WO2008092950A1

WO2008092950A1 - Fermentation process for preparing pravastatin

Info

Publication number: WO2008092950A1
Application number: PCT/EP2008/051264
Authority: WO
Inventors: Matej Bizjak; Saso Kranjc; Peter Mrak; Gizela Stampar; Robert Mencigar; Danijel Smodis
Original assignee: Lek Pharmaceuticals D.D.
Priority date: 2007-02-02
Filing date: 2008-02-01
Publication date: 2008-08-07
Also published as: EP2115152A1; EP1953233A1

Abstract

The present invention relates to the improved production of pravastatin by fermentation using unpurified or partially purified raw materials or precursors.

Description

FERMENTATION PROCESS FOR PREPARING PRAVASTATIN

FIELD OF THE INVENTION

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.

BACKGOUND OF THE INVENTION

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. As 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. As also seen from 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 300μg/ml_ during the process.

In EP-A-O 776 974, a 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.

DISCLOSURE OF THE INVENTION

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.

We have developed a process where by careful selection of media and fermentation condition we were able to dispose of the step of isolation of mevastatin from the first fermentation broth. That is very beneficial from economical point of view. In spite of using impure mevastatin-containing raw materials in the form of a fermentation broth or a treated form derived therefrom, a surprisingly high yield of pravastatin substantially above 1 g/L, preferably 2g/L or higher, particularly 4g/L or higher and even up to 6g/L could be finally achieved, while at the same time the final amount of mevastatin in the final fermentation broth could be kept below 0.1 g/L. Although the new process is already more economic from the procedural point of view, yet a total conversion rate of higher than 80%, more preferably 90% and higher and in particular 95% and higher can be advantageously achieved, respectively determined as w/w by HPLC and referred to the amount of mevastatin feeded to the final (usually the second) fermentation broth. In such procedure 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.

In specific aspects, 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. Thus, while an untreated or treated broth can be effectively used, a process directed to provide mevastatin in crude, semi-purified, at least substantially purified or even isolated form, or eventually providing isolated/purified mevastatin in the form of a solution, can be advantageously omitted according to the present invention.

According to a preferred embodiment, it is especially effective to alkalize the first fermentation broth as it is when mevastatin is at an appropriate concentration, then acidifying or neutralizing it for lowering pH before being added to the second fermentation broth of the second microorganism capable of converting mevastatin to pravastatin. Preferably, 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 10⁰C and preferably to a range of about 12O⁰C or above. When the first fermentation broth treated as mentioned above is at an increased temperature, it is subsequently allowed to cool or actively cooled to a range below, for example, 37⁰C and preferably below 30⁰C before being added to the second fermentation broth.

In specific aspect said mevastatin producing microorganism is Penicillium citrinum, and a microorganism capable of converting mevastatin to pravastatin belongs the genus Streptomyces, more specifically it is Streptomyces Carbophylus.

In more specific aspect 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. During further fermentation process, it is preferred that the concentration of mevastatin is maintained below 3 g/L, more preferably between 0,1 and 1 g/L.

In a process aspect 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.

Specifically this is performed in a process which comprises steps: culturing Penicillium citrinum in a first medium containing glycerol, glucose, yeast extract, soy meal, NaNO₃ , MgSO₄ at temperature around 25⁰C 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 3O⁰C; (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 28⁰C while controlling the pH of the fermentation broth to be in the range between 7.2 and 8 by addition of an aqueous solution of glucose; and isolating pravastatin. 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.

Aspect 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.

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. The present invention will be described in more detail by referring to the following illustrative preferred embodiments and examples, the present invention however not being limited thereto.

In accordance with a particular embodiment of the present invention, 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.

In a further specific embodiment, a seed medium containing glycerol 0.5-5%, glucose 2-8%, yeast extract 1 -3%, soy meal 2-8%, NaNO₃ 0.1 -1%, MgSO₄ 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%, NaNO₃ 0.1 -1%, MgSO₄ 0.1 -0.5%, antifoaming agent 0.05-0.2%, where it is grown at 24+/-2⁰C 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/cornsteep powder 0-15%, NaNO₃ 0-0.3%, ZnSO₄ 0-0.1%, antifoaming agent 0.05-0.2% where it is grown at 24+/-2 ⁰C for up to 350 hours .

To increase yield during the fermentation to the medium is continuously added 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 ⁰C for half hour and cooled. Thereafter or before the sterilisation the pH of said broth is optionally adjusted to around 8.

At the same time medium in 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+/-2⁰C. In case of large scale production also 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). In both cases 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 ⁰C. During the growth 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.

During the fermentation 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.

In the first step of pravastatin isolation 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.

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.

Example

A seed medium containing glycerol, glucose, yeast extract, soy meal, NaNO₃ , MgSO₄ is inoculated with a culture of Penicilium citrinum for 2 days hours at 25⁰C, 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, NaNO₃, ZnSO₄, where it is grown at 25 ⁰C 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 ⁰C for half hour and cooled.

At the same time 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

3O⁰C. Thereafter 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 ⁰C.

Upon raise of pH from around 6,5 to above 7,1 , which indicates that glucose has been consumed the above cooled mevastatin containing broth is in batches added to the fermentor, taking care that the concentration of mevastatin in fermentor is bellow 1 g/l. The fermentation is continued for 5 days maintaining the pH of fermentation broth between 7,2 and 8 with aqueous solution of glucose .

