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(11) EP 2 236 513 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 1/32 (2006.01) C07K 7/56 (2006.01) 02.10.2013 Bulletin 2013/40

(21) Application number: 10157468.9

(22) Date of filing: 24.03.2010

(54) An improved purification process for lipopeptides Verbessertes Reinigungsverfahren für Lipopeptide Procédé de purification amélioré pour lipopeptides

(84) Designated Contracting States: (74) Representative: Jones, Helen M.M. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Gill Jennings & Every LLP HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL The Broadgate Tower PT RO SE SI SK SM TR 20 Primrose Street London EC2A 2ES (GB) (30) Priority: 24.03.2009 IN MU06762009 (56) References cited: (43) Date of publication of application: US-A- 5 166 135 US-B1- 6 183 736 06.10.2010 Bulletin 2010/40 • ARIMA K ET AL: "Surfactin, a crystalline (73) Proprietor: CADILA HEALTHCARE LIMITED peptidelipid surfactant produced by Bacillus Ahmedebad 380 015, subtilis: Isolation, characterization and its Gujarat (IN) inhibition of fibrin clot formation" BIOCHEMICAL AND BIOPHYSICAL RESEARCH (72) Inventors: COMMUNICATIONS, ACADEMIC PRESS INC. • Gurnani, Menka ORLANDO, FL, US LNKD- DOI: 10.1016/0006-291X 380 015, Gujarat (IN) (68)90503-2, vol. 31, no. 3, 10 May 1968 • Maurya, Rajkumar (1968-05-10), pages 488-494, XP024830992 ISSN: 380 015, Gujarat (IN) 0006-291X [retrieved on 1968-05-10]

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 236 513 B1

Printed by Jouve, 75001 PARIS (FR) EP 2 236 513 B1

Description

Field of the Invention

5 [0001] The present invention relates to the improved process for purification of lipopeptide compounds, and in particular certain Echinocandin-type compounds, by using salt-adsorbent complex complex(s). In a further embodiment, it also includes appropriate combination of solvents for binding and elution of compound from the salt-adsorbent complex complex(s).

10 Background

[0002] Echinocandin lipopeptides are natural products that have shown potent fungicidal activity against Aspergillus and Candida sp. and protect animals from fungicidal infection by inhibiting the enzyme β (1, 3)- D- Glucan synthase, thereby disturbing the integrity of the fungal cell wall. Echinocandin family compounds are natural products such as 15 Echinocandin B, Echinocandin C, Mulundocandin, Sporiofungin A, Pneumocandin A0, and Pneumocandin B0. Their isolation, structure elucidation and biological evaluation have been reported by Schmartz et al in "Cutaneous Antifungal Agents, 1993, pp375- 394". The natural Echinocandin products are used as a precursor for semi- synthetic lipopeptides such as Anidulafungin, Micafungin, Caspofungin. [0003] Anidulafungin is a semisynthetic lipopeptide Echinocandin B derivative developed by Eli Lilly/Versicor as an 20 antifungal agent for intravenous administration. Anidulafungin is disclosed in U.S. Pat. Nos. 5,965,525 and 6,384,013. Cilofungin is an Echinocandin lipopeptide disclosed by Eli Lilly in U.S. Pat. No. 4,293,489 for use as an antifungal agent. Micafungin (FUNGARD) is an Echinocandin-like lipopeptide developed by Fujisawa, as an antifungal agent for intrave- nous administration. Micafungin is disclosed in U.S. Pat. No. 6,107,. Daptomycin (CIDECIN) is a semisynthetic lipopeptide derivative developed by Cubist as an antibacterial agent. Daptomycin is disclosed by Eli Lilly in U.S. Pat. No. 4,537,717. 25 [0004] Pneumocandin B0 is produced as a secondary metabolite by fermentation of the fungusGlarea lozoyensis (U.S. Patent 5, 194, 377 and 5, 202, 309) which is precursor for Caspofuingin acetate (CANCIDAS), a semisynthetic lipopeptide Echinocandin B derivative currently being sold in US as an antifungal agent for intravenous administration. [0005] US5, 166, 135 discloses a procedure for the purification of Echinocandin B, comprising extraction of Echinoc- andin B from the growth broth using an organic solvent and a further purification step. 30 [0006] ARIMA et al. (1968) BIOCHEM. BIOPHYS. RES. COMM. 31,488-494 discloses a method purification of a lipopeptide, surfactin, from a nutrient broth. Surfactin is produced in a fair amount in the culture fluids of several strains of B. subtilis. [0007] US6, 183, 736 discloses two lipopeptides A1 and A2 produced by Bacillus subtilis and their use as an antifungal agent against Aspergillus flavus. Both peptides are cyclic, acidic and have a broad range of antifungal and antimicrobial 35 activity. Both peptides belong to the Bacillomycin D family. A purification method of these lipopeptides is disclosed. [0008] The purification of Echinocandin lipopeptide compounds is a technical challenge despite intense research effort directed at controlling distribution and amount of various analogues produced by organism during fermentation. The organism can produce, in addition to desired compound, 20 or more Echinocandin- type compounds including Pneumo- candin A0 and C0. Furthermore, these lipopeptides have unique solubility properties; they are essentially insoluble in 40 water and most pure solvents, but dissolve in alcohols and some aqueous/ organic solvent mixture. Moreover, it is difficult, if not impossible, to purify them by crystallization. [0009] Therefore, the separation of any desired product like Pneumocandin B0 from its key analog impurities like pneumocandin C0, pneumocandin B5 and pneumocandin E0 is very cumbersome and expensive because all of them comprise a cyclic hexapeptide coupled with dimethylmyristate side chain. 45 [0010] Several efforts have been made to separate desired Pneumocandins from culture broth and to purify it by using various processes to obtain good productivity of highly purified Pneumocandins. [0011] US 4,874,843 describes the use of non- functional resin in reversed mode to purify Echinocandin- type products. Even though the process improved the purity of products derived from fermentation process, further improvements are still needed to remove contaminants that are difficult to separate from both the intermediate and final pharmaceutical 50 compounds. [0012] The use of SP-207, HP-20 resin and reverse phase HPLC are described for the separation of pneumocandin B0. (U.S. 5,162,211 and Robert E. Schwartz, The Journal of Antibiotics, Vol. 45, No. 12 1853-1866, 1992). R. E. Schwartz et.al.(1992) describe large scale whole broth methanol extract purification by use of fluidized bed of SP- 207 followed by HP 20 column and then again use SP- 207 for further material concentration and then purification of the same over silica 55 column. This process has disadvantage that the purification method is very complicated because of being multi-step process. Furthermore, SP-207 and HP-20 are capital expensive to use at large scale production. Another disadvantage is that process is very time consuming and a major amount of desired product is lost in the transfer of sample from one column to another that decreases the yield of product of interest and makes the recovery of product poor.

