Process for the Production of Lipstatin and Tetrahydrolipstatin

Europäisches Patentamt *EP000803576B1* (19) European Patent Office

Office européen des brevets (11) EP 0 803 576 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: C12P 17/02 of the grant of the patent: // (C12P17/02, C12R1:04) 04.12.2002 Bulletin 2002/49

(21) Application number: 97106445.6

(22) Date of filing: 18.04.1997

(54) Process for the production of lipstatin and tetrahydrolipstatin Verfahren zur Herstellung von Lipstatin und Tetrahydrolipstatin Procédé de préparation de lipstatine et de tétrahydrolipstatine

(84) Designated Contracting States: • Weber, Wolfgang AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL 79639 Grenzach-Wyhlen (DE) PT SE (74) Representative: Witte, Hubert, Dr. et al (30) Priority: 26.04.1996 EP 96106598 124 Grenzacherstrasse 4070 Basel (CH) (43) Date of publication of application: 29.10.1997 Bulletin 1997/44 (56) References cited: EP-A- 0 129 748 (73) Proprietor: F. HOFFMANN-LA ROCHE AG 4070 Basel (CH) • CHEMICAL ABSTRACTS, vol. 107, no. 21, 23 November 1987 Columbus, Ohio, US; abstract (72) Inventors: no. 196439, WEIBEL, E. K. ET AL: "Lipstatin, an • Bacher, Adelbert inhibitor of pancreatic lipase, produced by 85748 Garching (DE) Streptomyces toxytricini. I. Producing • Stohler, Peter organism, fermentation isolation and biological 4415 Lausen (CH) activity" XP002094769 & J. ANTIBIOT. (1987), 40(8), 1081-5 CODEN: JANTAJ;ISSN: 0021-8820,

