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`EP 2 918 324 A1
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`(11)
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`EUROPEAN PATENT APPLICATION
`
`(43) Date of publication:
`16.09.2015 Bulletin 2015/38
`
`(21) Application number: 15165834.1
`
`(22) Date of filing: 13.04.2012
`
`(51) Int Cl.:
`B01D 15/32 (2006.01)
`C07K 7/56 (2006.01)
`
`A61K 31/4164 (2006.01)
`
`(84) Designated Contracting States:
`AL AT BE BG CH CY CZ DE DK EE ES FI FR GB
`GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO
`PL PT RO RS SE SI SK SM TR
`
`(30) Priority: 20.04.2011 US 201161477190 P
`
`• DALE, Eli Karin
`N-0750 OSLO (NO)
`• HANSEN, Espen Fridtjof
`N-3050 LOMMEDALEN (NO)
`• MELDAHL EIDE, Kjersti
`0192 OSLO (NO)
`
`(62) Document number(s) of the earlier application(s) in
`accordance with Art. 76 EPC:
`12713998.8 / 2 699 327
`
`(74) Representative: Onsagers AS
`P.O. Box 1813 Vika
`0123 Oslo (NO)
`
`(71) Applicant: Xellia Pharmaceuticals ApS
`2300 Copenhagen S (DK)
`
`(72) Inventors:
`• AASSVEEN, Lene
`N-0171 OSLO (NO)
`
`Remarks:
`This application was filed on 30-04-2015 as a
`divisional application to the application mentioned
`under INID code 62.
`
`(54)
`
`METHOD FOR PURIFICATION OF MICAFUNGIN
`
`(57)
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`The present invention relates to a method for the purification of Micafungin.
`
`Printed by Jouve, 75001 PARIS (FR)
`
`EP2 918 324A1
`
`AMNEAL EX. 1013
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`Description
`
`Technical filed
`
`[0001] The present invention relates to an improved method for purification of a Micafungin salt, in particular Micafungin
`sodium.
`
`Background
`
`[0002] Micafungin is the active pharmaceutical ingredient in Mycamine®. According to the FDA label, the chemical
`structure of Micafungin sodium is represented by formula (I):
`
`[0003] Micafungin is also known as Pneumocandin A0, 1-[(4R,5R)-4,5-dihydroxy-N2-[4-[5-[4-(pentyloxy)phenyl]-3-
`isoxazolyl]benzoyl]-L-ornithine]-4-[(4S)-4-hydroxy-4-[4-hydroxy-3-(sulfooxy)phenyl]-L-threonine]. Micafungin sodium is
`furthermore known as FK-463. The assigned Registry No’s by Chemical Abstracts are 235114-32-6 for Micafungin and
`208538-73-2 for Micafungin sodium.
`[0004] Micafungin is an echinocandin which inhibits 1,3-β-D-glucan synthase and thus leads to fungal cell lysis. Mi-
`cafungin is thus useful as an antifungal agent in the treatment of infections caused by strains of e.g. Aspergillus, Cryp-
`tococcus, Candida, Mucor, Actinomyces, Histoplasma, Dermatophyte, Malassezia, and Fusarium. Micafungin is the
`active ingredient in the approved drugs Mycamine® and Funguard® which are used in the treatment and prophylaxis
`of infections caused by Candida.
`[0005] Various methods for the preparation and purification of Micafungin are known to the skilled person, see e.g.
`US 6,107,458 and US 7,199,248. More particularly, US 7,199,248 discloses a method wherein a crude DIPEA salt of
`Micafungin is purified by filtration and chromatographic separation using a regenerated y Alumina in a 1350-L column
`and eluting Micafungin DIPEA with Methanol. The Micafungin containing fraction is further purified and transferred to a
`sodium salt of Micafungin, inter alia by ion exchange chromatography using a regenerated ion exchange resin UBK510L.
`Micafungin sodium is eluted with aqueous Methanol. Acetone and ethyl acetate is finally used for precipitation of Mi-
`cafungin sodium.
