Method for producing organic compound

Abstract

Disclosed is a method for producing a target organic compound, the method including: (1) a step for causing an immobilization carrier to adsorb a target organic compound; (2) a step for passing an eluent through the immobilization carrier on which the target organic compound has been adsorbed; (3) a step for dividing an elution fraction containing the target organic compound into (a) a fraction which contains the eluent at a first concentration and (b) a fraction in which the concentration of the eluent is lower than the first concentration; (4) a step for concentrating the fraction (a) so as to obtain a concentrate; and (5) a step for adding the fraction (b) to the concentrate and concentrating the concentrate. This method makes it possible to efficiently remove an eluent.

Description

Methods for producing organic compounds

[0001] This disclosure relates to a method for producing organic compounds. More specifically, it relates to a method for efficiently removing eluents used in purification using immobilized carriers such as ion exchange resins, synthetic adsorption resins, activated carbon, or silica gel from an eluted fraction containing a target organic compound.

[0002] Volatile organic acids are frequently used as eluents to extract compounds adsorbed onto anion exchange resins (Patent Documents 1 and 2). Typically, the eluent cannot be completely separated from the target organic compound during elution and is mixed into the fraction of the target organic compound as an impurity. If the eluent is volatile, it is possible to separate the two by vacuum concentration using the difference in volatility. However, eluents with boiling points higher than water are less volatile, and in some cases, the eluent cannot be sufficiently removed without adding water and performing vacuum concentration. In this case, the time required for vacuum concentration is extended, making it impossible to prevent the decomposition of the target organic compound by heating, leading to a deterioration in quality, such as an increase in the impurity content. Patent Document 2 discloses a method for obtaining a fraction of reduced glutathione without acetic acid contamination by chromatographic separation. However, this merely discards the fraction contaminated with acetic acid in the latter half of elution, and does not describe a method for efficiently evaporating and removing acetic acid from the acetic acid-contaminated fraction.

[0003] Japanese Patent Publication No. 5-33715 Patent No. 3315158

[0004] The object of this disclosure is to provide a method for efficiently removing eluents used in purification using a stationary carrier.

[0005] The present inventors have discovered that the eluent can be efficiently removed by separating a fraction containing the target organic compound eluted from a solid carrier into a fraction with a high concentration of the eluent and a fraction without the eluent or with a lower concentration, and by first concentrating the fraction with a high concentration of the eluent. This has led to the completion of the present invention. That is, the present disclosure is as follows: [1] A method for producing a target organic compound, comprising the steps of: (1) adsorbing the target organic compound onto a stationary carrier; (2) passing an eluent through the stationary carrier on which the target organic compound has been adsorbed; (3) dividing the eluted fraction containing the target organic compound into (a) a fraction containing the eluent at a first concentration and (b) a fraction in which the concentration of the eluent is less than the first concentration; (4) concentrating the fraction from (a) to obtain a concentrate; and (5) adding the fraction from (b) to the concentrate to concentrate it. [2] A method for purifying a target organic compound, comprising the steps of: (1) adsorbing the target organic compound onto a stationary carrier; (2) passing an eluent through the stationary carrier on which the target organic compound has been adsorbed; (3) dividing the eluted fraction containing the target organic compound into (a) a fraction containing the eluent at a first concentration and (b) a fraction in which the concentration of the eluent is less than the first concentration; (4) concentrating the fraction from (a) to obtain a concentrate; and (5) adding the fraction from (b) to the concentrate to concentrate it. [3] A method for removing an eluent, comprising the steps of: (1) adsorbing a target organic compound onto a stationary carrier; (2) passing an eluent through the stationary carrier on which the target organic compound has been adsorbed; (3) dividing the eluted fraction containing the target organic compound into (a) a fraction containing the eluent at a first concentration and (b) a fraction in which the concentration of the eluent is less than the first concentration; (4) concentrating the fraction from (a) to obtain a concentrate; and (5) adding the fraction from (b) to the concentrate to concentrate it. [4] The method according to any one of [1] to [3], wherein the concentration in steps (4) and (5) above is reduced-pressure concentration. [5] The method according to any one of [1] to [4], wherein the eluent contains at least one of a volatile acid, a volatile base, and a volatile organic solvent. [6] The method according to any one of [1] to [4], wherein the target organic compound is a compound having a solubility of 10 to 800 g / L at 20 ± 5°C, and the solvent of the eluent is water.The method according to any one of [1] to [5]. [7] The method according to [6], wherein the solubility is the solubility in water. [8] The method according to any one of [4] to [7], wherein the temperature when concentrating under reduced pressure in steps (4) and (5) is 80°C or lower. [9] The method according to any one of [1] to [8], wherein the target organic compound is a peptide, nucleotide or derivative thereof, fatty acid, statin, vitamin, sugar or terpenoid.

[10] The method according to [9], wherein the peptide is a peptide having 2 to 10 amino acid residues, a peptide having 11 to 50 amino acid residues, or a protein having 51 or more amino acid residues.

[11] The method according to [9], wherein the fatty acid is a carboxylic acid with a carbon chain of 6 or more.

[12] The method according to any one of [1] to [8], wherein the target organic compound is a cytidine diphosphate choline compound, oxidized glutathione, flavin adenine dinucleotide, or pravastatin.

[13] The method according to any one of [1] to [8], wherein the target organic compound is a cytidinediphosphate choline compound or oxidized glutathione.

[14] The method according to any one of [1] to

[13] , wherein the immobilization support is an ion exchange resin, a synthetic adsorption resin, activated carbon, or silica gel.

[15] The method according to

[14] , wherein the ion exchange resin is a strongly basic anion exchange resin, a weakly basic anion exchange resin, a strongly acidic cation exchange resin, or a weakly acidic cation exchange resin.

[16] The method according to

[15] , wherein the ion exchange resin is a strongly basic anion exchange resin having either a quaternary ammonium of type I having a trimethylammonium group or a triethylammonium group as an exchange group, or a type II having a dimethylethanolammonium group, and the resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[17] The method according to

[15] , wherein the ion exchange resin is a weakly basic anion exchange resin having primary to tertiary amino groups or polyamine groups as exchange groups, and the resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[18] The ion exchange resin is a strongly acidic cation exchange resin having sulfonic acid groups as exchange groups, and the resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.The method according to

[15] .

[19] The method according to

[15] , wherein the ion exchange resin is a weakly acidic cation exchange resin having a carboxylate group as an exchange group, and the resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[20] The method according to

[14] , wherein the synthetic adsorption resin has a hydrophobic substituent or an unsubstituted type as an exchange group, and is an aromatic resin, an acrylic acid-based methacrylic resin, or an acrylonitrile aliphatic resin.