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 803 576 B1

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Description uct, lipstatin, in a much higher concentration. [0006] The biosynthetic pathway leading to lipstatin is [0001] The invention relates to a novel fed-batch proc- subject to a series of general control mechanisms, such ess for the fermentative production of lipstatin, which as nitrogen repression. The addition of readily available process comprises 5 nitrogen sources, especially amino acids and their de- rivatives, including L-leucine or N-Formyl-L-leucine, to a) aerobically cultivating a micro-organism of the or- the medium strongly represses the formation of lipstatin. der of actinomycetes which produces lipstatin, in an In the present invention this is overcome by starting the aqueous culture medium which is substantially free addition of these amino acids only after the biosynthetic of fats and oils, and which contains suitable carbon 10 enzymes have been formed and thus are no longer re- and nitrogen sources and inorganic salts, until the pressed on a genetical level. In addition, L-leucine or N- initial growth phase is substantially finished and suf- formyl-L-leucine is added in such a way, that their con- ficient cell mass has been produced, and centration in the broth remains low. [0007] Conveniently, the actinomycete producing lip- b) adding to the broth linoleic acid, optionally togeth- 15 statin is grown in a suitable aqueous basal medium con- er with caprylic acid, [wherein part or the totality of taining one ore more carbon sources, such as starch, the linoleic acid and/or of the caprylic acid can be starch hydrolysates and sugars, e.g., glucose and/or su- replaced by the corresponding ester(s) and/or salt crose, glycerol, phospholipids, as well as one or more (s)], and N-formyl-L-leucine or preferably L-leucine, nitrogen sources, such as soybean flour, cotton seed the linoleic acid or its ester(s) or salt(s) being stabi- 20 flour, corn steep powder or corn steep liquor and yeast lized by an antioxidant. extract. Both carbon and nitrogen sources are supplied in such amounts that an abundant growth is enabled, [0002] Lipstatin, a fermentative process for its produc- typically in a range of 5 to 50 grams of each carbon tion, a process for its isolation from the broth and a proc- source and of each nitrogen source per liter of medium. ess for its hydrogenation to tetrahydrolipstatin (THL, or- 25 Further, macro- and micro-elements are added to the listat, an anti-obesity agent) are described in US Pat. medium. They might be contained in complex media 4,598,089. components or added as inorganic salts. [0003] The organism producing lipstatin as described [0008] The aqueous culture medium is substantially in US Pat. 4,598,089 is Streptomyces toxytricini Preo- free of fats and oils and contains less than 10 grams of brazhenskaya & Sveshnikova (see Bergey's Manual of 30 triglycerides per liter of medium. Conveniently, linoleic Determinative Bacteriology, 8th edition, page 811). It acid and/or caprylic acid and/or their esters or salts are was deposited on June 14, 1983, at the Agricultural Re- fed at such a rate as to be freely available in the broth search Culture Collection, Peoria, Ill. under the desig- but so that their accumulation is prevented, particularly nation NRRL 15443. The process of the instant inven- at a rate of 10 to 1000, preferably of 100 to 300 mg per tion can be performed with this organism or with any oth- 35 liter and hour. The feeding of linoleic acid and caprylic er strain derived of it, such as a mutant strain selected acid and/or their salts or of their esters is preferably con- for a better productivity. It can also be performed with ducted so that their concentration in the broth remains other lipstatin producing organisms of the order actino- inferior to 1000, preferably inferior to 300 mg per liter, mycetes, either belonging to the family of streptomyc- and discontinued so that the broth is practically free of etes or belonging to another family within the order of 40 said fatty acid(s) and/or its esters or salts before lipstatin actinomycetes. is isolated. Conveniently, the linoleic acid and caprylic [0004] When applying a fermentation system as de- acid are added to the broth in a ratio of 1 to 10, preferably scribed in US Patent 4,598,089, the fermentation broth 1.5 to 3 parts per weight of linoleic acid being added for after cultivation contains lipstatin in very small amounts 1 part of caprylic acid. Conveniently, N-formyl-L-leucine of a few milligram per liter and it is difficult to isolate it 45 or preferably L-leucine is added to the broth at a rate of by economically and technically feasible methods. 1 to 100, preferably 5 to 50 mg per liter of broth per hour, [0005] The present invention provides an improved so that its concentration remains less than 25 millimolar. process for the fermentative production of lipstatin, oc- [0009] Examples of salts and esters which can be curring in the fermentation broth with a higher concen- substituted for a part (or for the totality) of the linoleic tration by using the fed-batch process described above. 50 acid or of its mixture with caprylic acid are alkaline or In the first step a) of this process the cells of the lipstatin alkaline earth metal salts, e.g. sodium, potassium, cal- producing micro-organism are grown in a basal medi- cium or magnesium salts, and lower alkyl esters, e.g. um. In the second step b) of this process, to this basal methyl esters, or glycerides. medium certain components are added, which either [0010] In order to prevent the oxidation of linoleic acid serve directly as biochemical precursors or undergo a 55 [or of its ester(s) or salt(s)] it is mixed with an antioxidant, short biochemical conversion and then serve as precur- such as ascorbyl palmitate, tocopherol, lecithin, or mix- sors of the biosynthetic pathway. By this system the mi- tures thereof, and/or a radical trapping agent, such as cro-organism is enabled to synthesise the desired prod- BHA (tert.-butyl-4-hydroxy-anisol) or BHT (2,6-ditert.-