`[0006]
`It is well known in the art that the diisopropylethylamine (DIPEA) salt of Micafungin is more stable than the
`sodium salt of Micafungin. Therefore, the prior art methods as the method disclosed above often make use of the DIPEA
`salt as an intermediate when preparing Micafungin sodium. This is e.g. described in the General Thesis, "Process
`Development of Micafungin, a Novel Lipopeptide Antifungal Agent" by Ohigashi et al. in Journal of Synthetic Organic
`Chemistry, Japan, vol. 64, No. 12, Dec. 2006. In Ohigashi et al., a method is disclosed wherein impurities present in a
`DIPEA salt of Micafungin are removed by the use of resins. The DIPEA salt purified by the use of an alumina resin
`column is according to Ohigashi et al. then subjected to ion exchange chromatography for converting the DIPEA salt of
`Micafungin to a sodium salt of Micafungin.
`[0007]
`It is clear that the prior art methods for the preparation of Micafungin sodium as mentioned above involves two
`consecutive chromatography steps, i.e. firstly the purification of Micafungin DIPEA on a Reverse Phase Chromatography
`(RPC) resin and secondly the transformation of the DIPEA salt to the sodium salt, i.e. a salt swap, on a ion-exchange
`resin. The use of two subsequent chromatography steps for the obtainment of the desired sodium salt of Micafungin is
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`labour-intensive and the providing of an improved method involving fewer processing steps, less chemicals and equip-
`ment would be beneficial both from an economical aspect as well as from an environmental and labour-saving point of
`view.
`[0008]
`It is thus still a need for more efficient processes for the preparation of a purified Micafungin and derivates
`thereof, such as the sodium salt of Micafungin.
`
`Summary of the invention
`
`[0009] The object of the present invention is to provide an improved process for the purification of a salt of Micafungin,
`such as Micafungin sodium.
`[0010] As mentioned above, the prior art methods is inter alia attended with the disadvantage of two consecutive
`chromatography steps. The present invention is based on the surprising findings that the two steps (salt swap and
`purification) can be performed in the one and same operation. The process of the present invention thus involves several
`self-evident advantages, both of environmental and economical character, such as reduced consumption of chemicals,
`shorter process times and the use of less hardware requirements.
`[0011] According to one embodiment of the invention, a method for producing a pharmaceutically acceptable Micafun-
`gin salt is provided wherein said process comprises the steps of:
`
`a) applying a Micafungin starting material to a hydrophobic adsorbent resin support;
`
`b) exposing the bound Micafungin to an aqueous solution of a dissolved pharmaceutically acceptable salt;
`
`c) eluting the dissolved pharmaceutically acceptable salt of Micafungin with a solution comprising a water miscible
`organic solvent;
`
`provided that at least one of
`
`i) the starting material in step a)
`ii) or the aqueous solution in step b)
`
`comprises a water miscible organic solvent.
`[0012] According to one aspect of the above embodiment, a method is provided which further comprises a step wherein
`the bound Micafungin of step b) is further exposed to an aqueous solution comprising a water miscible organic solvent
`one or more times for the removal of impurities.
`[0013] According to another aspect of the above embodiment, a method is provided wherein the starting material in
`step a) comprises a water miscible organic solvent in the range of 0 - 30 % v/v.
`[0014] According to another aspect of the above embodiment, a method is provided wherein the starting material in
`step a) comprises a water miscible organic solvent in the range of 5-15 % v/v.
`[0015] According to another aspect of the above embodiment, a method is provided wherein the starting material in
`step a) comprises a water miscible organic solvent in the range of 10 % v/v.
`[0016] According to another aspect of the above embodiment, a method is provided wherein the aqueous solution in
`step b) comprises a water miscible organic solvent in the range of 0 - 40 % v/v.
`[0017] According to another aspect of the above embodiment, a method is provided wherein the aqueous solution in
`step b) comprises a water miscible organic solvent in the range of 20 % v/v.
`[0018] According to another aspect of the above embodiment, a method is provided wherein the water miscible organic
`solvent is selected from the group consisting of branched or non-branched C1-C3 alcohols, C3-C6 ketones and polar
`aprotic organic solvents.
`[0019] According to another aspect of the above embodiment, a method is provided wherein the water miscible organic
`solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, acetone and acetonitrile.
`[0020] According to another aspect of the above embodiment, a method is provided wherein the aqueous solution in
`step c) comprises at least 30% v/v acetonitrile as a water miscible organic solvent.
`[0021] According to another aspect of the above embodiment, a method is provided wherein the water miscible organic
`solvent is methanol.
`[0022] According to another aspect of the above embodiment, a method is provided wherein the aqueous solution in
`step c) comprises at least 70 % v/v methanol, such as at least 90 %v/v methanol, such as at least 95 % v/v methanol
`as a water miscible organic solvent.