[21] The above-mentioned immobilized carrier may be, specifically, a strongly basic anion exchange resin such as Dow Chemical's 1×2, 1×4, 1×8, 22, MSA-2, or DuPont's Amberlite (e.g., FPA400J Cl, IRA404J Cl, FPA420OH, FPA900UPS Cl, HPR4580 Cl, SCAV4 Cl, FPA410J Cl, IRA411 Cl, FPA22UPS Cl, HPR4700 Cl, HPR4700OH, HPR4780 Cl, IRA400J Cl, IRA402BL Cl, IRA900J Cl, HPR4002 Cl, IRA410J Cl, IRA910CT Cl, HPR4010 Cl, HPR4100 Cl, HPR9200 Cl, HPR550 Cl, HPR550 OH, etc.), Purolite A400, A600, SGA550, A200, A300, A500, A501P, A502PS, A503, A510, A520E, A850, A860, A870, PFA520E, Purolite SST series (e.g., SSTA63, SSTA64), Mitsubishi Chemical Diaion PA series (e.g., PA306S, PA308, PA308L, PA312, PA312L, PA312LOH, PA312LTU, PA312LTUMB, PA 316, PA316L, PA318L, PA318LOH, PA408, PA412, PA412M, PA418, PA418L, PA418LL, PAF308L, HPA25L, HPA25M, HPA512L, HPA716, etc.), Mitsubishi Chemical's Diaion NSA100, UMA130J, Mitsubishi Chemical's Diaion SA series (e.g., SA10A, SA10AL, SA10ALLP, SA10AOH, SA10AP, SA10DL, SA11A, SA11AL, SA12A, SA12AL,SA12ALL, SA20A, SA20ALL, SA20ALLLP, SA20AP, SA20AP2, SAF11AL, SANUPB, SAT10L, SAT20L, etc.), Mitsubishi Chemical's Diaion UBA series (e.g., UBA100, UBA100OH, UBA100OHUP, UBA120, UBA120A, UBA120OH, UBA120OHUP, UBA150, UBA200, etc.), Mitsubishi Chemical's Rewrite JA series (e.g., J Examples of weakly basic anion exchange resins include A100, JA200, JA400, JA420, JA450, etc., Lanxess's Levatit Monoplus series (e.g., M500, M800, MP800, M600, MP600, etc.), and specifically, Dow Chemical's DowX Monosphere series (e.g., Monosphere 77), and DuPont's Amberlite (e.g., FPA95, FPA77UP, XE583GF, FPA53, HPR4780). Cl, IRA67, IRA96SB, IRA98, FPA95, FPA77UPS, FPA53, etc.), Purolite's A100, A103S, A110, A111S, A133S, A830, A830W, A845, A847, Mitsubishi Chemical Corporation's Diaion WA series (e.g., WA10, WA20, WA21J, WA30, WA30C, WA30LL, WA55, etc.), Mitsubishi Chemical Corporation's Sepabeads FPDA13, Mitsubishi Chemical Corporation's Rewrite JA series (e.g., JA300, JA310, JA450, JA830, etc.), Lanxess's Levatit MP62WS, Lanxess's Levatit Monoplus MP64, and as for strong acid cation exchange resins, specifically, for example, Dow Chemical's DOWEX88, DOWEX88MB, DOWEX Monosphere 88, XUS40232-01, DuPont Amberlite (e.g., FPC16UPS Na, FPC88MB Na, FPC240H, CR3220 Ca, CR1310 Ca,Na, CR1360 Na, CR99K / 350, HPR1100Na, etc.), Purolite C100, C100E, C120E, C100x10, C100x16MBH, C145S, C150, C160, SGC650, Purolite SST series (e.g., SSTC60, SSTC60H,(e.g., SSTC80C), Mitsubishi Chemical Corporation's Diaion SK series (e.g., SK1B, SK1BH, SK1BL, SK1BLH, SKL10, SKT10L, SK104, SK110, SKT110, SKT110L, SK110L, SK112, SK112L, SK116, SKT20L, etc.), Mitsubishi Chemical Corporation's Diaion PK series (e.g., PK208, PK208LH, PK212, PK212L, PK212LH, PK216, PK216L, PK216H, PK216LH, PK220, PK220L) , PK228, PK228L, PK228LH, etc.), Mitsubishi Chemical Corporation's Diaion RCP series (e.g., RCP145H, RCP160M, etc.), Mitsubishi Chemical Corporation's Diaion HPK25, Mitsubishi Chemical Corporation's Diaion UBK series (e.g., UBK16, UBK14, UBK12, UBK10, UBK10H, UBK10HUP, UBK08, UBK08A, UBK08H, UBK08HUP, UBK04, UBK02, UBKN1U, UBKN1UMB, UBK522M, UBK530, UBK530J) UBK530K, UBK535, UBK535J, UBK535K, UBK535L, UBK550, UBK555, etc.), Mitsubishi Chemical Corporation's Relight JC series (e.g., JC600, JC603, etc.), LANXESS's Levatit S1668, LANXESS's Levatit Monoplus series (e.g., S108, S108H, SP112, etc.), and as weakly acidic cation exchange resins, specifically, for example, DuPont's Amberlite (e.g., FPC76J, FPC3500, etc.), and Purolite's C104, C106, C107 E, C115E, Purolite SSTC104 from Purolite Corporation, Diaion WK series from Mitsubishi Chemical Corporation (e.g., WK10, WK100, WK10S, WK11, WK40, WK60, WK60L, etc.), Diaion WT01S from Mitsubishi Chemical Corporation, Levatit CNP80WS from Lanxess, and as synthetic adsorbent resins, specifically, for example, Amberlite from DuPont (e.g., XAD2000, XAD4, XAD1180N, XAD7HP, XAD-2, FPX66, etc.), PuroSorb PAD series from Purolite Corporation (e.g., PAD400, PAD500, PAD600, PAD900,PAD1200, PAD610, PAD950, etc.), Purolite Macronet MN series (e.g., MN200, MN202, MN270, MN100, MN102, MN502, etc.), Mitsubishi Chemical Diaion HP series (e.g., HP20, HP20SS, HP21, etc.), Mitsubishi Chemical Diaion HP2MGL, Mitsubishi Chemical Sepabeads SP800 series (e.g., SP825L, SP850, etc.), Mitsubishi Chemical Sepabeads SP207, SP207SS, SP20SS, SP70, SP700, Mitsubishi Chemical MCIGEL The method according to any one of [1] to

[20] , wherein at least one of CHP20 / P20, CHP20 / P30, CHP20 / P70, CHP20 / P120, and CHP07 / P120.

[22] The method according to any one of [5] to

[21] , wherein the volatile acid is an inorganic acid or an organic acid having 1 to 3 carbon atoms and a carboxyl group.

[23] The method according to

[22] , wherein the volatile acid is hydrochloric acid, nitric acid, formic acid, acetic acid, or propionic acid.

[24] The method according to

[22] , wherein the volatile acid is formic acid, acetic acid, or propionic acid.

[25] The method according to any one of [5] to

[21] , wherein the volatile organic solvent is a protic polar solvent.

[26] The method according to any one of [5] to

[21] , wherein the volatile organic solvent is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, or ethyl acetate.

[27] The method according to any one of [5] to

[21] , wherein the volatile organic solvent is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, or 2-butanol.

[28] The method according to any one of [1] to

[27] , wherein the concentration of the eluent in the (a) fraction separated in step (3) is 100 g / L or less, and the fraction contains the target organic compound.

[29] The method according to any one of [1] to

[27] , wherein the concentration of the eluent in the (a) fraction separated in step (3) is 100 g / L or less, and the fraction contains 10 g / L or more of the target organic compound.

[30] The fraction separated in step (3),The method according to any one of [1] to

[29] , wherein the ratio of the mass of the eluent to the mass of the target organic compound in the above (a) fraction (eluent / target organic compound) is 0.06 or more.

[31] The method according to any one of [1] to

[30] , wherein the ratio of the mass of the eluent to the mass of the target organic compound in the above (b) fraction separated in step (3) is less than 0.06.