2 EP 2 236 513 B1

[0013] US 6,610,822 describes the purification process for Pneumocandin B0 where two phase, multi solvent system and back extraction steps have been used to maintain the polarity balance of the solution in which the compound is purified. The process comprises use of multiple solvents like isobutyl alcohol (IBA), methanol, n-heptane, water and acetonitrile, which is used in the extraction and purification of Pneumocandin B0. The problem associated with the said 5 process is that recovery and reuse of multisolvent system involves critical monitoring as the process depends upon exact ratio of solvents to bring about polarity shift as described earlier. The process has low productivity, overall yield 69% and purity obtained is 61.4%. [0014] US 2008/0108806 also describes purification of Echinocandin-type compounds such as pneumocandins, by two-phase system, first to remove non polar impurities and then extraction of compound followed by crystallization and 10 precipitation The first extraction is performed using non- polar or weakly polar water immiscible organic solvent followed by second extraction using water immiscible alcohols. The second extraction is followed by concentration and crystal- lization/precipitation. The crystallization/precipitation process by addition of antisolvent is facilitated using solid carriers. The process describes use of solid carriers to precipitate compound from the concentrated solution. This process also has low productivity. 15 [0015] US patent no. 6,506,726 and 6,590,073 describe purification of echinocandin type compounds, especially of deacylated echinocandin B by chromatography. [0016] EP1157030 describes formation of crystalline complex between echinocandin- type compounds and carbohy- drate. The patent describes enhanced stability and water solubility for these complexes. [0017] Since the potency of the final pharmaceutical product is dependent upon the purity of intermediates used to 20 make the final product, therefore the improvements in purity at any stage of the manufacturing process is highly desirable. Ideally, the contaminants should be removed at the earliest stage possible in the manufacturing process. [0018] Accordingly, conventional technologies of purifying pneumocandin B0 from the culture broth are problematic in that highly purified Pneumocandin Bo is not readily produced, stability of compound is not maintained, the recovery yield is relatively low and production cost are relatively high. Hence, there remains a long felt need to develop an improved 25 purification process, which provides better resolution and productivity of Pneumocandins and other Echinocandin- type compounds at economical cost. Therefore, the object of the present invention is to develop an improved & economical purification process to obtain good yield and purity of certain Echinocandin- type compounds by using salt- adsorbent complex such as calcium phosphate. Moreover, the present process is simpler and provides improved productivity and purity than some of the existing process for the purification of Echinocandin- type compounds. 30 [0019] The present process is simple, cost effective, rapid, and scalable at industrial scale and provide highly purified yield of Echinocandin-type compounds at low cost as compared to prior art. The method allows for the removal of impurities by using economical salt- adsorbent complex and provide highly purified echinocandin type compounds which is highly improved in terms of purity than prior art and sufficient for further processing to obtain an active pharmaceutical ingredient such as, for example the antifungals caspogungin, anidulafungin, and micafungin. 35 [0020] Additionally this method reduces overall solvent and energy consumption making the process economically viable for commercial use and does not require chromatography steps involving expensive resins which are used in prior art processes for the isolation and purification of similar natural products.

Objects of the Invention 40 [0021] In one embodiment, the present invention provides a simple, rapid and an efficient method for the purification of Echinocandin type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5. In another embodiment of the present invention provides the use of salt- adsorbent complexes to purify and recover the Echinocandin type compound selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5 from the process solvents. 45 [0022] In another embodiment, the present invention also reduces the related structural analogue impurities from the Echinocandin compound of interest. [0023] In yet another embodiment the anions of the salt- adsorbent complex comprises phosphates. [0024] In yet another embodiment of the present invention describes the binding, appropriate washing and elution conditions for improved productivity. 50 [0025] In further embodiment the present invention provides a cost effective purification process compared to those known in the prior art.

Detailed Description

55 [0026] The present invention provide a process, which is easily scalable, cost effective, and with improved purity. [0027] The present invention provides a process for separation and purification of compounds related to the class of Echinocandin-type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5. The present process is simple, cost effective, rapid, and scalable at industrial scale and provide highly purified yield of compounds at low cost.