2 3 EP 0 803 576 B1 4 butyl-p-cresol). of this medium is filled into a 500 ml Erlenmeyer [0011] The invention further relates to a process for flask, closed with a cotton plug and sterilised. It is the production of tetrahydrolipstatin, which process then inoculated with a loopful of spores of Strepto- comprises myces toxytricini strain NRRL 15443 and subse- 5 quently incubated under shaking at 27°C for 24 a) aerobically cultivating a micro-organism of the or- hours. der of actinomycetes which produces lipstatin, in an aqueous culture medium which is substantially free b) 100 ml of this seed culture is used to inoculate a of fats and oils, and which contains suitable carbon fermentor with a vessel size of 14 l containing 8 l of and nitrogen sources and inorganic salts, until the 10 a production medium containing per liter: 32 g of initial growth phase is substantially finished and suf- defatted soybean flour, 20 g of glycerol, 14 g of lec- ficient cell mass has been produced, ithin, 0.25 ml of polypropylene glycol as an antifoam agent, whereas the pH is adjusted to 7.4 with NaOH b) adding to the broth linoleic acid, preferably to- 28 %. The medium contains less than 5 grams per gether with caprylic acid, [wherein part or the totality 15 liter of triglycerides. of the linoleic acid and/or of the caprylic acid can be replaced by the corresponding ester(s) and/or salt c) After a growth phase of 47 hours the feeding is (s)], and N-formyl-L-leucine or preferably L-leucine, started. It consists of the fatty acids linoleic acid and the linoleic acid or its ester(s) or salt(s) being stabi- caprylic acid, whereby linoleic acid is stabilized by lized by an antioxidant, 20 the addition of 0.2 % (w/w) of an antioxidant (RON- OXAN A) consisting of 70% (w/w) of lecithin, 25% c) isolating lipstatin from the broth and hydrogenat- of ascorbyl palmitate and 5% of tocopherol. These ing lipstatin to tetrahydrolipstatin. fatty acids are added at a rate of 136 to 190 mg per liter and hour, and the rate of addition is adjusted in [0012] The isolation of the lipstatin from the fermen- 25 such a way that the concentration of each linoleic tation broth can be carried out according to methods acid and caprylic acid remains below 70 mg per liter. which are known per se and which are familiar to any Totally added in the course of the fermentation are person skilled in the art. For example, it can be carried 108 grams of linoleic acid and 54 g of caprylic acid. out as follows: The L-leucine is added at a rate of 14.4 mg per liter [0013] After completion of the fermentation, the fer- 30 and hour, as an aqueous solution containing 80 g mentation broth is centrifuged. The resulting cell mass of L-leucine per l of feed, the pH being adjusted to can then be treated with a lower alkanol such as meth- 11 with NaOH 28 % . A total of 10.2 grams of L-leu- anol or ethanol, and extracted with the same solvent. cine is added. The centrifugate can be extracted with a suitable organ- ic solvent (e.g. with methylene chloride or ethyl acetate). 35 [0016] The culture medium after seeding with the The material produced from the extracts contains the aforementioned seed culture and while feeding with the desired lipstatin and can be enriched and purified by aforementioned fatty acids and L-leucine is incubated chromatographic methods, e.g. as described in US Pat. under stirring, and aeration at a rate of 4 l of air per 4,598,089. minute to keep the culture aerobic. The pH is maintained [0014] The hydrogenation of lipstatin to tetrahydrolip- 40 in a range of 6.1 to 7.3 by the automated addition of statin can be carried out according to methods which sulfuric acid or sodium hydroxide solution. The dis- are known per se, e.g. as described in US Pat. 4598 solved oxygen concentration is prevented to be less 089, in the presence of a suitable catalyst. Examples of than 10% of the saturation concentration by adjusting catalysts which can be used are palladium/carbon, plat- the stirrer speed. At harvest time, the culture is practi- inum oxide, palladium and the like. Suitable solvents 45 cally free of linoleic acid and caprylic acid. The titer of are, for example, lower alcohols such as methanol and lipstatin, i.e. its concentration in the culture medium, is ethanol. The hydrogenation is preferably carried out at 150 mg/l after an incubation period of 138 hours. low hydrogen pressures and at room temperatures. Example 2 Example 1 50 [0017] The same seed culture as in Example 1a) [0015] above is used to inoculate a 14 l fermentor with a production medium as described in US Pat. 4,598,089, a) A seed culture is prepared consisting of the fol- namely the production medium N 7 in Example 1 there- lowing pre-culture medium: 10 g of defatted soy 55 of. This medium contains, per 8 liters: 80 g of potato flour, 10 g of glycerol, 5 g of yeast extract and water starch, 40 g of glucose, 80 g of ribose, 40 g of glycerol, to make 1 l. The pH is adjusted to 7.0 with NaOH 16 of peptone, 160 g of soybean flour, 16 g of ammonium 28 % , giving a pH of 6. 8 after sterilisation. 100 ml sulfate. An antifoam agent (0.25 ml of polypropylene gly-

