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Method of Purifying Cytidine Diphosphate Choline Verfahern Zur Reinigung Von Cytidin-Diphosphat-Cholin Procédé De Purification De La Cytidine Diphosphate Choline

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Method of Purifying Cytidine Diphosphate Choline Verfahern Zur Reinigung Von Cytidin-Diphosphat-Cholin Procédé De Purification De La Cytidine Diphosphate Choline (19) TZZ_¥ _Z_T (11) EP 1 939 210 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07H 19/10 (2006.01) C12P 19/32 (2006.01) 03.12.2014 Bulletin 2014/49 (86) International application number: (21) Application number: 06782606.5 PCT/JP2006/315802 (22) Date of filing: 10.08.2006 (87) International publication number: WO 2007/018259 (15.02.2007 Gazette 2007/07) (54) METHOD OF PURIFYING CYTIDINE DIPHOSPHATE CHOLINE VERFAHERN ZUR REINIGUNG VON CYTIDIN-DIPHOSPHAT-CHOLIN PROCÉDÉ DE PURIFICATION DE LA CYTIDINE DIPHOSPHATE CHOLINE (84) Designated Contracting States: (74) Representative: Duckworth, Timothy John et al AT BE BG CH CY CZ DE DK EE ES FI FR GB GR J A Kemp HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI 14 South Square SK TR Gray’s Inn London WC1R 5JJ (GB) (30) Priority: 10.08.2005 JP 2005231958 (56) References cited: (43) Date of publication of application: EP-A1- 1 502 956 GB-A- 1 305 539 02.07.2008 Bulletin 2008/27 JP-A- 62 016 497 (73) Proprietor: Kyowa Hakko Bio Co., Ltd. • FUJIO TATSURO ET AL: "Construction of a Chiyoda-ku plasmid carrying both CTP synthetase and a Tokyo 100-8185 (JP) fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes (72) Inventors: and its application to industrial CDP-choline • MURATA, Hideki production: Enzymatic production of CDP- 1-1, Kyowa-cho, Hofu-shi, Yamaguchi 747-8522 choline from orotic acid (Part II)", BIOSCIENCE (JP) BIOTECHNOLOGY AND BIOCHEMISTRY, vol. 61, • MOKUDAI, Tsuyoshi no. 6, 1997, pages 960-964, XP55009405, ISSN: 1-1, Kyowa-cho, Hofu-shi, Yamaguchi 747-8522 0916-8451 (JP) • SHIOMI, Michio Yamabu-gun, Chiba 289-1726 (JP) 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 1 939 210 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 1 939 210 B1 2 Description prises contacting a CDP-choline solution containing a nucleic acid analogue and having a pH of not less [0001] The present invention relates to a method of than 0.5 and not more than 5.0 with an H-type strong- purifying cytidine diphosphate choline useful as a starting ly acidic cation exchange resin, and eluting CDP- material of pharmaceutical products and a starting ma- 5 choline adsorbed onto the resin with water or an terial of nutritious foods. aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify CDP- Background Art choline. (2) The method of the above-mentioned (1), wherein [0002] As the method of purifying cytidine diphosphate 10 the CDP-choline solution is prepared from a resulting choline (hereinafter to be abbreviated as CDP-choline), medium containing CDP-choline, which has been chemical synthetic methods (patent reference 1, patent produced and accumulated therein by placing a bio- reference 2) and methods using a culture of a microor- catalyst having an activity to produce CDP-choline ganism or an enzyme (patent reference 3, patent refer- from a precursor of UTP and choline or phosphoryl- ence 4) are known. As the method of purifying CDP-15 choline in an aqueous medium together with the pre- choline produced by a chemical synthetic method, a cursor of UTP and choline or phosphorylcholine or method using an anion exchange resin (patent reference a salt thereof. 1) and a method using both of a strongly acidic ion ex- (3) The method of the above-mentioned (2), wherein change resin and a weakly basic ion exchange resin (pat- the biocatalyst comprises a culture or a treated cul- ent reference 2) are known. In the latter method, two20 ture of a microorganism capable of producing UTP kinds of ion exchange resins are used. Moreover, while from a precursor of UTP, and a culture or a treated phosphorylcholine and cytidine-5’-monophosphate culture of a microorganism capable of producing (hereinafter to be abbreviated as CMP) contained in a CDP-choline from UTP and choline or phosphoryl- CDP-choline solution can be separated, uracil or uridine- choline. 