3 EP 2 236 513 B1

As used herein unless mentioned otherwise, the term "Compound-salt-adsorbent complex" refers to a unit where a compound is adsorbed on the salt either strongly or weakly. [0028] As used herein unless mentioned otherwise, the term "Echinocandin-type compounds" refers to compounds which belong to Echinocandin family or share common structural features of Echinocandins or analogues of Echinoc- 5 andins. [0029] As used herein unless mentioned otherwise, the term "salt-adsorbent complex" refers to those salts which adsorb the Echinocandin-type compounds. The salts are selected from salts of phosphates, carbonates and sulphates and comprise phosphates. Preferred are those salts which are highly insoluble or weakly soluble in process solvent (s) and at least comprise one divalent cation. 10 [0030] In one embodiment, the present invention provides a new process for separation and purification of Echinoc- andin-type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5. In preferred embodiment, the present invention provides a new process for separation and purification of Pneumocandins B0, A0, C0, D0, E0, B2 and B5, from fermentation broths or partially purified process streams. In such embodiment, the present invention uses suitable salt-adsorbent complex complex to bind compound from solution or whole broth which is followed by physical 15 separation of the compound- salt-adsorbent complex from the medium comprising solution or whole broth and thereafter recovering the Echinocandin-type compounds by elution in presence of appropriate solvent system from salt adsorbent. [0031] The present invention uses salt adsorbents, which adsorb the Echinocandin-type compounds. In preferred embodiment Salt-adsorbent complex are insoluble compounds and comprise at least one anion and divalent cation. [0032] The present invention uses suitable salt-adsorbent complex selected from but not limited to phosphates and 20 at least comprises one divalent cation. In preferred embodiment, the salt-adsorbent complex is calcium phosphate. In another embodiment, the salt adsorbent binds the Echinocandin-type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5, which is followed by physical separation of the compound- salt-adsorbent complex from the medium containing solution or broth and thereafter recovered the Echinocandin- type compounds by elution in presence of appropriate solvent system from salt adsorbent. 25 [0033] In one embodiment, the process for separation and purification of lipopeptide compounds which are Echinoc- andin-type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5, comprises following steps -

a) addition of salt adsorbent (s), such as calcium phosphate in whole fermentation broth containing selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5 Echinocandin- type compound and/or in situ formation of similar 30 salt- adsorbent complex. b) separation of cake which comprise salt-compound adsorbent and mycelia; c) extraction of compound of interest from the cake by using suitable organic solvent (s); d) concentration of the solvent e) further addition of salt adsorbents such as calcium phosphate to bind the compound of interest from solution 35 where compound of interest again forms salt-compound adsorbent complex; f) further separation of compound salt adsorbent; g) optionally, washing of separated compound-salt adsorbent by the using suitable washing system; h) elution of compound of interest from adsorbent by using appropriate eluting system; i) concentration of eluate; 40 j) precipitation of the product.

[0034] The present invention describes the process for the purification of Pneumocandins selected from Pneumocandin B0, Pneumocandin A0, Pneumocandin C0, Pneumocandin D0, E0, B2 and B5. In preferred embodiment the present invention describes purification of Pneumocandin B0 from its structural analogues. 45 [0035] Pneumocandin B0. is produced by the process known in the art by cultivatingGlarea losoyeinsis (formerly identified as Zalerion arboricola), ATCC 74030 in a suitable nutrient medium, similar to those described in US 5,306,708. [0036] In one embodiment, the appropriate concentration of Pneumocandins is obtained by carrying out the fermen- tation process and the salt-adsorbent complex such as calcium phosphate is added in the broth. [0037] In preferred embodiment, the salt-adsorbent complex such as calcium phosphate is formed in situ in step (a) 50 of the process by the addition of chemicals such as calcium chloride in a phosphate containing fermentation broth. [0038] In another preferred embodiment, salt-adsorbent complex such as calcium phosphate is added extraneously or formed in-situ during or after fermentation process. [0039] In another embodiment of the present invention, addition or in situ formation of salt-adsorbent complex such as calcium phosphate in the fermentation whole broth helps to recover extracelullarly released compounds. The process 55 allows removing of the impurities as well as remaining medium components, which are soluble in water, and thereafter the solid cake is obtained comprising compound-salt-adsorbent complex and mycelium. Therefore, it was surprisingly found that the addition and/ or formation of salt-adsorbent complex in step (a) not only prevents loss of extracellular product but also significantly reduces the processing volume. In one embodiment, the compound of interest is extracted

4 EP 2 236 513 B1

(step c) from the above solid cake by the use of suitable solvent system. [0040] In another preferred embodiment the Echinocandin type compound selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5 is extracted from the whole broth or compound obtained from any other stream of recovery process which are known in the art. Thereafter salt adsorbents such as calcium phosphate is added for binding of the Echinocandin 5 type compound from solution where Echinocandin type compound of interest forms salt- compound adsorbent complex in suitable solvent system. [0041] In one embodiment, the suitable solvents used either alone or in combination with each other are selected from