3 5 EP 0 803 576 B1 6 col pro liter of medium) is added as in example 1b) L-leucine or preferably L-leucine is added to the above. The pH is adjusted to 7.0 with NaOH 28% before broth at a rate of 1 to 100, preferably 5 to 50 mg per sterilization. Incubation is carried out aerobically, while liter of broth per hour, so that its concentration re- stirring at 400 rpm and with an aeration rate of 4 l of air mains less than 25 millimolar. per minute. 5 [0018] After incubation, a concentration of lipstatin of 7. A process for the production of tetrahydrolipstatin, less than 10 mg/l was found in the culture medium. which process comprises

a) aerobically cultivating a micro-organism of Claims 10 the order of actinomycetes which produces lipstatin, in an aqueous culture medium which is 1. A process for the fermentative production of lipsta- substantially free of fats and oils, and which tin, which process comprises contains suitable carbon and nitrogen sources and inorganic salts, until the initial growth a) aerobically cultivating a micro-organism of 15 phase is substantially finished and sufficient the order of actinomycetes which produces lip- cell mass has been produced, statin, in an aqueous culture medium which is substantially free of fats and oils, and which b) adding to the broth linoleic acid, preferably contains suitable carbon and nitrogen sources together with caprylic acid, [wherein part or the and inorganic salts, until the initial growth 20 totality of the linoleic acid and/or of the caprylic phase is substantially finished and sufficient acid can be replaced by the corresponding es- cell mass has been produced, and ter(s) and/or salt(s)], and N-formyl-L-leucine or preferably L-leucine, the linoleic acid or its ester b) adding to the broth linoleic acid, optionally (s) or salt(s) being stabilized by an antioxidant, together with caprylic acid, [wherein part or the 25 totality of the linoleic acid and/or of the caprylic c) isolating lipstatin from the broth and hydro- acid can be replaced by the corresponding es- genating lipstatin to tetrahydrolipstatin. ter(s) and/or salt(s)], and N-formyl-L-leucine or preferably L-leucine, the linoleic acid or its ester (s) or salt(s) being stabilized by an antioxidant. 30 Patentansprüche

2. A process as in claim 1, wherein the micro-organ- 1. Verfahren zur Herstellung von Lipstatin durch Gä- ism of the order of actinomycetes is of the family of rung, umfassend: streptomycetes, and the aqueous culture medium, substantially free of fats and oils, contains less than 35 (a) aerobes Züchten eines Mikroorganismus 10 grams of triglycerides per liter of medium. der Ordnung Aktinomyceten, der Lipstatin her- stellt, in einem wässrigen Kulturmedium, das 3. A process as in claim 1 or 2, wherein the linoleic im Wesentlichen frei von Fetten und Ölen ist, acid or its mixture with caprylic acid and/or their es- und das geeignete Kohlenstoff- und Stickstoff- ters or salts are added at such a rate as to be freely 40 quellen und anorganische Salze enthält, bis die available in the broth but so that their accumulation anfängliche Wachstumsphase im Wesentli- is prevented, preferably at a rate of 10 to 1000, spe- chen beendet ist und genügend Zellmasse her- cially of 100 to 300 mg per liter and hour. gestellt worden ist, und (b) Zusetzen von Linolsäure zur Kulturbrühe, 4. A process as in claim 3, wherein the addition of li- 45 gegebenenfalls zusammen mit Caprylsäure, noleic acid and caprylic acid and/or their salts or of [wobei ein Teiloder die gesamte Menge der Lin- their esters is conducted so that their concentration olsäure und/oder der Caprylsäure durch den/ in the broth remains inferior to 1000, preferably in- die entsprechenden Ester und/oder Salz(e) er- ferior to 300 mg per liter, and discontinued so that setzt werden kann], und N-Formyl-L-leucin the broth is practically free of said fatty acid(s) and/ 50 oder vorzugsweise L-Leucin, wobei die Linol- or its esters or salts before lipstatin is isolated. säure oder ihr(e) Ester oder Salz(e) durch ein Antioxidans stabilisiert werden. 5. A process as in claim 3 or 4, wherein linoleic acid and caprylic acid are added to the broth in a ratio of 2. Verfahren nach Anspruch 1, wobei der Mikroorga- 1 to 10, preferably 1.5 to 3 parts per weight of lino- 55 nismus der Ordnung Aktinomyceten aus der Fami- leic acid being added for 1 part of caprylic acid. lie der Streptomyceten ist, und das wässrige Kultur- medium, im Wesentlichen frei von Fetten und Ölen, 6. A process as in claim 3, 4 or 5, wherein N-formyl- weniger als 10 g Triglyceride pro Liter Medium ent-