5’-triphosphate (hereinafter to be abbreviated as UTP) 25 (4) The method of the above-mentioned (2), wherein cannot be separated efficiently by this method. the biocatalyst comprises an enzyme that catalyzes a reaction to produce CDP-choline from a precursor [patent reference 1] Japanese Published Examined of UTP and choline or phosphorylcholine. Patent Application No. 6558/1988 (5) The method of the above-mentioned (4), wherein [patent reference 2] Japanese Published Examined 30 the enzyme that catalyzes a reaction to produce Patent Application No.31306/1994 CDP-choline is an enzyme selected from the group [patent reference 3] Japanese Patent No.3369236 consisting of orotate phosphoribosyl transferase, [patent reference 4] WO99/49073 orotidine-5’-monophosphate decarboxylase, uridine phosphorylase, uracil phosphoribosyltransferase, EP 1 502 956 discloses a process for producing cytidine 35 uridine kinase, uridylate and cytidylate kinases, nu- 5’-diphosphate choline. GB1305539 disloses a process cleoside diphosphate kinase, cytidine-5’-triphos- for the preparation of nucleoside diphosphate esters, phate synthase, choline phosphate cytidyltrans- which include cytidine diphosphate choline. Fujio et al ferase and choline kinase. (Bioscience Biotechnology and Biochemistry, 61 (6) (6) The method of any of the above-mentioned (1) 960-964, 1997) discloses a method for enzymatic pro- 40 to (5), wherein the nucleic acid analogue is selected duction of cytidine diphosphate choline from orotic acid from uracil and UTP. and choline chloride. Effect of the Invention Disclosure of the Invention 45 [0005] The present invention provides CDP-choline Problems to be Solved by the Invention and a salt thereof at a low cost. [0003] An object of the present invention is to provide Best Mode for Carrying Out the Invention a method of purifying CDP-choline, in which method CDP-choline can be separated from a nucleic acid ana- 50 [0006] The CDP-choline solution to be used in the logue conveniently. present invention may be prepared by any method as long as it contains a nucleic acid analogue and has a pH Means of Solving the Problems of not less than 0.5 and not more than 5.0. When the prepared CDP-choline solution has a pH of 0.5 - 5.0, it [0004] The present invention relates to the following 55 is directly used. When the pH is higher than 5.0, an acid (1) - (6). such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid is added, and when the pH is lower than (1) A method of purifying CDP-choline, which com- 0.5, an alkali such as sodium hydroxide or potassium 2 3 EP 1 939 210 B1 4 hydroxide is added to adjust to pH 0.5 - 5.0, preferably may be subjected to an activated carbon treatment or 1.0 - 3.5, before use. decolorization treatment using a nonpolar porous syn- [0007] The H-type strongly acidic cation exchange res- thetic adsorbent, for example, Diaion HP series (e.g., in may be of a gel type or a porous type, as long as it is HP20, HP21 etc.) manufactured by Mitsubishi Chemical, an H-type strongly acidic cation exchange resin. Specific 5 Diaion SP800 series (e.g., SP825, SP850 etc.) manu- examples thereof include the Dowex series (e.g., HCR- factured by Mitsubishi Chemical, Diaion SP200 series S, HCR-W2, Marathon C, Monosphere 650C, MSC-1, (e.g., SP207 etc.) manufactured by Mitsubishi Chemical, Monosphere 88, 50Wx2, 50W34, 50W38 etc.) manu- Amberlite XAD series (e.g., XAD4, XAD7HP, XAD16HP, factured by Dow Chemical Company, Diaion SK series XAD1180, XAD2000 etc.) manufactured by Rohm and (e.g., SK1B, SK104, SK110, SK112 etc.) manufactured 10 Haas. by Mitsubishi chemical, Diaion PK series (e.g., PK208, [0014] The above-mentioned CDP-choline-containing PK212, PK220, PK228 etc.) manufactured by Mitsubishi solution or decolorized solution is adjusted to pH 2.0 - Chemical, Amberlite series (e.g., IR120B, IR124 etc.) 4.0 with acid or alkali. After concentration is carried out manufactured by Rohm and Haas. as necessary, the concentration of CDP-choline is ad- [0008] Degree of crosslinking of the H-type strongly 15 justed to 50 - 800 g/L, preferably 100 - 700 g/L, and crys- acidic cation exchange resin is not particularly limited as tals of CDP-choline can be obtained using an organic long as CDP-choline and nucleic acid analogue can be solvent, preferably a hydrophilic organic solvent such as separated at the degree of crosslinking. It is preferably acetone, ethanol, methanol or propanol. 2 - 10%, more preferably 4 - 6%. [0015] In addition, the above-mentioned CDP-choline- [0009] In the present invention, the H-type strongly 20 containing solution or decolorized solution is adjusted to acidic cation exchange resin is preferably used in the pH 5.0 - 9.5 with sodium hydroxide. After concentration form of a packed column, and as the column to be used is carried out as necessary, the concentration of CDP- in the present invention, any column may be used. choline is adjusted to 50 - 800 g/L, preferably 100 - 700 [0010] The CDP-choline solution containing a nucleic g/L, and crystals of CDP-choline sodium salt can be ob- acid analogue and having a pH of not less than 0.5 and 25 tained using an organic solvent, preferably a hydrophilic not more than 5.0 is contacted with the H-type strongly organic solvent such as acetone, ethanol, methanol or acidic cation exchange resin by applying the solution to propanol.
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