but not limited to (C1- C6) alcohols, such as methanol, ethanol, isopropyl alcohol, isobutyl alcohol and n- butanol, preferably methanol, isobutyl alcohol; (C2- C6) ketones such as acetone, acetophenone, methyl ethyl ketone, methyl 10 isopropyl ketone preferably acetone; and acetonitrile [0042] The extraction step (c) can be repeated to further increase the yield of compound of interest in the solvent. In one embodiment the compound of interest is obtained in the aqueous phase by evaporating the solvent, when the extraction solvent is water miscible such as methanol. In another embodiment, the compound of interest is obtained in the solvent phase by concentrating under vacuum, when the extraction solvent is water immiscible such as isobutyl 15 alcohol or n-butanol. The compound of interest may further be purified by repeating the use of salt- adsorbent. [0043] In another embodiment the compound is extracted from whole broth using suitable organic solvent. In one

embodiment, the solvent used either alone or in combination with each other are selected from but not limited to C 1- C6 alcohols, such as methanol, ethanol, isopropyl alcohol, isobutyl alcohol and n- butanol, preferably methanol, isobutyl alcohol; (C2- C6) ketones selected from acetone, acetophenone, methyl ethyl ketone, methyl isopropyl ketone preferably 20 acetone; and acetonitrile. In preferred embodiment water immiscible organic solvent is C 4- C6 alcohols. In most preferred embodiment, water immiscible organic solvent is isobutanol, n- butanol. [0044] The compound of interest is further purified in step (c) by the use of the salt- adsorbent complex. [0045] The present invention further includes but not limited to the extraction of Echinocandin type compound selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5 from the whole broth or compound obtained from any other stream 25 of recovery process in the presence of suitable solvent selected from methanol or isobutanol and thereafter the solvent containing Echinocandin type compounds is treated with the salt adsorbents such as calcium phosphate for binding of the Echinocandin type compound from solvent where Echinocandin type compound of interest forms salt-compound adsorbent complex in suitable solvent system. [0046] The inventors of the present invention surprisingly found that the use of salt- adsorbent complex such as calcium 30 phosphate easily separated the compound of interest from the water/ organic solvent, which makes the process simple, rapid, cost effective and provides easy recovery of solvents at industrial scale. [0047] In another embodiment, the Echinocandin type compound selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5 is obtained from any other stream of purification process, thereafter the compound of interest is purified through appropriate salt- absorbent in the presence of suitable solvent. In such embodiment, the solvent used either 35 alone or in combination with each other are selected from but not limited to C1- C6 alcohols, such as methanol, ethanol,

isopropyl alcohol, Isobutyl alcohol and n- butanol, preferably methanol, isobutyl alcohol; (C 2- C6) ketones selected from acetone, acetophenone, methyl ethyl ketone, methyl isopropyl ketone preferably acetone; and acetonitrile [0048] In one embodiment of the invention, the salt- adsorbent complex complex is used for adsorption of Echinocandin type compound selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5 on salt-adsorbent complex complex. In 40 such embodiment, the anions of salts, includes but are not limited to phosphates.. In another embodiment, the cations of the salts, include but are not limited to calcium (Ca), copper (Cu), iron (Fe), Magnese (Mn), Magnesium (Mg), Zinc (Zn), Barium (Ba). In preferred embodiment the cation of salt is calcium. In one embodiment the salt- absorbents can be synthesized chemically such as calcium phosphate or obtained naturally such as hydroxy apatite. In preferred embodiment, anions and cations are used for in-situ formation of salt-absorbents and it further makes 45 adequate salt-compound complex with Echinocandin type compound, selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5. [0049] In another preferred embodiment, the salt-adsorbent may be directly added to liquid containing Echinocandin type compound selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5, in order to achieve adequate com- pound.-salt-adsorbent complex. 50 [0050] In such an embodiment, the salt-absorbent are used in powder or particulate form. In preferred embodiment, powder form is used and the salt-adsorbent complex and compound adsorption process is performed (Step e) in batch or continuous mode, preferably, batch mode at industrial scale. [0051] Without wishing to be bound to any addition concentration of salt adsorbent, the addition concentration of salt adsorbent can be varied to get efficient compound-salt-adsorbent complex complex. In one embodiment at least 0.1 55 times or more amount of salt adsorbent quantity than measured Pneumocandin B0 is added to liquid containing Pneu- mocandin B0, in order to obtain adequate adsorption between salt and Pneumocandin B0. At least 0.5% (w/v) or higher concentrations of the salt-absorbent is used. In such an embodiment, the adsorption of compound on salt-adsorbent complex starts immediately as the salt-adsorbent and compound are mixed, but the degrees of adsorption gradually