`[0023] According to another aspect of the above embodiment, a method is provided wherein the starting material in
`step a), the aqueous solution in step b) and the washing solution in step c) comprises a water miscible organic solvent.
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`[0024] According to another embodiment of the invention, a method for producing a pharmaceutically acceptable
`Micafungin salt is provided wherein said process comprises the steps of:
`
`a) applying a Micafungin starting material to a hydrophobic adsorbent resin support;
`
`b) exposing the bound Micafungin to an aqueous solution of a dissolved pharmaceutically acceptable salt;
`
`c) optionally exposing the bound salt of Micafungin obtained in b) to an aqueous solution comprising a water miscible
`organic solvent one or more times for the removal of impurities;
`
`d) eluting the dissolved pharmaceutically acceptable salt of Micafungin with a solution comprising a water miscible
`organic solvent;
`
`provided that when step c) is not performed, then the Micafungin starting material and/or the aqueous solution in step
`b) comprises a water miscible organic solvent.
`[0025] According to one embodiment, the Micafungin starting material or the aqueous solution in step b) of the present
`method comprises 2-30% v/v of a water miscible organic solvent.
`[0026] According to one embodiment, the Micafungin starting material and the aqueous solution in step b) of the
`present method comprises 2-30% v/v of a water miscible organic solvent.
`[0027] According to one embodiment, the aqueous solution in step c) of the present method comprises 30-50% v/v
`of a water miscible organic solvent.
`[0028] According to another embodiment, the cation present in the Micafungin starting material of the present invention
`is DIPEA.
`[0029] According to yet another embodiment, the resin used according to the present method is HP20SS.
`[0030] According to another embodiment, the Micafungin starting material and the aqueous solution in step b) of the
`present method comprises a water miscible organic solvent.
`[0031] According to yet another embodiment, the Micafungin starting material and the aqueous solution in step b) of
`the present method comprises Methanol.
`[0032] According to yet another embodiment of the present method, the water miscible organic solvent present in the
`Micafungin starting material and/or the aqueous solution in step b) is Methanol.
`[0033] According to yet another embodiment of the present method, the aqueous solution in step b) comprises Meth-
`anol.
`[0034] According to a final embodiment of the present invention, a method is provided wherein the pharmaceutically
`acceptable salt of Micafungin is the sodium salt, and the resin is HP20SS, and the Micafungin starting material comprises
`Micafungin DIPEA and 10% v/v Methanol, and the solution in step b) comprises 20% v/v Methanol and 80% v/v 3M
`NaCl, 0.1M NaCH3COO and the solution in step c) comprises 40% v/v Methanol and the solution in step d) comprises
`90% v/v Methanol.
`
`Brief description of the figures
`
`[0035] Figure 1-9 illustrate the elution profile of DIPEA and Micafungin sodium from HP20SS according to the corre-
`sponding Examples 1-9. The pertaining tables show the level of DIPEA in eluted fractions (CV = column volumes).
`
`Detailed description
`
`[0036] According to the present invention, Micafungin is any compound comprising the structure
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`or salts thereof. The expression "salts thereof" is meant to embrace any salts of Micafungin that may be useful for the
`purpose of preparation and/or purification of Micafungin or any pharmaceutically acceptable salts of Micafungin useful
`as an active ingredient in a medicinal formulation. In this respect, a non-limiting list of salts of Micafungin is the sodium
`salt, the potassium salt, the diisopropylethylamine (DIPEA) salt, etc.
`[0037] Micafungin is a semisynthetic derivative of FR901379, a fermentation product obtained from Coleophoma
`empetri F-11899 (deposited as FERM BP-2635).
`[0038] Micafungin may be prepared by deacylation of the fermentation product FR901379, obtaining a compound
`named the Micafungin peptide core herein, and subsequent coupling of the Micafungin side chain, 4-[5-(4-pentyloxy-
`phenyl)isoxazole-3-yl]benzoic acid, to said peptide core. The Registry Number of the said side chain assigned by Chem-
`ical abstracts is 179162-55-1. It is also known by the name FR195752.