[32] The method according to any one of [1] to

[31] , wherein the ratio of the mass of the eluent to the mass of the target organic compound in the solution obtained after concentration in step (5) is less than 0.06.

[33] The method according to [4], wherein the target organic compound is a cytidinediphosphate choline compound, the immobilization support is a strongly basic anion exchange resin, the eluent is formic acid, and the temperature when concentrating under reduced pressure in steps (4) and (5) is 40°C or less.

[34] The method according to

[33] , wherein the concentration of formic acid in the fraction of step (3) (b) is less than 1.49 mg / L.

[35] The method according to [4], wherein the target organic compound is a cytidinediphosphate choline compound, the immobilization support is a strongly basic anion exchange resin, the eluent is propionic acid, and the temperature when concentrating under reduced pressure in steps (4) and (5) is 50°C or less.

[36] The method according to

[35] , wherein the concentration of propionic acid in the fraction of step (3) (b) is less than 1.15 mg / L.

[37] The method according to [4], wherein the target organic compound is a cytidinediphosphate choline compound, the immobilization support is a weakly basic anion exchange resin, the eluent is acetic acid, and the temperature when concentrating under reduced pressure in steps (4) and (5) is 50°C or less.

[38] The method according to

[37] , wherein in the fraction of step (3) (b) above, the concentration of acetic acid is less than 0.947 mg / L.

[39] The method according to [4], wherein the target organic compound is a cytidinediphosphate choline compound, the immobilization support is a strongly basic anion exchange resin, the eluent is acetic acid, and the temperature when concentrating under reduced pressure in steps (4) and (5) above is 50°C or less.

[40] In the fraction of step (3) (b) above, the concentration of acetic acid is less than 0.947 mg / L.The method according to

[39] .

[41] The method according to [4], wherein the target organic compound is oxidized glutathione, the immobilization support is a weakly basic anion exchange resin, the eluent is propionic acid, and the temperature when concentrating under reduced pressure in steps (4) and (5) is 40°C or less.

[42] The method according to

[41] , wherein the concentration of propionic acid in the fraction of step (3) (b) is less than 1.15 mg / L.

[43] The method according to [4], wherein the target organic compound is oxidized glutathione, the immobilization support is a strongly acidic cation exchange resin, the eluent is ammonia, and the temperature when concentrating under reduced pressure in steps (4) and (5) is 40°C or less.

[44] The method according to

[43] , wherein the concentration of ammonia in the fraction of step (3) (b) is less than 0.08 mg / L.

[45] The method according to [4], wherein the target organic compound is flavin adenine dinucleotide, the immobilization carrier is a synthetic adsorbent resin, the eluent is acetone, and the temperature during vacuum concentration in steps (4) and (5) is 40°C or lower.

[46] The method according to

[45] , wherein the concentration of acetone in the fraction of step (3) (b) is less than 1.5 g / L.

[47] The method according to [4], wherein the target organic compound is pravastatin, the immobilization carrier is a synthetic adsorbent resin, the eluent is ethyl acetate, and the temperature during vacuum concentration in steps (4) and (5) is 40°C or lower.

[48] The method according to

[47] , wherein the concentration of ethyl acetate in the fraction of step (3) (b) is less than 0.095 mg / L.

[49] The method according to [4], wherein the target organic compound is flavin adenine dinucleotide, the immobilization support is a synthetic adsorption resin, the eluent is 2-butanol, and the temperature when concentrating under reduced pressure in steps (4) and (5) is 40°C or lower.

[50] The method according to

[49] , wherein the concentration of 2-butanol in the fraction of step (3) (b) is less than 50 mg / L.

[51] The method according to any one of [1] to [3], wherein the concentration in steps (4) and (5) includes membrane concentration.

[52] The method according to

[51] , wherein the eluent comprises at least one of an acid, a base, and an organic solvent.

[53] The target organic compound isThe method according to

[51] or

[52] , wherein the compound has a solubility of 10 to 800 g / L at 20 ± 5°C, and the solvent of the eluent is water.

[54] The method according to

[53] , wherein the solubility is the solubility in water.

[55] The method according to any one of

[51] to

[54] , wherein the temperature at which the membrane is concentrated in steps (4) and (5) is 80°C or lower.

[56] The method according to any one of

[51] to

[55] , wherein the target organic compound is a peptide, a nucleotide or its derivative, a fatty acid, a statin, a vitamin, a sugar, or a terpenoid.

[57] The method according to

[56] , wherein the peptide is a peptide having 2 to 10 amino acid residues, a peptide having 11 to 50 amino acid residues, or a protein having 51 or more amino acid residues.

[58] The method according to

[56] , wherein the fatty acid is a carboxylic acid with a carbon chain of 6 or more carbon atoms.

[59] The method according to any one of

[51] to

[55] , wherein the target organic compound is a cytidinediphosphate choline compound, oxidized glutathione, flavin adenine dinucleotide, or pravastatin.

[60] The method according to any one of

[51] to

[55] , wherein the target organic compound is a cytidinediphosphate choline compound or oxidized glutathione.

[61] The method according to any one of

[51] to

[60] , wherein the immobilization support is an ion exchange resin, a synthetic adsorption resin, activated carbon, or silica gel.

[62] The method according to

[61] , wherein the ion exchange resin is a strongly basic anion exchange resin, a weakly basic anion exchange resin, a strongly acidic cation exchange resin, or a weakly acidic cation exchange resin.

[63] The method according to

[61] , wherein the ion exchange resin is a strongly basic anion exchange resin having either a quaternary ammonium of type I having a trimethylammonium group or a triethylammonium group as an exchange group, or a type II having a dimethylethanolammonium group, and the resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[64] The method according to

[61] , wherein the ion exchange resin has primary to tertiary amino groups or polyamine groups as exchange groups, and the resin matrix is ​​a weakly basic anion exchange resin of porous type, macroporous type, gel type, styrene type or acrylic type.

[65] The ion exchange resin has sulfonic acid groups as exchange groups, and the resin matrix is ​​a porous type,The method according to

[61] , wherein the strongly acidic cation exchange resin is of the macroporous, gel, styrene, or acrylic type.

[66] The method according to

[61] , wherein the ion exchange resin is a weakly acidic cation exchange resin having a carboxylate group as an exchange group, and the resin matrix is ​​of the porous, macroporous, gel, styrene, or acrylic type.

[67] The method according to

[61] , wherein the synthetic adsorption resin has a hydrophobic substituent or an unsubstituted type as an exchange group, and is an aromatic resin, an acrylic acid-based methacrylic resin, or an acrylonitrile aliphatic resin.

[68] The method according to any one of

[51] to

[67] , wherein the immobilizing carrier is at least one of the carriers described in

[21] .

[69] The method according to any one of

[52] to

[68] , wherein the acid is an inorganic acid or an organic acid having 1 to 3 carbon atoms and a carboxyl group.

[70] The method according to

[69] , wherein the acid is hydrochloric acid, nitric acid, formic acid, acetic acid, or propionic acid.

[71] The method according to

[69] , wherein the acid is formic acid, acetic acid, or propionic acid.

[72] The method according to any one of

[52] to

[68] , wherein the organic solvent is a protic polar solvent.

[73] The method according to any one of

[52] to

[68] , wherein the organic solvent is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, or ethyl acetate.