5 EP 2 236 513 B1

increase with time. However, increasing the quantity of salt- adsorbent also decreases the time for major adsorption of compound on it. [0052] The salt-adsorbent is mixed with Echinocandin type compounds and suitable system selected from Pneumo- candins B0, A0, C0, D0, E0, B2 and B5 is obtained for optimum adsorption of Echinocandin type compounds on salt 5 adsorbent [0053] In such embodiment, the selection of the suitable system depends upon the decreased solubility of compound. The system is selected from aqueous system or organic solvent or combination thereof for the formation of adequate compound-salt-adsorbent complex. The organic solvent includes both water miscible and water immiscible solvents. The solvent used are selected from but not limited to fully aqueous system or C1- C6 lower alcohols such as methanol, 10 ethanol, isopropyl alcohol, Isobutyl alcohol and n-butanol, preferably methanol and iso butyl alcohol; ketones such as acetone, acetophenone, methyl ethyl ketone, methyl isopropyl ketone preferably acetone and acetonitrile In one em- bodiment, the concentration of water miscible organic solvents are maintained below 25%, preferably 10%, more pref- erably 5% even more preferably free of organic solvent.. [0054] In another embodiment, the water concentration is maintained below 10%, preferably 5%, more preferably 1%., 15 even more preferably free of water in water immiscible solvent system. In another embodiment the salt adsorbent is dry free of liquid. [0055] The appropriate ratio of water and solvent can be obtained by addition or removal of solvents to have system for optimum formation compound-salt-adsorbent complex. The appropriate ratio of water and solvent can be obtained by addition or removal of solvents to have system for optimum formation of compound- salt-adsorbent complex. In another 20 embodiment, another organic solvent is added, which decreases the polarity of the system. In such embodiement, the solvents are selected from but not limited to C5-8 aliphatic alkanes such as heptane, hexane, cyclohexane or octane; C6-8 aromatic hydrocarbons such as toluene. [0056] Withoutwishing tobe bound to anytheory inventers believe that decreasing solvent content increases adsorption of compound with salt- adsorbent in aqueous system. In organic solvent system the increasing content of water decreases 25 the adsorption of compound with salt-adsorbent. [0057] Compound salt-adsorbent complex is then separated (step f) from the liquid for further processing. [0058] The salt-compound absorbent obtained from the above said process is washed (step g) to remove bound impurities. The washing is performed using appropriate system, which removes the color and other impurities but not the compound of interest. 30 [0059] In one embodiment, the washing is performed using water or organic solvents or mixture thereof, in which the

compound is less or not soluble. In such embodiment, the organic solvents are selected from but not limited to, C5 to C8 aliphatic alkanes, C6 to C8 aromatic hydrocarbons, C4 to C8 ethers and C3 to C6, ketones and acetonitrile. [0060] In preferred embodiment, C5-8 aliphatic alkanes are selected from heptane, hexane, cyclohexane or octane. In another preferred embodiment, C6-8 aromatic hydrocarbons are selected from toluene & preferred 4C-8 ether is 35 selected from diethyl ether, diso propyl ether, dibutyl ether. In preferred embodiment C 3-6 ester is selected from isobutyl acetate, n-butyl acetate, n-propyl acetate, isopropyl acetate or ethyl acetate. In most preferred embodiment, the non polar or weakly polar water immiscible organic solvent is ethyl acetate, iso butyl acetate, butyln- acetate, iso propyl acetate and n-propyl acetate. [0061] The above-describedwashing step is optional. TheEchinocandin type compound selectedfrom Pneumocandins 40 B0, A0, C0, D0, E0, B2 and B5 may be eluted (step i) directly from compound-salt-adsorbent complex, but washing helps in improving quality of the desired product. Without wishing to be bound to any theory the applicants believe that the aqueous washing using water removes the polar impurities and non-polar solvent such as ethyl acetate washing removes the non-polar impurities. Hence, washing is the useful step for the removal of the both polar and non polar impurity. 45 [0062] In one embodiment, the suitable eluting system has superior affinity for Echinocandin- type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5. The elution of Echinocandin-type compounds Pneumocandins B0, A0, C0, D0, E0, B2 and B5 is performed by using organic solvents which are selected from lower alcohols preferably

(C1-C6) alcohols, in preferred embodiment, methanol n-butanol and isobutyl alcohol, dimethyl formamide, dimethyl sulfoxide, ketones preferably, methyl-ethyl ketone, acetonitrile and/or water mixture. 50 [0063] In such embodiment, the eluate is processed to obtain purified Pneumocandins by methods well known in prior art. In preferred embodiment, the eluate is concentrated under vacuum to obtain purified Pneumocandins. In such embodiment, acetonitrile and/or acetone arc added to concentrate to precipitate the Pneumocandins. The precipitate is separated and dried to obtain purified Pneumocandins. [0064] In one embodiment the present process carries out at 0°C to 60 °C. In preferred embodiment the present 55 process carries out at 5 °C to 35 °C and in most preferred embodiment the process carries out at below room temperature. [0065] The above adsorption of compound on salt-adsorbent complex formation step and elution can be performed more than once or repeated at any stream of purification process. [0066] Following examples illustrate some of the preferred mode of carrying out the invention, including the best mode

6 EP 2 236 513 B1

known to the inventors. These examples are provided for the purpose of illustration and should not be construed as limiting the scope of the invention.

Example 1 5 Isolation and purification of Pneumocandin B0

[0067] 13 L fermentation broth comprising Pneumocandin B0 and remaining soluble phosphates from medium com- ponents, in which, 260 gm of the calcium chloride solution is added. (100g potassium phosphate may be added in case 10 the medium is free of it.). The mixture was stirred for 3 hrs. The solid cake was separated from aqueous medium by centrifugation or filtration. The filtrate substantially free of Pneumocandin was discarded. The compound from solid cake was extracted using 9 L methanol. The methanol extraction process was repeated. The methanol was separated from the solid cake by centrifugation and was evaporated in order to obtain aqueous concentrate. when 2 L of concentrate

was obtained, salt adsorbent CaPO 4 was (100g of CaPO 4, prepared by mixing Cacl2 and potassium phosphate solutions 15 and neutralizing the solution; then filtering the precipitate obtained) added and stirred for 3hrs .The mixture was con- centrated under vacuum to obtained aqueous solution. The mixture was filtered to obtain the compound: salt- adsorbent complex. The obtained compound: salt-adsorbent complex was washed using water, thereafter, further it was washed using ethyl acetate. The purified compound was eluted from the salt adsorbent by using methanol. The elute was concentrated under vacuum to obtain purified Pneumocandin B0.The yield of the process is 70%. The concentrated 20 Pneumocandin B0 was further precipitated using acetonitrile. The precipitate obtained is filtered and dried to obtain purified powder of Pneumocandin B0 having purity of more than 88% by HPLC analysis.

