115152 A1 EP 2115152A1
Authority
EP
European Patent Office
Prior art keywords
mevastatin
pravastatin
fermentation broth
broth
microorganism
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP08708571A
Other languages
German (de)
English (en)
Inventor
Matej Bizjak
Saso Kranjc
Peter Mrak
Gizela Stampar
Robert Mencigar
Danijel Smodis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lek Pharmaceuticals dd
Original Assignee
Lek Pharmaceuticals 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 Pharmaceuticals dd filed Critical Lek Pharmaceuticals dd
Priority to EP08708571A priority Critical patent/EP2115152A1/fr
Publication of EP2115152A1 publication Critical patent/EP2115152A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • 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
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the invention in general relates to production of fermentation products using unpurified or partially purified raw materials or precursors. Raw materials or precursors for those fermentations are also gained from fermentations. More specifically the present invention from a field of pharmaceutics relates to a novel process for preparing pravastatin by means of a microbiological process.
  • Pravastatin and the salts thereof, in particular sodium salt are HMG-CoA reductase inhibitor: disclosed in GB Pat. No. 1 ,555,831.
  • Pravastatin is generally produced by two step fermentation, where first mevastatin (also known as ML-236B or compactin) is produced anc isolated and added as a substrate to a medium where various microorganisms capable of converting mevastatin to pravastatin are grown, which subsequently convert mevastatin into pravastatin, which is subsequently isolated by known methods.
  • microorganisms capable of converting mevastatin to pravastatin usually of the genera Gilbertella, Streptomyces, Circinella, Monascus, Nocardia, Amycolata, Mucor or Penicilliumare used, and specifically Streptomyces Carbophylus, Saccharopolyspora hirsute, Amycolata autotrophica, Streptomyces carbophylus, Streptomyces californicus, Amycolata hydrocarbon-noxydans, Amycolatopsis fastidiosa, Streptomyces roseochromogenes are accessible in public culture collections.
  • the fermentations are biotransformation processes where organisms are grown in medium while producing an industrially useful substance from simple precursors, like carbon and nitrogen sources, or converting a substance in their feed into another industrially useful substance.
  • the two step fermentation processes for the production of pravastatin are, for example, mentioned in WO 98/45410 and WO 99/10499.
  • the second fermentation step may conventionally involve bacteria as described for example by EP-A-O 649 907.
  • the ring-opened compactin salt is produced by alkalization of the isolated lactone form, and compactin salt or acid form is added as substrate to the second fermentation process.
  • WO 99/10499 proposes a fermentation process with fungal host cells transformed by a foreign hydroxylase gene so that pravastatin is produced directly in one culture system.
  • WO 98/45410 uses an isolated Strepromyces exfoliatus strain said to have strong tolerance to relatively high ML-236B concentrations.
  • WO 98/06867 is specifically directed to the production of pravastatin percursor ML-236B and for this purpose uses a microorganism classified as a Gliocladium species. After fermentation process under described fermentation conditions, ML-236B is obtained purified or isolated as free acid form, lactone form or, after saponification of the lactone, as sodium salt form.
  • WO 2004/087935 performs the second fermentation step under conditions that the concentration of added compactin is maintained at a level not less than 300 ⁇ g/ml_ during the process.
  • cytochrome P-450 promoter was isolated from Streptomyces carbophilus and used to produce transformed S. carbophilus or S. lividans capable of converting ML-236B to pravastatin by the promoter activity which is substrate inducible.
  • Substrate induction is performed in a working example (Example 3) by adding isolated ML- 236B to media containing S. lividans strains.
  • ML-236B-producing Penicillium citrinum may be co-cultivated with the transformed Streptomyces strain, thereby requiring co-cultivation of different microorganisms while still allowing plasmid-based cytochrome P-450 expression through substrate induction.
  • Mevastatin is preferably a product of fermenting a fungus Penicillium citrinum as accessible in public culture collections, such as ATCC 38065, while to produce pravastatin, one preferably ferments a bacteria Streptomyces carbophylus as accessible in public culture collections such as SANK 62585.
  • the conditions to optimally grow fungi and bacteria, or in general mevastatin producing organisms and mevastatin-to-pravastatin converting organisms are usually different, and they require different media.
  • the usual approach to produce pravastatin is thus to isolate mevastatin from first fermentation broth of first microorganism, usually in form of a salt and feed the solution of this salt to the second fermentation broth of second microorganism.
  • mevastatin-containing broth can be added also in form of permeate, retentate, extract or similar. According to a preferred embodiment, it is most efficient when the fermentation broth is added as it is, or as processed only by alkalization and neutralization, optionally also by sterilization.
  • An aspect of the invention is a process for preparing pravastatin which process comprises contacting fermentation broth (first fermentation broth) of a mevastatin producing microorganism (first organism) with a microorganism (second organism) capable of converting mevastatin to pravastatin.