[74] The method according to any one of

[52] to

[68] , wherein the organic solvent is methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, or 2-butanol.

[75] The method according to any one of

[51] to

[74] , wherein the concentration of the eluent in the fraction (a) separated in step (3) is 100 g / L or less, and the fraction contains the target organic compound.

[76] The method according to any one of

[51] to

[74] , wherein the concentration of the eluent in the (a) fraction separated in step (3) above is 100 g / L or less, and the fraction contains 10 g / L or more of the target organic compound.

[77] The method according to any one of

[51] to

[76] , wherein the ratio of the mass of the eluent to the mass of the target organic compound in the (a) fraction separated in step (3) above (eluent / target organic compound) is 0.06 or more.

[78] The method according to any one of

[51] to

[76] ,The method according to any one of

[51] to

[77] , wherein the ratio of the mass of the eluent to the mass of the target organic compound in the (b) fraction (eluent / target organic compound) is less than 0.06.

[79] The method according to any one of

[51] to

[78] , wherein the ratio of the mass of the eluent to the mass of the target organic compound in the solution obtained after concentration in step (5) is less than 0.06.

[80] The method according to

[51] , wherein the target organic compound is a cytidinediphosphate choline compound, the immobilization support is a strongly basic anion exchange resin, and the eluent is acetic acid.

[81] The method according to

[80] , wherein the concentration of acetic acid in the (b) fraction of step (3) is less than 0.947 mg / L.

[82] The method according to any one of [1] to [3], wherein the concentration of the eluent in the (b) fraction is 1 / 2 or less of the concentration in the (a) fraction.

[83] The method according to

[82] , wherein the eluent is formic acid and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[84] The method according to

[82] , wherein the eluent is acetic acid and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[85] The method according to

[82] , wherein the eluent is propionic acid and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[86] The method according to

[82] , wherein the eluent is ammonia and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[87] The method according to

[82] , wherein the eluent is acetone and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[88] The method according to

[82] , wherein the eluent is ethyl acetate, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[89] The method according to

[82] , wherein the eluent is 2-butanol, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[90] The target organic compound is a cytidinediphosphate choline compound, and the eluent is formic acid, and the concentration of the eluent in fraction (b) isThe method according to

[82] , wherein the concentration in fraction (a) is 1 / 2 or less.

[91] The method according to

[82] , wherein the target organic compound is a cytidinediphosphate choline compound, the eluent is acetic acid, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[92] The method according to

[82] , wherein the target organic compound is a cytidinediphosphate choline compound, the eluent is propionic acid, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[93] The method according to

[82] , wherein the target organic compound is oxidized glutathione, the eluent is propionic acid, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[94] The method according to

[82] , wherein the target organic compound is oxidized glutathione, the eluent is ammonia, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[95] The method according to

[82] , wherein the target organic compound is flavin adenine dinucleotide, the eluent is acetone, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[96] The method according to

[82] , wherein the target organic compound is pravastatin, the eluent is ethyl acetate, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[97] The method according to

[82] , wherein the target organic compound is flavin adenine dinucleotide, the eluent is 2-butanol, and the concentration of the eluent in fraction (b) is 1 / 2 or less of the concentration in fraction (a).

[0006] The method described herein makes it possible to efficiently remove the eluent. By efficiently removing the eluent, the energy and time required for concentration can be reduced. Furthermore, even when vacuum concentration is employed as the concentration method, the thermal load can be reduced.

[0007] Figure 1 is a graph showing the results of Preparation Example 1, representing the concentrations of oxidized glutathione and propionic acid in the eluate obtained by eluting oxidized glutathione using propionic acid as the eluent. Figure 2 is a graph showing the results of Preparation Example 2, representing the concentrations of cytidinediphosphate choline and formic acid in the eluate obtained by eluting cytidinediphosphate choline using formic acid as the eluent. Figure 3 is a graph showing the results of Preparation Example 2, representing the concentrations of cytidinediphosphate choline and formic acid in the eluate obtained by eluting cytidinediphosphate choline using formic acid as the eluent. Figure 4 is a graph showing the results of Preparation Example 3, representing the concentrations of cytidinediphosphate choline and propionic acid in the eluate obtained by eluting cytidinediphosphate choline using propionic acid as the eluent. Figure 5 is a graph showing the results of Preparation Example 4, representing the concentrations of cytidinediphosphate choline and acetic acid in the eluate obtained by eluting cytidinediphosphate choline using acetic acid as the eluent. Figure 6 is a graph showing the results of Preparation Example 5, representing the concentrations of cytidinediphosphate choline and acetic acid in the eluate obtained by eluting cytidinediphosphate choline using acetic acid as the eluting agent. Figure 7 is a graph showing the results of Preparation Example 6, representing the concentrations of flavin adenine dinucleotide and acetone in the eluate obtained by eluting flavin adenine dinucleotide using acetone as the eluting agent. Figure 8 is a graph showing the results of Preparation Example 7, representing the concentrations of pravastatin and ethyl acetate in the eluate obtained by eluting pravastatin using ethyl acetate as the eluting agent. Figure 9 is a graph showing the results of Preparation Example 8, representing the concentrations of flavin adenine dinucleotide and 2-butanol in the eluate obtained by eluting flavin adenine dinucleotide using 2-butanol as the eluting agent. Figure 10 is a graph showing the results of Preparation Example 9, representing the concentrations of oxidized glutathione and ammonia in the eluate obtained by eluting oxidized glutathione using ammonia as the eluting agent. Figure 11 is a graph showing the results of preparation example 10, and represents the concentrations of cytidinediphosphate choline and acetic acid in the eluate obtained by eluting cytidinediphosphate choline using acetic acid as the eluent.

[0008] The following is a detailed description of this disclosure, but these are merely examples of desirable embodiments and are not limiting to these.

[0009] The "~" in a numerical range indicates a range that includes the numbers before and after it. For example, "0 mass% to 100 mass%" means a range that is 0 mass% or greater and 100 mass% or less.

[0010] A method for producing an organic compound according to this disclosure includes: (1) a step of adsorbing a target organic compound onto a stationary carrier; (2) a step of passing an eluent through the stationary carrier on which the target organic compound has been adsorbed; (3) a step of dividing the eluted fraction containing the target organic compound into (a) a fraction containing the eluent at a first concentration and (b) a fraction in which the concentration of the eluent is less than the first concentration; (4) a step of concentrating the fraction from (a) to obtain a concentrate; and (5) a step of adding the fraction from (b) to the concentrate to concentrate it.

[0011] The target organic compound is an organic compound obtained by the production method of this disclosure, which has a saturated vapor pressure of 300 Pa or less at 20±5°C and a solubility of 1 to 1000 g / L at 20±5°C, preferably 10 to 800 g / L, and more preferably 100 to 800 g / L. Solubility refers to the solubility in the eluent or the solubility of the eluent in the solvent, and in one example the solvent of the eluent is water.

[0012] Specifically, the target organic compounds include peptides, nucleotides or their derivatives, fatty acids, statins, vitamins, sugars, and terpenoids.

[0013] Examples of peptides include peptides with 2 to 10 amino acid residues, peptides with 11 to 50 amino acid residues, and proteins with 51 or more amino acid residues. Specifically, examples of peptides include reduced glutathione (γ-L-glutamyl-L-cysteinyl-glycine, hereinafter also referred to as GSH), oxidized glutathione (glutathione disulfide, hereinafter also referred to as GSSG), and glutathione trisulfide (hereinafter also referred to as GSSSG), with GSSG being preferred.