`[0039] Deacylation of FR901379 can be performed by enzymes produced by certain microorganisms of the Actino-
`planaceae, for example, Actinoplanes utahensis lF0-13244, Actinoplanes utahensis ATCC 12301, Actinoplanes mis-
`sourienses NRRL 12053. The deacylated peptide core is represented here as formula II. The sodium salt of the deacylated
`peptide core was named FR133303 (see EP462531).
`[0040] Reacylation of the peptide core represented by formula II can be performed as disclosed in US 7,199,248.
`[0041] The Micafungin peptide core is represented by the formula II.
`
`[0042] According to the present invention, a method for the preparation of a purified salt of Micafungin is provided.
`According to one embodiment of the invention, the salt of Micafungin obtained according to the present invention is the
`sodium salt of Micafungin. It is to be understood that also other pharmaceutically acceptable salts of Micafungin may
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`be obtained by the present method, such as e.g. the potassium salt of Micafungin.
`[0043] According to the present invention, a method is provided for the purification and preparation of a pharmaceutically
`acceptable salt of Micafungin wherein a Micafungin starting material is adsorbed to a solid support, subjecting and
`washing the adsorbed Micafungin with an aqueous solution comprising a dissolved salt and finally eluting the so obtained
`pharmaceutically acceptable salt of Micafungin using an elution solution comprising a water miscible organic solvent.
`[0044] According to the present invention, the term "Micafungin starting material" is an aqueous solution comprising
`a dissolved salt of Micafungin. The Micafungin starting material enables the adsorption of the Micafungin salt to a solid
`support in such a way that both salt swap and purification may be performed in one step.
`[0045] The Micafungin starting material comprises a salt of Micafungin suitable for the adsorption to a solid support.
`When said salt is adsorbed to a solid support and upon the further exposure to a solution of a dissolved pharmaceutically
`acceptable salt, a salt swap is enabled. The initial cations associated with Micafungin in the Micafungin starting material
`can then be removed by washing with an aqueous solution optionally comprising a water miscible organic solvent. Finally,
`the pharmaceutically acceptable Micafungin salt can be eluted by an aqueous solution comprising a water miscible
`organic solvent.
`[0046] The Micafungin salt present in the starting material is thus different from the salt of Micafungin being eluted
`from the solid support after the salt swap and purification. The Micafungin salt present in the Micafungin starting material
`is according to a preferred embodiment of the invention the DIPEA salt of Micafungin.
`[0047] According to one aspect of the present invention, a method is provided, wherein the final salt of Micafungin is
`substantially free of the cation of the Micafungin salt present in the Micafungin starting material. The term "substantially
`free" means in the context of the present invention that the levels of said cation in the final Micafungin salt are not
`detectable. The term "not detectable" means in the context of the present invention that the cation of the Micafungin
`starting material, e.g. DIPEA cannot be detected by the following HPLC assay:
`
`System Parameters
`
`[0048]
`
`Column
`Flow rate
`Column temperature
`Back pressure
`
`Kinetex C18, 2.6 m, 100 mm x 2.1 mm. 100A
`0.8 mL/min
`30°C
`approximately 3500 psi or 250bar
`
`Mobile phase A
`
`Mobile phase B
`
`99% MilliQ water,
`1% Acetonitrile,
`0.1% (v/v) TFA
`
`1% MilliQ water,
`99% Acetonitrile,
`0.1 % (v/v) TFA
`
`Gradient
`
`Time (min)
`
`%B
`
`0
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`6
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`7
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`8
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`15
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`0
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`15
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`50
`
`50
`
`0
`
`0
`
`Run Time
`
`15 min
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`(continued)
`
`Injection volume
`Auto sampler temperature
`Integrator setting
`
`5mL
`5 °C
`Peak area %.
`
`Retention time, Dipea: approx. 4.6 minutes
`
`Detector: Corona Cad detector
`
`Sample preparation:
`
`Accurately weigh 50 mg of the sample into a 10 ml volumetric flask. Dissolve in and dilute to volume with dilution
`solution (water with 0.1% TFA). Inject. 5ml.
`
`[0049] According to another aspect of the present invention, a method is provided, wherein the final salt of Micafungin
`is sufficiently free of the cation of the Micafungin salt present in the Micafungin starting material. The term "sufficiently
`free" means in the context of the present invention that the level of the cation from the Micafungin starting material
`remaining in the final Micafungin salt is below 300 ppm. E.g. according to one embodiment of the present method, the
`level of DIPEA in the produced Micafungin sodium is less than 300 ppm.