  • the first fermentation is a portion or all of untreated or treated broth of the mevastatin producing microorganism, but preferably it is a treated form of broth including but not limited to used broth, partially purified broth, broth permeate, broth retentate, broth extract, alkalized broth, acidified broth and sterilized broth, respectively alone or in combination.
  • the broth of the mevastatin-producing microorganism in addition to containing mevastatin at an appropriate level, may thus further contain unpurified or partially purified raw materials of the first fermentation broth and is contacted with a microorganism capable of converting mevastatin to pravastatin.
  • the first fermentation broth is sterilized after alkalization and before lowering pH, or after lowering pH. Sterilization may be accomplished by a raise in temperature, for example to a range above 1 10 0 C and preferably to a range of about 12O 0 C or above.
  • a raise in temperature for example to a range above 1 10 0 C and preferably to a range of about 12O 0 C or above.
  • said mevastatin producing microorganism is Penicillium citrinum
  • a microorganism capable of converting mevastatin to pravastatin belongs the genus Streptomyces, more specifically it is Streptomyces Carbophylus.
  • said first fermentation broth is sterilized prior to contacting with said second organism.
  • Contacting with said second organism is specifically done in second fermentation broth.
  • the concentration of mevastatin is maintained below 3 g/L, more preferably between 0,1 and 1 g/L.
  • the invention provides for preparation of pravastatin comprising steps: culturing Penicillium citrinum in a first medium sustaining growth thereof under condition capable of producing mevastatin giving a fermentation broth containing mevastatin; feeding said fermentation broth containing mevastatin to a second medium where a microorganism capable of converting mevastatin to pravastatin is grown; further growing said microorganism capable of converting mevastatin to pravastatin in said second medium under condition capable of producing pravastatin; and isolating pravastatin.
  • this is performed in a process which comprises steps: culturing Penicillium citrinum in a first medium containing glycerol, glucose, yeast extract, soy meal, NaNO 3 , MgSO 4 at temperature around 25 0 C for up to 3 days giving a fermentation broth containing mevastatin; alkalizing said fermentation broth to pH between 10 and 10,5; (optionally) sterilizing said fermentation broth; (optionally) cooling said sterilized fermentation broth to below 3O 0 C; (optionally) acidifying said fermentation broth to pH around 8 (where this step can be before or after sterilizing and/or cooling step); feeding said sterilized fermentation broth containing mevastatin to a second medium containing glucose, glycerol, soy meal, cornsteep liquor and cornsteep powder, cotton seed meal, and yeast extract where a microorganism Streptomyces carbophylus has been grown; further growing said microorganism capable of converting mevastatin to pravastatin in said second medium at temperature around 28 0
  • Alternative aspect of the invention relates to the growth of an organism capable of converting mevastatin to pravastatin, preferably Streptomyces carbophylus in medium comprising glucose characterized by that after glucose has been consumed, to said medium the fermentation broth containing mevastatin is added continuously or in batches in a manner that the concentration of mevastatin in said broth is between 0.1 and 3g/L.
  • aspects of the invention is also the use of the broth, specifically use of sterilized broth, or alkalized broth or alkalized, sterilized, cooled and acidified broth, produced during fermentation of Penicillium citrinum under condition capable of producing mevastatin, as a substrate in fermentation of a microorganism capable of converting mevastatin to pravastatin, specifically where a microorganism capable of converting mevastatin to pravastatin is selected from Saccharopolyspora hirsute, Amycolata autotrophica, Streptomyces carbophylus (preferred), Streptomyces californicus, Amycolata hydrocarbon- noxydans, Amycolatopsis fastidiosa, Streptomyces roseochromogenes.
  • a further aspect of the invention is a fermentation medium capable to sustain growth of bacteria (preferably Streptomyces), containing between 1 and 30% vol/vol, preferably up to 10% vol/vol of broth, (preferably containing at least 1% of mevastatin) produced during fermentation of Penicillium citrinum under condition capable of producing mevastatin.
  • bacteria preferably Streptomyces
  • containing between 1 and 30% vol/vol preferably up to 10% vol/vol of broth, (preferably containing at least 1% of mevastatin) produced during fermentation of Penicillium citrinum under condition capable of producing mevastatin.
  • a further aspect is a fermentation broth which comprises a microorganism capable of converting mevastatin to pravastatin; pravastatin at a concentration of 2g/L or higher, particularly 4g/L or higher and even up to 6g/L; while at the same time the final amount of mevastatin in the final fermentation broth is kept below 0.1 g/L.
  • This final fermentation broth obtainable by the process according to the present invention, which fermentation broth may still contain ingredients from the first fermentation broth of mevastatin producing microorganism, is particularly useful as a starting material for isolating and/or purifying pravastatin.
  • the remarkable high yield in pravastatin combined with a very low mevastatin in the final fermentation broth, and thus the remarkably high total conversion rate of higher than 80%, more preferably 90% or higher and even 95% or higher achieved by the process according to the present invention enables a much easier purification and isolation scheme to be subsequently performed from the final fermentation broth, for finally obtaining pravastatin in a form directly suitable for use in a pharmaceutical preparation, such as in salt form or in free acid form.