[0014] Nucleotides are compounds composed of a base, a sugar, and a phosphate group, including ribonucleotides such as adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), and cytidine triphosphate (CTP); deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dAMP), deoxyadenosine tri Examples of deoxyribonucleotides include xyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), thymidine monophosphate (dTMP), thymidine diphosphate (dTDP), and thymidine triphosphate (dTTP). Specifically, examples of nucleotide derivatives include cytidine diphosphate choline compounds and nicotinamide adenine dinucleotide (hereinafter, the oxidized form is NAD). ＋ Also expressed as NADH in its reduced form), nicotinamide adenine dinucleotide phosphate (hereinafter, the oxidized form is NADP ＋ Also represented as , the reduced form is also represented as NADPH), nicotinamide mononucleotide (hereinafter also represented as NMN), flavin adenine dinucleotide (hereinafter, the oxidized form is also represented as FAD, and the reduced form is FADH) ２ Examples include cytidinediphosphate choline compounds and NAD (also expressed as ). ＋ Or NADH, or FAD or FADH ２ It is preferable that this be the case.

[0015] A cytidine-5'-diphosphate compound is a cytidine-5'-diphosphate compound having a cationic substituent. Examples of cytidine-5'-diphosphate compounds having a cationic substituent include compounds in which cytidine-5'-diphosphate is bonded to a cationic substituent. More specifically, a cytidine-5'-diphosphate compound having a cationic substituent is a compound in which a cationic substituent is bonded to the β-phosphate group of cytidine-5'-diphosphate. Here, the positions of the phosphate groups in cytidine-5'-diphosphate are, in order from the cytidine side, the α-phosphate group and the β-phosphate group (Chemistry and Education, Vol. 46, No. 6, 1998, pp. 334-337). Examples of cationic substituents include substituents having an onium ion such as ammonium, oxonium, phosphonium, sulfonium, iminium, nitrilium, nitrosonium, diazonium, nitronium, or diazenium, and among these, those having an ammonium ion are preferred. Examples of cytidine-5'-diphosphate compounds having a cationic substituent include cytidine-5'-diphosphate choline (also expressed as cytidine-5'-diphosphate choline) and cytidine-5'-diphosphate ethanolamine. Among these, from the viewpoint of purification efficiency, cytidine-5'-diphosphate choline or cytidine-5'-diphosphate ethanolamine are preferred, and cytidine-5'-diphosphate choline is more preferred. When the target organic compound is a cytidine-5'-diphosphate choline compound, the eluted fraction may contain only one type of cytidine-5'-diphosphate choline compound, or it may contain two or more types of cytidine-5'-diphosphate choline compounds.

[0016] The fatty acids are preferably monovalent carboxylic acids having a carboxyl group in the hydrocarbon chain, and having six or more carbon atoms. Specifically, examples include medium-chain fatty acids with 6 to 12 carbon atoms (such as caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, and lauric acid), and long-chain fatty acids with 13 to 21 carbon atoms (such as myristic acid, pentadecyl acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eleostearic acid, arachidic acid, meadic acid, and arachidonic acid).

[0017] Statins are compounds (HMG-CoA reductase inhibitors) having a function as a pharmaceutical used for lowering cholesterol by inhibiting the action of HMG-CoA reductase. Specific examples thereof include pravastatin, atorvastatin, lovastatin, simvastatin, fluvastatin, rosuvastatin and pitavastatin, with pravastatin being preferred.

[0018] Vitamins are compounds having a function as nutrients necessary for the body that exhibit specific biological activities. Specific examples thereof include vitamin A (retinol, retinal, retinoic acid), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), vitamin B12 (cyanocobalamin, hydroxocobalamin, methylcobalamin), vitamin C (ascorbic acid), vitamin D (cholecalciferol (D3), ergocalciferol (D2)), vitamin E (tocopherol, tocotrienol), and vitamin K (phylloquinone, menaquinone (such as menaquinone-4 and menaquinone-7); hereinafter also referred to as VKT), with menaquinone being preferred.

[0019] Saccharides include monosaccharides, oligosaccharides, polysaccharides, and derivatives thereof. Specific examples thereof include: monosaccharides and derivatives thereof such as glucose, galactose, fructose, mannose, fucose, xylose, glucosamine, glucuronic acid, N-acetylgalactosamine, neuraminic acid, and sialic acid; disaccharides and derivatives thereof such as sucrose, lactose, maltose, trehalose, cellobiose, and lactulose; and polysaccharides and derivatives thereof such as starch, glycogen, cellulose, dextran, agarose, carrageenan, heparin, alginic acid, hyaluronic acid, and chondroitin sulfate.

[0020] Terpenoids refer to a group of compounds with isoprene as the basic unit, including squalene; carotenoids such as α-carotene, β-carotene, γ-carotene, δ-carotene, lycopene, lutein, zeaxanthin, cryptoxanthin, and astaxanthin; and steroids such as cholesterol, cortisol, aldosterone, testosterone, estradiol, and progesterone.

[0021] The target organic compound is preferably cytidine diphosphate choline, cytidine diphosphate ethanolamine, oxidized glutathione, flavin adenine dinucleotide, or pravastatin. These compounds are easily decomposed by heating at 20°C or higher, so they can benefit from the shortening of heating time accompanying concentration, which makes them desirable. More preferably, the compounds that are easily decomposed by heating are cytidine diphosphate choline, cytidine diphosphate ethanolamine, and oxidized glutathione.

[0022] Examples of the immobilized carrier include ion exchange resins, synthetic adsorption resins, activated carbon, and silica gel, and ion exchange resins or synthetic adsorption resins are preferred.

[0023] Examples of the ion exchange resin include basic anion exchange resins (such as weakly basic anion exchange resins and strongly basic anion exchange resins) and acidic cation exchange resins (such as strongly acidic cation exchange resins and weakly acidic cation exchange resins).

[0024] The strongly basic anion exchange resin, for example, has a quaternary ammonium group that is either type I having a trimethylammonium group or triethylammonium group as an exchange group, or type II having a dimethylethanolammonium group, and examples thereof include strongly basic anion exchange resins whose resin matrix is porous type, macroporous type, gel type, styrene type, or acrylic type.