`[0050] According to one aspect of the present invention, a method for producing Micafungin sodium is provided,
`wherein the obtained Micafungin sodium is substantially free of DIPEA.
`[0051] According to one aspect of the present invention, a method for producing Micafungin sodium is provided,
`wherein the obtained Micafungin sodium is sufficiently free of DIPEA.
`[0052] According to yet another aspect of the invention, the Micafungin starting material is an aqueous solution of a
`dissolved salt of Micafungin which in addition comprises a water miscible organic solvent. The presence of a water
`miscible organic solvent will further facilitate the adsorption of the Micafungin salt present in the Micafungin starting
`material to a solid support in such a way that both purification and salt swap may be performed in one step. More
`particularly, the Micafungin starting material may comprise an aqueous solution of dissolved Micafungin diisopropyl-
`ethylamine (DIPEA) and a water miscible organic solvent.
`[0053] The salt of Micafungin present in the Micafungin starting material may be prepared according to methods well
`known to the skilled person. For example, Micafungin DIPEA of the formula
`
`may be prepared according to the methods disclosed in US 6,170,458 or US 7,199,248. A Micafungin salt useful as a
`starting material according to the present invention may also be prepared as outlined in Ohigashi et al., supra.
`[0054]
`It is to be understood that the term "a water miscible organic solvent" that may optionally be present in the
`Micafungin starting material represents a solvent that further facilitates the binding of the Micafungin present in the
`Micafungin starting material in such a way that a salt swap may be performed in addition to the purification of Micafungin
`in one chromatographic step only. A non-limiting list of suitable water miscible organic solvent comprises branched or
`non-branched C1-C3 alcohols, C3-C6 ketones and polar aprotic organic solvents (e.g. Methanol, Ethanol, Propanol,
`Isopropanol, Acetone and Acetonitrile). According to one embodiment of the present invention, the water miscible organic
`solvent is Methanol.
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`[0055] The skilled person will acknowledge the proper amount of the water miscible organic solvent to be present in
`the Micafungin starting material based on the teaching of the present invention and his/her common general knowledge.
`More particularly, the skilled person will understand that any amount of the water miscible organic solvent that facilitates
`the binding of the Micafungin salt comprised in the starting material and the further salt swap, purification and eluting of
`the desired Micafungin salt may be used.
`[0056] The amount of water miscible organic solvent present in the starting material will depend on the type of solvent
`used. According to one embodiment of the present invention, the amount of the water miscible organic solvent that may
`be present in the Micafungin starting material used according to the present invention is from 0-30% v/v. According to
`another embodiment of the present invention, the amount of the water miscible organic solvent that may be present in
`the Micafungin starting material used according to the present invention is from 5-15% v/v. According to yet another
`embodiment of the present invention, the amount of the water miscible organic solvent in the Micafungin starting material
`used according to the present invention is about 10% v/v.
`[0057] A solid support according to the present invention may be any material enabling the adsorption of the Micafungin
`salt present in the starting material to said support, i.e. binding said salt through hydrophobic interaction in such a way
`that the bound Micafungin salt may be transferred to another salt, e.g. facilitating the transformation of Micafungin DIPEA
`to Micafungin sodium. Such a transformation of one salt of a compound to another salt of said compounds is commonly
`known as a "salt swap".
`[0058] Furthermore, the solid support must also provide binding of the Micafungin salt present in the starting material
`in such a way that impurities may be removed in addition to said salt swap prior to the elution of the desired pharma-
`ceutically acceptable salt of Micafungin.
`[0059] Yet another characteristic of the solid support in addition to the above is that it finally must facilitate a reversible
`binding of the Micafungin salt at the option of the skilled person, i.e. enabling elution of the desired salt obtained after
`the salt swap and purification of Micafungin.
`[0060] More particularly, the solid support is according to the invention a hydrophobic adsorbent resin support including
`but not limited to modified silica, polystyrene or acrylic materials, and including e.g. the polyamide adsorbent Polyamide
`C6 (Macherey Nagel), the acrylic adsorbent XAD7HP® (Rohm & Haas) or HP2MG® (Diaion), and the polystyrene divinyl
`benzene adsorbents XAD1600 (Rohm & Haas), HP20SS (Diaion), SP20SS (Diaion). The most preferred resin is HP20SS.