  • a pharmaceutical preparation such as in salt form or in free acid form.
  • mevastatin producing fungi is fermented in first medium containing carbon source such as glycerol, glucose, nitrogen source such as yeast extract, soy meal, minerals, antifoaming agent.
  • Mevastatin containing broth of this fermentation is alkalized, optionally sterilized and cooled and added as feed to the second medium containing carbon source such as glucose; nitrogen source such as soy meal, proflo, cotton seed meal; antifoaming agent wherein bacteria able to hydroxylate mevastatin to pravastatin has been grown for some time.
  • the concentration of mevastatin in said second medium is kept between 0.1 and 3g/L.
  • the pravastatin produced is isolated from this broth.
  • a seed medium containing glycerol 0.5-5%, glucose 2-8%, yeast extract 1 -3%, soy meal 2-8%, NaNO 3 0.1 -1%, MgSO 4 0.1 -0.5%, antifoaming agent 0.05-0.2% is inoculated with a culture of Penicilium citrinum for up to 120 hours at 24+/-2°C, whereupon the matured seed culture is grown in a prefermentor containing glycerol 0.5-5%, glucose 2-8%, yeast extract 1 -3%, soy meal 2-8%, NaNO 3 0.1 -1%, MgSO 4 0.1 -0.5%, antifoaming agent 0.05-0.2%, where it is grown at 24+/-2 0 C for up to 50 hours and from preferementor added to a fermentor containing sucrose 5-30%, glycerol 0-10%, glucose 0- 30%, yeast extract 0-1 .5%, soy meal 1 -6%, cornsteep liquor/cornste
  • an aqueous solution of carbon source such as sucrose, glucose, glycerol.
  • the amount of produced mevastatin is monitored by HPLC and the fermentation is stopped when the concentration is 10 to 25g/L,
  • the mevastatin containing broth is thereafter added an alkali (NaOH) to pH 10 -1 1 ,0, optionally sterilized at 121 0 C for half hour and cooled. Thereafter or before the sterilisation the pH of said broth is optionally adjusted to around 8.
  • NaOH alkali
  • prefermentor containing glucose 0-3%, glycerol 0-3%, soy mealO.5-1.5, yeast extract 0.1 -1% and antifoaming agent 0.05-0.2% is inoculated with a culture of Streptomyces carbophilus and grown for up to 80 hours at 28+/-2 0 C.
  • two step seed phase can be used. Where in first propagator phase the same seed medium as for prefermentor is used. After about 50 h cultivation at 28+/-2°C the culture is transferred to prefermentor phase.
  • the prefermentor phase in case of two step process the prefermentor phase is shorter (up to 4Oh).
  • the grown biomass is transferred to fermentor containing glucose 0-2%, glycerol 0-2%, soy meal 1 -4%, CSL (cornsteep liquor)/CSP (cornsteep powder) 0-0.5%, cotton seed meal 0-0.5%, yeast extract 0-0.5%, antifoaming agent 0.05-0.2% and grown for about 15 to 40 hours at 28 ⁇ 2 0 C.
  • the pH is monitored and upon raise of pH the cooled above mevastatin containing broth is continuously or in batches added to the fermentor, taking care that the concentration of mevastatin in fermentor is bellow 3 g/l, more preferably 1 g/l.
  • the fermentation is continued for up to 160 hours, where an aqueous solution of glucose is added to maintain the pH of fermentation broth between 7,2 and 8,2. Alternatively if the pH of mevastatin contained broth is not adjusted to around 8, an acid is used to control the pH.
  • mevastatin is converted to pravastatin, with a remarkably high final yield up to 6g/L in spite of adding not isolated mevastatin but only a crude first microorganism fermentation broth containing mevastatin, where the final amount of mevastatin is below 0.1 g/L, and after the fermentation the pravastatin is isolated from the second broth.
  • the second fermentation broth is charged to a microfiltration unit, where the mycelium is separated from the filtrate.
  • the permeate is led to reverse osmosis unit to be additionally concentrated.
  • the obtained concentrate (retentate) is led to extraction, where it is acidified and extracted to organic solvent.
  • the obtained Pravastatin extract is collected and concentrated.
  • Pravastatin TBA salt After concentration the tert butyl amine is added to obtain Pravastatin TBA salt. Some solvents, like water, methanol, acetone may be added to obtain pure Pravastatin TBA crystals. The obtained crystals are filtered and dried, and if desired converted to free acid or sodium salt. The pure Pravastatin in free acid or salt form can be used to prepare a pharmaceutical formulation together with suitable pharmaceutically acceptable carriers or excipients.
  • a seed medium containing glycerol, glucose, yeast extract, soy meal, NaNO 3 , MgSO 4 is inoculated with a culture of Penicilium citrinum for 2 days hours at 25 0 C, whereupon the matured seed culture is grown in a refermentor containing same medium, where it is grown same temperature for another 2 days and from preferementor added to a fermentor containing sucrose, glycerol, glucose, yeast extract, soy meal, cornsteep liquor/cornsteep powder, NaNO 3 , ZnSO 4 , where it is grown at 25 0 C for 10 days. During the fermentation to the medium is continuously added an aqueous solution of sacharose.
  • the mevastatin containing broth is thereafter added an alkali to pH 10 -10,5, sterilized at 121 0 C for half hour and cooled.
  • medium in prefermentor containing glucose, glycerol, soy meal, and yeast extract is inoculated with a culture of Streptomyces Carbophylus and grown for 3 days at
  • the grown biomass is transferred to fermentor containing glucose, glycerol, soy meal, CSL /CSP, cotton seed meal, and yeast extract and grown for one day at 28 ⁇ 2 0 C.
  • the pravastatin is isolated from the broth, which is first charged to the microfiltration unit, where the mycelium is separated from the filtrate which is acidified and extracted to ethyl acetate. After concentration the tert butyl amine is added to obtain Pravastatin tert butyl ammonium salt, which is isolated, and trans-salified to sodium salt.