[0025] Examples of strongly basic anion exchange resins include, specifically, Dow Chemical's 1x2, 1x4, 1x8, 22, MSA-2, and DuPont's Amberlite (e.g., HPR4700 Cl, HPR4700 OH, FPA400J Cl, IRA404J Cl, FPA420 OH, FPA900UPS Cl, HPR4580 Cl, SCAV4 Cl, FPA410J Cl, IRA411 Cl, FPA22UPS Cl, HPR4780 Cl, IRA400J Cl, IRA402BL Cl, IRA900J Cl, HPR4002 Cl, IRA410J Cl, IRA910CT Cl, HPR4010 Cl, HPR4100 Cl, HPR9200, Cl, HPR550, Cl, HPR550, OH, etc.), Purolite A400, A600, SGA550, A200, A300, A500, A501P, A502PS, A503, A510, A520E, A850, A860, A870, PFA520E, Purolite SST series (e.g., SSTA63, SSTA64), Mitsubishi Chemical Diaion PA series (e.g., PA3 06S, PA308, PA308L, PA312, PA312L, PA312LOH, PA312LTU, PA312LTUMB, PA316, PA316L, PA318L, PA318 LOH, PA408, PA412, PA412M, PA418, PA418L, PA418LL, PAF308L, HPA25L, HPA25M, HPA512L, HPA716, etc.), Mitsubishi Diaion NSA100, UMA130J from Chemical Co., Ltd., and Diaion SA series from Mitsubishi Chemical Corporation (e.g., SA10A, SA10AL, SA10ALLP, SA10AOH, SA10AP, SA10DL, SA11A, SA11AL, SA12A, SA12AL, SA12ALL, SA20A, SA20ALL, SA20ALLP, SA20AP, SA20AP2, S AF11AL, SANUPB, SAT10L, SAT20L, etc.), Mitsubishi Chemical's Diaion UBA series (e.g., UBA100, UBA100OH, UBA100OHUP, UBA120, UBA120A, UBA120OH, UBA120OHUP, UBA150, UBA200, etc.), Mitsubishi Chemical's Rewrite JA series (e.g., JA100, JA200,JA400, JA420, JA450, etc.), and the Lewatit MonoPlus series from Lanxess (e.g., M500, M800, MP800, M600, MP600, etc.).

[0026] The ion type of quaternary ammonium as the exchange group in the strongly basic anion exchange resin is not particularly limited, and examples include hydroxide ion (OH － form), chloride ion (Cl － form), sulfate form (SO ４ ２－ form), phosphate form (PO ４ ３－ form), nitrate form (NO ３ － form) and a state bound to an organic acid used as an eluent described below, with hydroxide ion (OH － form) being preferable.

[0027] Weakly basic anion exchange resins have, for example, primary to tertiary amino groups or polyamine groups as exchange groups, and examples include weakly basic anion exchange resins whose resin matrix is of porous type, macroporous type, gel type, styrene type or acrylic type.

[0028] Specific examples of weakly basic anion exchange resins include the Dowex Monosphere series from Dow Chemical (e.g., Monosphere 77), Amberlite from DuPont (e.g., FPA95, FPA77UP, XE583GF, FPA53, HPR4780 Cl, IRA67, IRA96SB, IRA98, FPA95, FPA77UPS, FPA53, etc.), A100, A103S, A110, A111S, A133S, A830, A830W, A845, A847 from Purolite, the Diaion WA series from Mitsubishi Chemical Corporation (e.g., WA10, WA20, WA21J, WA30, WA30C, WA30LL, WA55, etc.), Sepabeads FPDA13 from Mitsubishi Chemical Corporation, the Relite JA series from Mitsubishi Chemical Corporation (e.g., JA300, JA310, JA450, JA830, etc.), Lewatit MP62WS from Lanxess, and Lewatit MonoPlus MP64 from Lanxess.

[0029] Furthermore, the ionic form of the amino group of the exchange group in the weakly basic anion exchange resin is not particularly limited, for example, hydroxide ion (OH) － (type), chloride ion (Cl － (SO type), sulfuric acid type (SO ４ ２－ (Type), Phosphate type (PO ４ ３－ type), nitric acid type (NO ３ － Examples include either the form (type) or a state in which an organic acid used as an eluent, as described later, is bound, and hydroxide ions (OH － It is preferable that it be of the type.

[0030] Examples of strongly acidic cation exchange resins include those having sulfonic acid groups as exchange groups, and whose resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[0031] Examples of strongly acidic cation exchange resins include, specifically, Dow Chemical's DOWEX 88, DOWEX 88MB, DOWEX Monosphere 88, XUS 40232-01, and DuPont's Amberlite (e.g., FPC16UPS Na, FPC88MB Na, FPC240H, CR3220 Ca, CR1310 Ca,Na, CR1360) Na, CR99K / 350, HPR1100Na, etc.), C100, C100E, C120E, C100x10, C100x16MBH, C145S, C150, C160, SGC650 from Purolite Corporation, Purolite SST series (e.g., SSTC60, SSTC60H, SSTC80C, etc.), Diaion SK series from Mitsubishi Chemical Corporation (e.g., SK1B, SK1BH, SK1BL, SK1B LH, SKL10, SKT10L, SK104, SK110, SKT110, SKT110L, SK110L, SK112, SK112L, SK116, SKT20L, etc.), Mitsubishi Chemical Corporation's Diaion PK series (e.g., PK208, PK208LH, PK212, PK212L, PK212LH, PK216, PK216L, PK216H, PK216LH, PK220, PK220L, PK228, PK228L, PK228LH, etc.), Mitsubishi Chemical Corporation's Diaion RCP series (e.g., RCP145H, RCP160M, etc.), Mitsubishi Chemical Corporation's Diaion HPK25, Mitsubishi Chemical Corporation's Diaion UBK series (e.g., UBK16, UBK14, UBK12, UBK10, UBK10H, UBK10HUP, UBK08, UBK08A, UBK08H, UBK08HUP, UBK04, UBK02, UBKN1U, Examples include UBKN1UMB, UBK522M, UBK530, UBK530J, UBK530K, UBK535, UBK535J, UBK535K, UBK535L, UBK550, UBK555, etc.), Mitsubishi Chemical Corporation's Rewrite JC series (e.g., JC600, JC603, etc.), LANXESS's Levatit S1668, and LANXESS's Levatit Monoplus series (e.g., S108, S108H, SP112, etc.).

[0032] The ionic form of the sulfonic acid group of the exchange group in the strongly acidic cation exchange resin is not particularly limited, for example, H ＋ Type, Na ＋ Type, K ＋ Type and NH ４ ＋ Examples include H ＋ Type or NH ４ ＋ It is preferable that it be a type.

[0033] Examples of weakly acidic cation exchange resins include those having a carboxylic acid group as an exchange group, and whose resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[0034] Examples of weakly acidic cation exchange resins include DuPont's Amberlite (e.g., FPC76J, FPC3500, etc.), Purolite's C104, C106, C107E, C115E, Purolite's PuroliteSSTC104, Mitsubishi Chemical Corporation's Diaion WK series (e.g., WK10, WK100, WK10S, WK11, WK40, WK60, WK60L, etc.), Mitsubishi Chemical Corporation's Diaion WT01S, and Lanxess's Levatit CNP80WS.

[0035] The ionic form of the carboxylic acid group of the exchange group in a weakly acidic cation exchange resin is not particularly limited, for example, H ＋ Type, Na ＋ Type, K ＋ Type and NH ４ ＋ Examples include H ＋ Type or NH ４ ＋ It is preferable that it be a type.

[0036] Examples of synthetic adsorption resins include those having hydrophobic substituents or unsubstituted types as exchange groups, and being aromatic resins, acrylic acid-based methacrylic resins, or acrylonitrile aliphatic resins.

[0037] Examples of synthetic adsorbent resins include DuPont's Amberlite (e.g., XAD2000, XAD4, XAD1180N, XAD7HP, XAD-2, FPX66, etc.), Purolite's PuroSorb PAD series (e.g., PAD400, PAD500, PAD600, PAD900, PAD1200, PAD610, PAD950, etc.), and Purolite's Macronet. Examples include the MN series (e.g., MN200, MN202, MN270, MN100, MN102, MN502, etc.), Mitsubishi Chemical's Diaion HP series (e.g., HP20, HP20SS, HP21, etc.), Mitsubishi Chemical's Diaion HP2MGL, Mitsubishi Chemical's Sepabeads SP800 series (e.g., SP825L, SP850, etc.), Mitsubishi Chemical's Sepabeads SP207, SP207SS, SP20SS, SP70, SP700, and Mitsubishi Chemical's MCIGEL CHP20 / P20, CHP20 / P30, CHP20 / P70, CHP20 / P120, CHP07 / P120.