`[0061] The salt swap is performed by exposing the Micafungin salt of the starting material being adsorbed to the solid
`support to an aqueous solution of the desired pharmaceutically acceptable salt, such as e.g. an aqueous solution of
`sodium or potassium salt. According to one embodiment of the invention, the adsorbed Micafungin salt, e.g. the DIPEA
`salt of Micafungin, is exposed to an aqueous dissolution of a NaCl and/or Na-acetate buffer with pH 4-6. The aqueous
`solution of a pharmaceutically acceptable salt may also include a water miscible organic solvent. The water miscible
`organic solvent may be the same solvent that may be present in the Micafungin starting material as defined above.
`[0062] According to one embodiment, the adsorbed Micafungin salt is exposed to an aqueous solution of a pharma-
`ceutical acceptable salt and 10% v/v methanol. According to another embodiment, the adsorbed Micafungin salt is
`exposed to an aqueous solution of a pharmaceutical acceptable salt comprising 20 % v/v methanol. The pH of the
`aqueous solution of the pharmaceutically acceptable salt of Micafungin to be obtained in this step should be selected
`so as to avoid degradation of said Micafungin salt. Preferably the pH of said solution is acidic. For example, the pH of
`the aqueous solution of the dissolved pharmaceutically acceptable salt may be about 4.5.
`[0063] Further purification of Micafungin may be performed by washing the solid support with a solution comprising a
`water miscible organic solvent either stepwise or using a gradient. E.g. a washing solution comprising 40% v/v Methanol,
`can remove significant amounts of impurities (such as by-products or degradation products) from the HP20SS resin. In
`general, by-products and degradation products with similar binding/elution properties as the desired Micafungin salt,
`can be selectively removed by applying a washing solution comprising a higher level of the water miscible organic
`solvent. In this context, "higher level" means that level of the water miscible organic solvent is higher compared with the
`level of water miscible organic solvent present in the Micafungin starting material or the solution comprising the phar-
`maceutically acceptable salt of Micafungin (i.e. step b) of the present method). The use of successive increasing levels
`of a water miscible organic solvent throughout the steps a) - d) of the present method presuppose that it is the same
`type of water miscible organic solvent used in all steps a)-d) whenever these steps includes the presence of said solvent.
`Reactants such as e.g. by-products and degradation products present in the starting material according to the present
`invention may thus be removed using higher concentrations of the same solvent as present in the Micafungin starting
`material.
`[0064] The resulting Micafungin salt, e.g. the sodium salt of Micafungin bound to the solid support obtained through
`the salt swapping and optional washing (purification) step, may be eluted by applying an aqueous solution comprising
`a water miscible organic solvent wherein the level of the water miscible organic solvent in the elution solution is higher
`compared with the amount of water miscible organic solvent present in the previous steps of the present method. The
`water miscible organic solvent may be of the same type as present in the Micafungin starting material. I.e., a non limiting
`list of suitable water miscible organic solvent to be used when eluting the desired Micafungin salt from the solid support
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`according to the present invention comprises branched or non-branched C1-C3 alcohols, C3-C6 ketones and polar
`aprotic organic solvents such as Methanol, Ethanol, Propanol, Isopropanol, Acetone, and Acetonitrile. According to one
`embodiment of the present invention, the water miscible organic solvent is Methanol.
`[0065] The amount of the water miscible organic solvent present in the elution solution is an amount being sufficient
`for elution of Micafungin. The amount of the water miscible organic solvent present in the elution solution will depend
`on the polarity of the solvent and the hydrophobicity of the hydrophobic adsorbent resin support. E.g. a pharmaceutically
`acceptable salt of Micafungin can be eluted from HP20SS by an aqueous solution comprising more than 30% v/v
`acetonitrile. A pharmaceutically acceptable salt of Micafungin can be eluted from HP20SS by an aqueous solution
`comprising more than 70% v/v Methanol. The skilled artisan will be able to determine the appropriate amount of the
`water miscible organic solvent in the aqueous elution solution based on his general knowledge of the various hydrophobic
`adsorbent resin support that are used according to the present invention and by routine experiments.
`[0066] According to a preferred embodiment of the present invention, Micafungin sodium is eluted by applying an
`aqueous solution comprising Methanol at a concentration of 70%-95% v/v.