Example 2

25 Isolation and purification of Pneumocandin B0

[0068] 13 L fermentation broth comprising Pneumocandin B0 and remaining soluble phosphates from medium com- ponents, in which, 260 gm of the calcium chloride solution is added. (100g potassium phosphate may be added in case the medium is free of it.). The mixture was stirred for 3 hrs. The solid cake containing compound: salt adsorbent with 30 mycelium were separated from aqueous medium by centrifugation or filtration. The compound from solid cake was extracted using 8 L iso-butyl alcohol (IBA). The iso butanol (IBA) extraction process is repeated using water saturated iso butanol (IBA). The isobutanol was separated from the solid cake by centrifugation and was evaporated under vacuum, in order to obtain concentrate.. When approximately 3 L of IBA concentrate was obtained, the salt adsorbent (100g of CaPO4, prepared by mixing Cacl2 and potassium phosphate solutions and neutralizing the solution; then filtering the 35 precipitate obtained) was added in concentrate and further concentration was continued to till the 500 ml of the IBA left. The compound: salt-adsorbent complex was obtained by filtering the remaining IBA. The obtained compound salt ad- sorbent complex was washed using water, thereafter, further it was washed using ethyl acetate. The purified compound was eluted from the salt adsorbent by using methanol. The eluate was concentrated under vacuum to obtain purified Pneumocandin B0. The yield of the process is 70% .The concentrated Pneumocandin B0 was further precipitated using 40 acetonitrile. The precipitate obtained is filtered and dried to obtain purified powder of Pneumocandin B0 having purity of more than 88% by HPLC analysis

Example 3

45 Isolation and purification of Pneumocandin B0

[0069] 14 L of fermentation broth comprising Pneumocandin B0 is extracted by iso butyl alcohol. The mycelial solids were separated by the filtration and extraction step with iso butyl alcohol was further repeated. The iso butyl alcohol was separated from the solid cake by filtration and was evaporated in order to obtain the concentrate. When approximately 50 3 L of IBA concentrate was obtained, the salt adsorbent (100g of CaPO4, prepared by mixing Cacl2 and potassium phosphate solutions and neutralizing the solution; then filtering the precipitate obtained) was added in concentrate and further concentration was continued to till the 500 ml of the IBA left. The obtained compound:salt adsorbent complex was washed using water, thereafter, further it was washed using ethyl acetate. The purified compound was eluted from the salt adsorbent by using methanol. The elute was concentrated under vacuum to obtain purified Pneumocandin B0. 55 The yield of the process is 75%. The concentrated Pneumocandin B0 was further precipitated using acetonitrile. The precipitate obtained is filtered and dried to obtain purified powder of Pneumocandin B0 having purity of more than 89% by HPLC analysis.

7 EP 2 236 513 B1

Example 4

Purification of crude Pneumocandin B0

5 [0070] 10g crude Pneumocandin B0 was dissolved in 70% methanol solution. 50g of calcium phosphate was added and concentrated the mixture under vacuum to remove the solvent. The compound salt adsorbent was recovered by filtration. It was further washed by water and subsequently with ethyl acetate. The washing was performed till the color was found in the washing. The compound was eluted from the complex by using methanol. The elute was concentrated under vacuum and precipitated by the acetonitrile. The off white colored purified Pneumocandin B0 obtained. 10 Example 5

[0071] 5g crude Pneumocandin B0 was dissolved in 70% acetone solution. 25g of calcium phosphate was added and concentrated the mixture under vacuum to remove the solvent. The compound salt adsorbent was recovered by filtration. 15 It was further washed by water and subsequently with ethyl acetate. The washing was performed till the colour is found in the washing. The compound was eluted from the complex by using 90% acetone. The elute was concentrated under vacuum and precipitated by the acetonitrile to recover the compound. The off white coloured purified pneumocandin Bo obtained.

20 Example 6

[0072] 5g crude Pneumocandin B0 was dissolved in 70% acetonitrile solution. 25g of calcium phosphate was added and concentrated the mixture under vacuum to remove the solvent. The compound salt adsorbent was recovered by filtration. It was further washed by water and subsequently with ethyl acetate. The washing was performed till the colour 25 isfound in thewashing. The compound was eluted from thecomplex byusing 88% acetonitrile. The elute wasconcentrated under vacuum and precipitated by the acetonitrile to recover the compound The off white coloured purified pneumocandin Bo obtained.

Example 7 30 [0073] 250 mg of each different insoluble phosphate salts of Fe, Cu, Ca, Zn, Mg, Mn were added in each 5 ml solution containing 100 mg of the crude Pneumocandin Bo. The solvent was evaporated under vacuum and the solution is centrifuged out to separate the salt - adsorbent. The compound is eluted from each salt adsorbent by using 10 ml methanol. The result of the experiment is summarized below in table 1. 35 No. Salt Adsorbent Pneumocandin B0(mg) Pneumocandin B0(mg) In supenatent In elute