4 7 EP 0 803 576 B1 8

hält. Revendications

3. Verfahren nach Anspruch 1 oder 2, wobei die Lin- 1. Procédé de production de lipstatine par fermenta- olsäure oder ihr Gemisch mit Caprylsäure und/oder tion, lequel procédé comprend : ihre Ester oder Salze in einer Rate zugesetzt wer- 5 den, dass sie in der Kulturbrühe frei verfügbar sind, a) la culture aérobie d'un microorganisme de aber so, dass ihre Anreicherung verhindert wird, l'ordre des actinomycètes qui produit la lipsta- vorzugsweise in einer Rate von 10 bis 1000, beson- tine, dans un milieu de culture aqueux qui est ders von 100 bis 300 mg pro Liter und Stunde. en grande partie exempt de graisses et d'hui- 10 les, et qui contient des sources appropriées de 4. Verfahren nach Anspruch 3, wobei der Zusatz von carbone et d'azote et des sels inorganiques, Linolsäure und Caprylsäure und/oder ihrer Salze jusqu'à ce que la phase initiale de croissance oder ihrer Ester so gesteuert wird, dass ihre Kon- soit en grande partie terminée, et qu'il y ait eu zentration in der Kulturbrühe unterhalb von 1000, production d'une masse cellulaire suffisante, et vorzugsweise unterhalb von 300 mg pro Liter bleibt, 15 b) l'addition, au bouillon, d'acide linoléique, und eingestellt wird, so dass die Kulturbrühe prak- éventuellement avec de l'acide caprylique [où tisch frei von Fettsäure(n) und/oder ihren Ester oder tout ou par-tie de l'acide linoléique et/ou de Salzen ist, bevor Lipstatin isoliert wird. l'acide caprylique peut être remplacée par le ou les esters et/ou le ou les sels correspondants], 5. Verfahren nach Anspruch 3 oder 4, wobei Linolsäu- 20 et de N-formyl-L-leucine ou de préférence de re und Caprylsäure in einem Verhältnis von 1:10, L-leucine, l'acide linoléique ou son ou ses es- vorzugsweise 1,5 bis 3 Gewichtsteile Linolsäure ters ou son ou ses sels étant stabilisés par un pro 1 Teil Caprylsäure zu der Kulturbrühe zugesetzt antioxydant. werden. 25 2. Procédé selon la revendication 1, dans lequel le mi- 6. Verfahren nach Anspruch 3, 4 oder 5, wobei N-For- croorganisme de l'ordre des actinomycètes est de myl-L-leucin oder vorzugsweise L-Leucin in einer la famille des streptomycètes, et le milieu de culture Rate von 1 bis 100, vorzugsweise 5 bis 50 mg pro aqueux en grande partie exempt de graisses et Liter Kulturbrühe pro Stunde zur Kulturbrühe zuge- d'huiles contient moins de 10 g de triglycérides par setzt wird, so dass seine Konzentration niedriger 30 litre de milieu. als 25 millimolar bleibt. 3. Procédé selon la revendication 1 ou 2, dans lequel 7. Verfahren zur Herstellung von Tetrahydrolipstatin, l'acide linoléique ou son mélange avec l'acide ca- umfassend: prylique et/ou leurs esters ou sels sont ajoutés à un 35 débit leur permettant d'être librement disponibles (a) aerobes Züchten eines Mikroorganismus dans le bouillon, mais empêchant leur accumula- der Ordnung Aktinomyceten, der Lipstatin her- tion, le débit étant de