[0038] Activated carbon mainly consists of materials such as coal, sawdust, and coconut shells, which are activated with gas or chemicals to create fine pores. Specifically, these include Osaka Gas Chemical's Granular Shirasagi G2c, Granular Shirasagi WH2c, Granular Shirasagi W2c, Granular Shirasagi WH5c, Granular Shirasagi W5c, Granular Shirasagi LGK-100, Granular Shirasagi LGK-400, Granular Shirasagi KL, Granular Shirasagi LH2c, Spherical Shirasagi X8100H, Spherical Shirasagi XS8100H, Granular Shirasagi G2x, Granular Shirasagi G5x, Granular Shirasagi S2x, Granular Shirasagi WH2x, Granular Shirasagi X2M, Granular Shirasagi C2c, and Granular Examples include Shirasagi C2x, Spherical Shirasagi X7000H, Spherical Shirasagi X7100H, Spherical Shirasagi XS7100H, Spherical Shirasagi X7000H-3, Spherical Shirasagi X7100H-3, Spherical Shirasagi LGK-700, Spherical Shirasagi DX7-3, Shirasagi M, Shirasagi P, Granular Shirasagi GM2X, Seitz AKSJ, BAC manufactured by Kureha Chemical Industries, and PN, Zn, SA, SA-SW, SX, CA, CN, CG, D-10, W, GL, HB PLUS manufactured by Nippon Norit Co., Ltd.

[0039] As silica gel, various functional groups can be bonded to the silanol groups on the silica gel surface, and normal-phase chromatography or reverse-phase chromatography can be selected according to the structure of the compound. Examples of normal-phase chromatography include silica gel with unmodified silanol groups on the surface and silica gel with modified aminopropyl groups.

[0040] The solvents used for adsorbing the target organic compound onto the immobilized support and for eluting it from the immobilized support may be the same or different water, methanol, ethanol, 1-propanol, 2-propanol, acetone, acetonitrile, ethyl acetate, n-hexane, benzene, toluene, chloroform, methylene chloride, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide, with water being preferred in all cases.

[0041] If the concentration process includes vacuum concentration, eluents used to elute the target organic compound adsorbed onto the immobilized carrier include volatile acids, volatile bases, and volatile organic solvents, and these may be used in mixture form.

[0042] Examples of volatile acids include inorganic acids or organic acids having 1 to 3 carbon atoms and a carboxyl group. Specifically, these include hydrochloric acid, nitric acid, formic acid, acetic acid, and propionic acid, with formic acid, acetic acid, or propionic acid being preferred.

[0043] Examples of volatile organic solvents include protic polar solvents, aprotic polar solvents, and nonpolar solvents. Examples of protic polar solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol. Examples of aprotic polar solvents include acetone, acetonitrile, ethyl acetate, dichloromethane, and tetrahydrofuran. Examples of nonpolar solvents include n-hexane, diethyl ether, and chloroform.

[0044] The volatile organic solvent is preferably methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, or ethyl acetate, and more preferably methanol.

[0045] Examples of volatile bases include ammonia, methylamine, dimethylamine, and trimethylamine.

[0046] When the concentration process includes membrane concentration, eluents used to elute the target organic compound adsorbed onto the stationary carrier include acids, bases, and organic solvents, which may be used in combination. Acids, bases, and organic solvents include not only the volatile acids, volatile bases, and volatile organic solvents mentioned above, but also non-volatile acids, non-volatile bases, and non-volatile organic solvents. Non-volatile acids are acids that do not evaporate or evaporate only partially when heated, for example, acids with a saturated vapor pressure of 300 Pa or less at 20 ± 5°C. Specific examples of non-volatile acids include sulfuric acid, phosphoric acid, boric acid, and citric acid. Non-volatile bases can be selected from the group consisting of alkali metal salts, alkali metal hydroxides, alkaline earth metal salts, alkaline earth metal hydroxides, and amino acids, or combinations thereof. Specific examples of non-volatile bases include sodium hydroxide, potassium hydroxide, and calcium hydroxide. Non-volatile organic solvents are those that do not evaporate or only partially evaporate when heated, for example, organic solvents with a saturated vapor pressure of 300 Pa or less at 20 ± 5°C. Specific examples of non-volatile organic solvents include glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF).

[0047] In this disclosure, steps (1) to (3) are also referred to as the first liquid passing step, and steps (4) to (5) are also referred to as the second concentration step.

[0048] (Preliminary step to the first liquid flow step) In the preliminary step to the first liquid flow step, a solution containing the target organic compound is prepared. The target organic compound may be produced by any of the following methods: fermentation, enzymatic methods, extraction from natural products, or chemical synthesis.

[0049] If the solution containing the target organic compound contains solid matter that would hinder purification by a stationary carrier, the solid matter may be removed by centrifugation, filtration, or other means.

[0050] If the solution containing the target organic compound contains water-soluble or hydrophobic impurities and salts that hinder purification by a stationary carrier, the water-soluble or hydrophobic impurities and salts may be removed by passing the solution through a column packed with ion exchange resin, synthetic adsorption resin, etc., or by electrodialysis, membrane filtration, activated carbon treatment, etc.

[0051] The concentration of the target organic compound in the solution containing the target organic compound is not particularly limited, but from the viewpoint of purification efficiency, it may be prepared to be usually 0.01 g / L or higher, preferably 0.1 g / L or higher, and more preferably 1 g / L or higher. In another embodiment, the above concentration may be prepared to be 0.01 to 100 g / L, preferably 0.1 to 50 g / L, and more preferably 1 to 20 g / L. In order to obtain a concentration of the target organic compound within the above range, the solution containing the target organic compound may be concentrated by a heating concentration method, a vacuum concentration method, or a general concentration method such as membrane concentration.

[0052] Step (1) In step (1), a solution containing the target organic compound (referred to as solution A) is passed through a column packed with a stationary support, thereby adsorbing the target organic compound onto the stationary support and allowing impurities to pass through, thus separating the target organic compound from the impurities. For example, if the target organic compound is produced by fermentation, impurities may include those originating from microorganisms. After passing solution A through the column, a solvent may be passed through the column to wash away any remaining impurities.

[0053] In this disclosure, the flow rate when a solution is passed through a stationary carrier is defined by space velocity (the ratio of the volume of solution passed through per hour when the volume of the stationary carrier is set to 1, hereinafter referred to as "SV").

[0054] The conditions for passing solution A through the system are as follows: the flow rate is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2; and the temperature is preferably 1 to 65°C, more preferably 1 to 45°C, and even more preferably 1 to 30°C.

[0055] Step (2) In step (2), the target organic compound adsorbed on the stationary carrier is eluted by passing a solution containing the eluent through the stationary carrier. The concentration of the eluent is not particularly limited, but from the viewpoint of purification efficiency, it may be prepared to be 0.01 mol / L or higher, preferably 0.1 mol / L or higher, and more preferably 0.4 mol / L or higher. In another embodiment, the concentration of the eluent in the above solution is preferably 0.01 to 4 mol / L, more preferably 0.1 to 1 mol / L, and even more preferably 0.4 to 0.6 mol / L.