1CaPo4 0.1 49 40 2FePo4 0.95 35

3ZnPo4 0.1 42

4CoPo4 0.09 35 5CuPo 0.3 39 45 4 6MgPo4 0.09 41

Claims 50 1. A process for the purification of Echinocandin type compounds selected from Pneumocandins B0, A0, C0, D0, E0, B2 and B5, comprising the binding of the Echinocandin type compound present in a solvent system on salt adsorbent, wherein the salt adsorbent comprises phosphate and at least one divalent cation; wherein the salt adsorbent is present in a concentration of at least 0.5% (w/v) of the liquid containing Pneumocandin; 55 and wherein the solvent system is an aqueous system or organic solvent or combination thereof, wherein the organic solvent is selected from the group consisting of C1- C6 lower alcohols; C2-C6 ketones and acetonitrile,

8 EP 2 236 513 B1

2. The process as claimed in claim 1 wherein the divalent cation is selected from Ca +2, Mg+2, Fe+2, Mn+2, Cu+2, Zn+2, Ba+2

3. The process as claimed in claim 2 wherein the divalent cation is Ca +2. 5 4. The process as claimed in any preceding claim, wherein the salt adsorbent is Calcium phosphate.

5. The process as claimed in claim 1 or claim 2, wherein the salt adsorbent is Copper phosphate.

10 6. A process for the purification of Echinocandin type compound as claimed in claim 1 comprising:

a. treating the Echinocandin type compound with salt adsorbent in a solvent system as defined in claim 1, to obtain a salt adsorbent compound complex; b. further separating Echinocandin type compound salt adsorbent complex from the solvent system. 15 c. optionally, washing of separated Echinocandin type compound-salt adsorbent complex by using suitable washing system; d. eluting the desired compound from the adsorbent by using appropriate eluting system

7. The process as claimed in claim 6 wherein the Echinocandin type compounds are selected from Pneumocandins 20 B0, A0, C0, D0

8. The process as claimed in claim 7 wherein the Echinocandin is Pneumocandin B0.

9. The process as claimed in any of claims 6 to 8, wherein the concentration of the water miscible solvents is maintained 25 below 20%.

10. The process as claimed in any of claims 6 to 8 wherein the concentration of water immiscible solvents is maintained below 10%.

30 11. The process as claimed in claim 6 wherein the solvent used step (c) is selected from one or more of water and/or water immiscible solvents, preferably, non polar or weakly polar water immiscible, organic solvents C 5 to C 8 aliphatic alkanes, C6 to C8 aromatic hydrocarbons, C4 to C8 ethers and C3 to C6 esters.

12. The process as claimed in claim 6 wherein the solvent used in step (d) is selected from one or more of dimethyl 35 sulphoxide, dimethyl formamides, C1-C6 lower alcohols, C2-C6 ketones and acetonitrile

Patentansprüche

40 1. Verfahren zur Aufreinigung von Echinocandin- Verbindungen, die aus Pneumocandinen B0, A0, C0, D0, E0, B2 und B5 ausgewählt sind, umfassend die Bindung der in einem Lösungsmittelsystem vorliegenden Echinocandin-Ver- bindung an ein Salz-Adsorbens, wobei das Salz-Adsorbens Phosphat und mindestens ein bivalentes Kation umfasst; wobei das Salz-Adsorbens in einer Konzentration von mindestens 0,5 % (w/v) der Pneumocandin enthaltenden 45 Flüssigkeit vorliegt; und wobei das Lösungsmittelsystem ein wässriges System oder organisches Lösungsmittel oder eine Kombination

davon ist, wobei das organische Lösungsmittel ausgewählt ist aus der Gruppe, bestehend aus niederen 1C- bis C6-Alkoholen; C2- bis C6-Ketonen und Acetonitril.

50 2. Verfahren nach Anspruch 1, wobei das bivalente Kation ausgewählt ist aus Ca +2, Mg+2, Fe+2, Mn+2, Cu+2, Zn+2 und Ba+2.

3. Verfahren nach Anspruch 2, wobei das bivalente Kation Ca+2 ist.

55 4. Verfahren nach einem der vorangehenden Ansprüche, wobei das Salz- Adsorbens Calciumphosphat ist.

5. Verfahren nach Anspruch 1 oder 2, wobei das Salz- Adsorbens Kupferphosphat ist.

9 EP 2 236 513 B1

6. Verfahren zur Aufreinigung einer Echinocandin- Verbindung nach Anspruch 1, umfassend:

a. Behandeln der Echinocandin- Verbindung mit Salz- Adsorbens in einem Lösungsmittelsystem wie nach Anspruch 1 definiert, zum Erhalt eines Salz- Adsorbens- Verbindung- Komplexes; 5 b. ferner Trennen des Echinocandin- Verbindung- Salz- Adsorbens- Komplexes aus dem Lösungsmittelsystem, c. gegebenenfalls Waschen des getrennten Echinocandin- Verbindung- Salz- Adsorbens- Komplexes unter Verwendung eines geeigneten Waschsystems; d. Eluieren der gewünschten Verbindung aus dem Adsorbens unter Verwendung eines geeigneten Elutionssy- stems. 10 7. Verfahren nach Anspruch 6, wobei die Echinocandin- Verbindungen ausgewählt sind aus Pneumocandinen B0, A0, C0, D0.

8. Verfahren nach Anspruch 7, wobei das Echinocandin Pneumocandin B0 ist. 15 9. Verfahren nacheinem der Ansprüche6 bis 8, wobei dieKonzentration der nicht mit Wasser mischbaren Lösungsmittel unter 20 % aufrechterhalten wird.

10. Verfahren nach einem der Ansprüche 6 bis 8, wobei die Konzentration von nicht mit Wasser mischbaren Lösungs- 20 mitteln unter 10 % aufrechterhalten wird.