After the fermentation 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.

Claims

1. A process for preparing pravastatin, which process comprises steps of: providing fermentation broth of a mevastatin producing microorganism, wherein said fermentation broth comprises mevastatin; contacting said fermentation broth comprising mevastatin with a microorganism capable of converting mevastatin to pravastatin.

2. The process according to claim 1 , wherein said fermentation broth of a mevastatin producing microorganism is a first fermentation broth comprising mevastatin, and wherein said first fermentation broth is added to another, second fermentation broth containing said microorganism capable of converting mevastatin to pravastatin.

3. The process according to claim 1 or 2, wherein said fermentation broth comprising mevastatin is selected from the group consisting of alkalized broth, acidified broth and sterilized broth.

4. A process according to any one of the previous claims, wherein mevastatin producing microorganism is Penicillium citrinum, and a microorganism capable of converting mevastatin to pravastatin belongs the genus Streptomyces.

5. A process according to any one of previous claims, wherein mevastatin producing microorganism fermentation broth contains mevastatin in concentration from 10 to 25 g/L and said fermentation broth has been sterilized prior to contacting with a microorganism capable of converting mevastatin to pravastatin.

6. A process according to any one of previous claims, wherein in the medium in which fermentation broth of a mevastatin producing microorganism is contacted with microorganism of the genus Streptomyces, the concentration of mevastatin is maintained below 3 g/L

7. A process according to claim 4 or 6, where a microorganism of the genus Streptomyces is Streptomyces carbophylus.

8. A process according to any one of the preceding claims, which comprises the steps of: a.) culturing Penicillium citrinum in a first medium sustaining growth thereof under condition capable of producing mevastatin, to provide a fermentation broth containing mevastatin; b.) optionally treating said fermentation broth containing mevastatin to obtain treated broth selected from the group consisting of alkalized broth, acidified broth and sterilized broth; c.) feeding fermentation broth containing mevastatin in untreated or treated form to a second medium where a microorganism capable of converting mevastatin to pravastatin is grown; d.) further growing said microorganism capable of converting mevastatin to pravastatin in said second medium under condition capable of producing pravastatin; and e.) isolating pravastatin.

9. A process according to any one of the preceding claims, which comprises the steps of: a.) culturing Penicillium citrinum in a first medium containing glycerol, glucose, yeast extract, soy meal, NaNO₃ , MgSO₄ at temperature around 25⁰C for up to 3 days giving a fermentation broth containing mevastatin; b.) alkalizing said fermentation broth to pH between 10 and 10,5; c.) optionally sterilizing said fermentation broth; d.) optionally cooling said sterilized fermentation broth to below 30⁰C e.) optionally acidifying said fermentation broth to pH around 8; f.) 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; g.) further growing said microorganism capable of converting mevastatin to pravastatin in said second medium at temperature around 28⁰C while controlling the pH of the fermentation broth to be in the range between 7.2 and 8 by addition of an aqueous solution of glucose or acid ; and h.) isolating pravastatin.

10. A process for growing an organism capable of converting mevastatin to pravastatin in a medium comprising glucose, characterized in that after glucose has been consumed, to said medium a fermentation broth containing mevastatin is added continuously or in batches in a manner that the concentration of mevastatin in said broth is kept between 0.1 and 3g/L

1 1. Use of a broth, produced during fermentation of Penicillium citrinum under condition capable of producing mevastatin, as a substrate in a fermentation process of a microorganism capable of converting mevastatin to pravastatin.

12. Use according to claim 1 1 , wherein a microorganism capable of converting mevastatin to pravastatin is selected from the group consisting of Saccharopolyspora hirsute, Amycolata autotrophica, Streptomyces carbophylus, Streptomyces californicus, Amycolata hydrocarbon-noxydans, Amycolatopsis fastidiosa and Streptomyces roseochromogenes.

13. A fermentation broth comprising: a microorganism capable of converting mevastatin to pravastatin; pravastatin at a concentration of 2g/L or higher; and a final amount of mevastatin of below 0.1 g/L.

14. The fermentation broth according to claim 13, comprising pravastatin at a concentration of 4g/L or higher.

15. The fermentation broth according to claim 13 or 14, wherein the ratio of pravastatin to mevastatin is defined by a total conversion rate of higher than 80%.

16. The fermentation broth according to claim 13 or 14, wherein the ratio of pravastatin to mevastatin is defined by a total conversion rate of higher than 90%.

17. The fermentation broth according to claim 13 or 14, wherein the ratio of pravastatin to mevastatin is defined by a total conversion rate of higher than 95%.

18. The fermentation broth according to any one of claims 13 to 17, wherein the microorganism capable of converting mevastatin to pravastatin is selected from the group consisting of Saccharopolyspora hirsute, Amycolata autotrophica, Streptomyces carbophylus, Streptomyces californicus, Amycolata hydrocarbon-noxydans, Amycolatopsis fastidiosa and Streptomyces roseochromogenes.

19. Use of a fermentation broth according to any one of claims 13 to 17 for producing purified or isolated pravastatin in salt form or in free acid form, optionally with a subsequent step of including the purified or isolated pravastatin into a pharmaceutical formulation.