préférence de 10 à 1000 et stellt, in einem wässrigen Kulturmedium, das en particulier de 100 à 300 mg par litre et par heure. im Wesentlichen frei von Fetten und Ölen ist, und das geeignete Kohlenstoff- und Stickstoff- 40 4. Procédé selon la revendication 3, dans lequel l'ad- quellen und anorganische Salze enthält, bis die dition de l'acide linoléique et de l'acide caprylique anfängliche Wachstumsphase im Wesentli- et/ou de leurs sels ou de leurs esters est mise en chen beendet ist und genügend Zellmasse her- oeuvre de façon que leur concentration dans le gestellt worden ist; bouillon reste inférieure à 1000, de préférence in- (b) Zusetzen von Linolsäure zur Kulturbrühe, 45 férieure à 300 mg par litre, et elle est interrompue vorzugsweise zusammen mit Caprylsäure, de façon que le bouillon soit en grande partie [wobei ein Teiloder die gesamte Menge der Lin- exempt dudit ou desdits acides gras et/ou de leurs olsäure und/oder der Caprylsäure durch den/ esters ou sels avant isolement de la lipstatine. die entsprechenden Ester und/oder Salz(e) er- setzt werden kann], und N-Formyl-L-leucin 50 5. Procédé selon la revendication 3 ou 4, dans lequel oder vorzugsweise L-Leucin, wobei die Linol- l'acide linoléique et l'acide caprylique sont ajoutés säure oder ihr(e) Ester oder Salz(e) durch ein au bouillon selon un rapport de 1 à 10, de préféren- Antioxidans stabilisiert werden; und ce de 1,5 à 3 parties en poids d'acide linoléique (c) Gewinnung von Lipstatin aus der Kulturbrü- ajouté par partie d'acide caprylique. he und Hydrierung von Lipstatin zu Tetrahydro- 55 lipstatin. 6. Procédé selon la revendication 3, 4 ou 5, dans lequel la N-formyl-L-leucine ou de préférence la L- leucine est ajoutée au bouillon à un débit de 1 à 100

5 9 EP 0 803 576 B1 10

et de préférence de 5 à 50 mg par litre de bouillon par heure, de façon que sa concentration reste in- férieure à 25 millimolaires.

7. Procédé de production de tétrahydrolipstatine, le- 5 quel procédé comprend :

a) la culture aérobie d'un microorganisme de l'ordre des actinomycètes qui produit la lipstatine, dans un milieu de culture aqueux qui est 10 en grande partie exempt de graisses et d'huiles, et qui contient des sources appropriées de carbone et d'azote et des sels inorganiques, jusqu'à ce que la phase initiale de croissance soit pour ainsi dire terminée, et qu'il y ait eu pro- 15 duction d'une masse cellulaire suffisante, et b) l'addition, au bouillon, d'acide linoléique, de préférence ceux de l'acide caprylique [où tout ou par-tie de l'acide linoléique et/ou de l'acide caprylique peut être remplacée par le ou les es- 20 ters et/ou le ou les sels correspondants], et de N-formyl-L-leucine ou de préférence de L-leucine, l'acide linoléique et son ou ses esters ou son ou ses sels étant stabilisés par un antioxydant, 25 c) l'isolement de la lipstatine à partir du bouillon, et l'hydrogénation de la lipstatine en tétrahydro- lipstatine.