11. Verfahren nach Anspruch 6, wobei das in Schritt (c) verwendete Lösungsmittel ausgewählt ist aus einem oder mehr von Wasser und/ oder nicht mit Wasser mischbaren Lösungsmitteln, bevorzugt nicht polaren oder schwach polaren

nicht mit Wasser mischbaren, organischen Lösungsmitteln: aliphatischen C5- bis C8- Alkanen, aromatischen C6- 25 bis C8- Kohlenwasserstoffen, C4- bis C8- Ethern und C3- bis C6- Estern.

12. Verfahren nach Anspruch 6, wobei das in Schritt (d) verwendete Lösungsmittel ausgewählt ist aus einem oder mehr von Dimethylsulfoxid, Dimethylformamiden, niederen C1- bis C6- Alkoholen, C2- bis C6- Ketonen und Acetonitril.

30 Revendications

1. Procédé de purification de composés de type échinocandine sélectionnés parmi les pneumocandines B0, A0, C0, D0, E0, B2 et B5, comprenant la liaison du composé de type échinocandine présent dans un système de solvants 35 sur un adsorbant de sel, dans lequel l’adsorbant de sel comprend du phosphate et au moins un cation divalent ; dans lequel l’adsorbant de sel est présent à une concentration d’au moins 0,5% (p/v) du liquide contenant la pneumocandine ; et dans lequel le système de solvants est un système aqueux ou un solvant organique ou une combinaison de ceux- 40 ci, dans lequel le solvant organique est sélectionné parmi le groupe constitué d’alcools inférieurs en C1 à C6 ; de cétones en C2 à C6 et d’acétonitrile,

2. Procédé selon le mode opératoire de la revendication 1, dans lequel le cation divalent est sélectionné parmi Ca +2, Mg+2, Fe+2, Mn+2, Cu+2, Zn+2, Ba+2. 45 3. Procédé selon le mode opératoire de la revendication 2, dans lequel le cation divalent est Ca +2.

4. Procédé selon le mode opératoire de l’une quelconque des revendications précédentes, dans lequel l’adsorbant de sel est du phosphate de calcium. 50 5. Procédé selon le mode opératoire de la revendication 1 ou de la revendication 2, dans lequel l’adsorbant de sel est du phosphate de cuivre.

6. Procédé destiné à la purification d’un composé de type échinocandine selon le mode opératoire de la revendication 55 1, comprenant les étapes consistant à :

a. traiter le composé de type échinocandine avec un adsorbant de sel dans un système de solvants comme défini dans la revendication 1, afin d’obtenir un complexe de composé adsorbant de sel ;

10 EP 2 236 513 B1

b. séparer en outre le complexe adsorbant de sel du composé de type échinocandine du système de solvants. c. éventuellement laver le complexe adsorbant de sel- composé de type échinocandine séparés en utilisant un système de lavage approprié ; d. éluer le composé souhaité de l’adsorbant en utilisant un système d’élution approprié. 5 7. Procédé selon le mode opératoire de la revendication 6, dans lequel les composés de type échinocandine sont sélectionnés parmi les pneumocandines B0, A0, C0, D0.

8. Procédé selon le mode opératoire de la revendication 7, dans lequel l’échinocandine est la pneumocandine B0. 10 9. Procédé selon le mode opératoire de l’une quelconque des revendications 6 à 8, dans lequel la concentration des solvants miscibles dans l’eau est maintenue en deçà de 20%.

10. Procédé selon le mode opératoire de l’une quelconque des revendications 6 à 8, dans lequel la concentration des 15 solvants immiscibles dans l’eau est maintenue en deçà de 10%.

11. Procédé selon le mode opératoire de la revendication 6, dans lequel le solvant utilisé à l’étape (c) est sélectionné parmi un ou plusieurs des solvants miscibles dans l’eau et/ou immiscibles dans l’eau, de préférence, des solvants

organiques immiscibles dans l’eau non polaires ou faiblement polaires, des alcanes aliphatiques en C5 à C8, des 20 hydrocarbures aromatiques en C6 à C8, des éthers en C4 à C8 et des esters en C3 à C6.

12. Procédé selon le mode opératoire de la revendication 6, dans lequel le solvant utilisé à l’étape (d) est sélectionné parmi un ou plusieurs du sulphoxyde de diméthyle, des diméthylformamides, des alcools inférieurs en C1 à C6, des cétones en C2 à C6 et de l’acétonitrile. 25

30

35

40

45

50

55

11 EP 2 236 513 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 5965525 A [0003] • US 4874843 A [0011] • US 6384013 A [0003] • US 5162211 A [0012] • US 4293489 A, Eli Lilly [0003] • US 6610822 B [0013] • US 6107 A [0003] • US 20080108806 A [0014] • US 4537717 A, Eli Lilly [0003] • US 6506726 B [0015] • US 5194377 A [0004] • US 6590073 B [0015] • US 5202309 A [0004] • EP 1157030 A [0016] • US 5166135 A [0005] • US 5306708 A [0035] • US 6183736 B [0007]

Non-patent literature cited in the description

• SCHMARTZ et al. Cutaneous Antifungal Agents, • ROBERT E. SCHWARTZ. The Journal of Antibiotics, 1993, 375-394 [0002] 1992, vol. 45 (12), 1853-1866 [0012] • ARIMA et al. BIOCHEM. BIOPHYS. RES. COMM., 1968, vol. 31, 488-494 [0006]

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