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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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur la production améliorée de pravastatine par fermentation au moyen de matières premières ou de précurseurs non purifiés ou partiellement purifiés
EP08708571A 2007-02-02 2008-02-01 Procédé de fermentation pour la préparation de pravastatine Withdrawn EP2115152A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP08708571A EP2115152A1 (fr) 2007-02-02 2008-02-01 Procédé de fermentation pour la préparation de pravastatine

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP07101628A EP1953233A1 (fr) 2007-02-02 2007-02-02 Procédé de fermentation pour la préparation de la pravastatine
PCT/EP2008/051264 WO2008092950A1 (fr) 2007-02-02 2008-02-01 Procédé de fermentation pour la préparation de pravastatine
EP08708571A EP2115152A1 (fr) 2007-02-02 2008-02-01 Procédé de fermentation pour la préparation de pravastatine

Publications (1)

Publication Number Publication Date
EP2115152A1 true EP2115152A1 (fr) 2009-11-11

Family

ID=38255815

Family Applications (2)

Application Number Title Priority Date Filing Date
EP07101628A Ceased EP1953233A1 (fr) 2007-02-02 2007-02-02 Procédé de fermentation pour la préparation de la pravastatine
EP08708571A Withdrawn EP2115152A1 (fr) 2007-02-02 2008-02-01 Procédé de fermentation pour la préparation de pravastatine

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP07101628A Ceased EP1953233A1 (fr) 2007-02-02 2007-02-02 Procédé de fermentation pour la préparation de la pravastatine

Country Status (2)

Country Link
EP (2) EP1953233A1 (fr)
WO (1) WO2008092950A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108977474A (zh) * 2018-04-20 2018-12-11 北大方正集团有限公司 一种美伐他汀的制备方法
CN111961615B (zh) * 2020-08-13 2021-07-27 江南大学 一株降低生物胺的糖多孢菌及其应用
JP7329221B2 (ja) * 2020-08-13 2023-08-18 江南大学 サッカロポリスポラ組成物及びその食品における使用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6043064A (en) * 1993-10-22 2000-03-28 Bristol-Myers Squibb Company Enzymatic hydroxylation process for the preparation of HMG-CoA reductase inhibitors and intermediates thereof
US5830695A (en) * 1995-11-29 1998-11-03 Sankto Company, Limited Actinomycete promoter
KR100186758B1 (ko) * 1996-08-09 1999-04-01 영진약품공업 주식회사 프라바스타틴(pravastatin)전구체의제조방법
KR100210482B1 (ko) * 1997-04-10 1999-07-15 김종인 스트렙토마이세스엑스포리아투스(streptomycesexfoliatus)yj-118과이를이용한프라바스타틴나트륨의제조방법
EP1015600A1 (fr) * 1997-08-22 2000-07-05 Dsm N.V. Production de statine par fermentation
EP1613760A2 (fr) * 2003-04-01 2006-01-11 Ranbaxy Laboratories, Ltd. Processus de fermentation pour la preparation de pravastatine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2008092950A1 *

Also Published As

Publication number Publication date
WO2008092950A1 (fr) 2008-08-07
EP1953233A1 (fr) 2008-08-06

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