`[0067] The Micafungin salt being prepared according to the present invention may be used in the manufacturing of
`pharmaceutical composition useful for the treatment and prevention of infectious diseases in mammals, including humans
`and animals. Said pharmaceutical composition may be prepared using standard techniques well known in the art. The
`pharmaceutical composition may also be subjected to conventional pharmaceutical operations such as sterilization.
`[0068] For example, a pharmaceutical composition may be prepared in form of a dosage unit containing a given
`amount of purified Micafungin, alone or in combination with another active ingredient, and together with pharmaceutically
`acceptable excipients. The term "pharmaceutical acceptable excipient" refers to one or more formulation materials
`suitable for accomplishing or enhancing the delivery of Micafungin contained in the pharmaceutical composition to the
`patients in need thereof. A "pharmaceutical acceptable excipient" may also be represented by the presence of adjuvants,
`such as preservatives, stabilizers, wetting agents, emulsifiers, buffers and the like. Lactose is an example of a suitable
`stabilizer for Micafungin, especially in lyophilized form. The skilled person will be well aware of the various pharmaceutical
`excipients available for the preparation of pharmaceutical formulations comprising antifungal compounds like Micafungin.
`The composition according to the present invention may be prepared to enable parenteral administration, e.g. intravenous
`administration.
`[0069] The patient in need for a composition according to the present invention may be administered a suitable dosage
`of Micafungin. A suitable daily dose for a human or mammal may vary widely depending on the condition of the patient
`and other factors. The daily dose may be determined by the skilled artisan using routine methods for determining the
`appropriate dose suitable for each patient, e.g. based on the doses commonly used for the treatment and prevention of
`infections when administering Mycamine®.
`[0070] A skilled person will from the following examples acknowledge the many advantages of the present invention.
`The experiments and results below are to be understood to serve as non-limiting examples only.
`
`EXAMPLES
`
`[0071]
`In the following examples, a hydrophobic resin, HP20SS (Diaion), was packed into a chromatography column
`(15ml, i.d 1.1cm). 1.125g Micafungin starting material (100mg Micafungin/ml) was added to the column (0.5 ml/min).
`The column was washed with a 3M NaCl/0.1M NaAc pH 5.5 solution with or without addition of methanol (150ml). The
`column was then washed with an aqueous solution comprising 40% methanol (90ml). Micafungin sodium was eluted
`from the column with 90% methanol.
`[0072] The 3M NaCl/0.1M NaAc pH 5.5 solution was prepared by dissolving 350.64g NaCl in 1988.5ml RO water and
`adding 11.44ml acetic acid. pH was adjusted to 5.5 with NaOH(s).
`
`Example 1: Organic solvent in load and wash
`
`[0073] An aqueous solution comprising crude Micafungin DIPEA salt and 10% v/v Methanol was applied to a HP20SS
`column.
`[0074] The column was first washed with an aqueous solution comprising 90% v/v 3M NaCI/O.1M NaAc pH 5.5 and
`10% v/v Methanol.
`[0075] The column was then washed with an aqueous solution comprising 40% v/v Methanol.
`[0076] Micafungin sodium was eluted with a solution comprising 90% v/v Methanol and 10% v/v water.
`[0077] As seen in Figure 1, the Micafungin sodium solution eluted at 18-20 CV is substantially free of DIPEA
`
`Example 2: Organic solvent in load
`
`[0078] An aqueous solution comprising crude Micafungin DIPEA salt and 10% v/v Methanol was applied to a HP20SS
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`column.
`[0079] The column was washed with an aqueous solution comprising 3M NaCI/O.1M NaAc pH 5.5.
`[0080] Micafungin sodium was eluted with a solution comprising 90% v/v Methanol and 10% v/v water.
`[0081] As seen in Figure 2, the Micafungin sodium solution eluted at 18-20 CV is substantially free of DIPEA
`
`Example 3: Organic solvent in load and wash
`
`[0082] An aqueous solution comprising crude Micafungin DIPEA salt and 10% v/v Methanol was applied to a HP20SS
`column.
`[0083] The column was first washed with an aqueous solution comprising 90% v/v3M NaCI/O.1M NaAc pH 5.5 and
`10% v/v Methanol.
`[0084] The column was then washed with an aqueous solution comprising 40% v/v Methanol.
`[0085] Micafungin sodium was eluted with a solution comprising 90% v/v Methanol and 10% v/v water.
`[0086] As seen in Figure 3, the Micafungin sodium solution eluted at 18-20 CV is suffi