[0056] The conditions for passing the eluent through the liquid are as follows: the flow rate is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2; and the temperature is preferably 1 to 60°C, more preferably 20 to 50°C, and even more preferably 30 to 45°C.

[0057] Steps (1) and (2) may be performed simultaneously. That is, the target organic compound may be eluted while adsorbing it onto the stationary support by passing a solution containing the target organic compound and the stationary support through a column packed with stationary support. It is more preferable if the adsorption force of the target organic compound to the stationary support is weaker than the adsorption force of the eluent.

[0058] In step (3), the target organic compound adsorbed onto the stationary carrier is eluted using an eluent to obtain (a) a fraction containing the eluent at a first concentration, and (b) a fraction in which the concentration of the eluent is less than the first concentration. The order in which these fractions are obtained is not particularly important, but generally, by passing a solution containing the eluent through a column packed with stationary carriers, an eluate containing the target organic compound is obtained at the initial stage of elution, then an eluate containing both the target organic compound and the eluent, and then an eluate containing only the eluent is obtained. These eluates are collected in predetermined volumes and divided into (a) a fraction containing the eluent at a first concentration (represented as solution B), and (b) a fraction in which the concentration of the eluent is less than the first concentration (represented as solution C). The concentration of the eluent in fraction (b) is defined as the second concentration. Fraction (b) may be a fraction that does not contain the eluent. That is, the second concentration may be 0. Solutions B and C may be collections of multiple fractions, or they may be collections of only a portion of a fraction. The concentration of the target organic compound or eluent refers to the amount (g) of the target organic compound or eluent contained per liter of solution, and can be expressed in units such as g / L.

[0059] The fraction belonging to solution B may or may not include the fraction containing the point where the eluent began to elute, provided that the concentration of the eluent is higher than that of the fraction belonging to solution C. The presence or absence of the target organic compound and the eluent in each fraction can be confirmed using a spectrophotometer, Brix meter, conductivity meter, pH meter, high-performance liquid chromatography (HPLC), or gas chromatography. There are no particular restrictions on the method for detecting the presence of the target organic compound and the eluent in the fraction where the eluent has begun to elute, but from the viewpoint of purification efficiency, for example, a spectrophotometer, Brix meter, conductivity meter, pH meter, high-performance liquid chromatography (HPLC), or gas chromatography can be used for monitoring. The concentration of the eluent in solution B, i.e., the first concentration, is not particularly limited, but may be 100 g / L or less, 75 g / L or less, 50 g / L or less, or 48.4 g / L or less, or 0.1 g / L or more, 1 g / L or more, 2 g / L or more, or 3 g / L or more, or 0.1 g / L or more and 100 g / L or less, 1 g / L or more and 75 g / L or less, 2 g / L or more and 50 g / L or less, or 3 g / L or more and 48.4 g / L or less. The concentration of the target organic compound in solution B is not particularly limited, but may be 1 g / L or more, 5 g / L or more, 10 g / L or more, or 10.9 g / L or more, and may be 150 g / L or less, 100 g / L or less, 80 g / L or less, or 77.9 g / L or less, and may be 1 g / L or more and 150 g / L or less, 5 g / L or more and 100 g / L or less, 10 g / L or more and 80 g / L or less, or 10.9 g / L or more and 77.9 g / L or less. The ratio of the mass of the eluent to the mass of the target organic compound in solution B (eluent / target organic compound) is preferably 0.1 or more, more preferably 0.06 or more, and even more preferably 0.02 or more. In another embodiment, for those for which the mass ratio cannot be calculated, a value calculated by an analytical method such as HPLC area can be applied. The ratio of the area of ​​the eluent to the mass of the target organic compound in solution B (eluent / target organic compound) is preferably 0.1 or higher, more preferably 0.06 or higher, and even more preferably 0.02 or higher.

[0060] The fraction belonging to solution C may or may not include the fraction containing the point at which the target organic compound begins to elute from the stationary support, provided that the concentration of the eluent is lower than that of the fraction belonging to solution B, and may or may not include the fraction containing the point at which the eluent begins to elute. The elution order of the fractions contained in eluate B and eluate C is not particularly important, but generally the fraction containing eluate C elutes first. The presence or absence of the target organic compound and the eluent in each fraction can be confirmed using a spectrophotometer, Brix meter, conductivity meter, pH meter, high-performance liquid chromatography (HPLC), or gas chromatography. There are no particular restrictions on the method for detecting the presence of the target organic compound and the eluent in the fraction at which the eluent begins to elute, but from the viewpoint of purification efficiency, it can be monitored using, for example, a spectrophotometer, Brix meter, conductivity meter, pH meter, high-performance liquid chromatography (HPLC), or gas chromatography. The concentration of the eluent in solution C, i.e., the second concentration, is not particularly limited, but may be 1 / 2 or less, 1 / 5 or less, 1 / 10 or less, 1 / 20 or less, 1 / 37.5 or less, 1 / 50 or less, or 1 / 100 or less of the first concentration. Furthermore, the second concentration is preferably less than 1.5 g / L, more preferably less than 50 mg / L, even more preferably less than 0.947 mg / L, even more preferably less than 0.20 mg / L, and most preferably less than 0.08 mg / L. The concentration of the target organic compound in solution C is not particularly limited, but may be 1 g / L or more, 2 g / L or more, 2.5 g / L or more, or 2.58 g / L or more, and may be 150 g / L or less, 100 g / L or less, 80 g / L or less, or 76.9 g / L or less, and may be 1 g / L or more and 150 g / L or less, 2 g / L or more and 100 g / L or less, 2.5 g / L or more and 80 g / L or less, or 2.58 g / L or more and 76.9 g / L or less. The ratio of the mass of the eluent to the mass of the target organic compound in solution C (eluent / target organic compound) is preferably less than 0.1, more preferably less than 0.06, and even more preferably less than 0.02. In another embodiment, for those for which the mass ratio cannot be calculated, a value calculated by an analytical method such as HPLC area can be applied.The ratio of the area of ​​the eluent to the mass of the target organic compound in solution C (eluent / target organic compound) is preferably less than 0.1, more preferably less than 0.06, and even more preferably less than 0.02.

[0061] The amount of the target organic compound recovered in solution B and solution C is preferably 50% or more, more preferably 70%, even more preferably 80% or more, and most preferably 90% or more, relative to the amount of the target organic compound contained in solution A.

[0062] Steps (4) and (5): Solution B obtained in step (3) is concentrated to obtain a concentrate (step (4)), and then solution C is added to the concentrate and further concentrated (step (5)) to efficiently remove the eluent and increase the concentration of the target organic compound, thereby obtaining a concentrate. Here, the concentrate in step (5) refers to either or both a solid matter from which the solvent has been removed by concentration, or a concentrated liquid in which the target organic compound has been concentrated in solution by solvent removal. The above concentration refers to one or all of the following: heating concentration, vacuum concentration, or membrane concentration.

[0063] Vacuum concentration is a method of concentrating a target organic compound by evaporating the solvent and eluent used in the solution by placing the solution under reduced pressure. Vacuum concentration is also called vacuum concentration, vacuum distillation, vacuum drying, etc., and can be carried out by commonly known methods, in which case various devices such as rotary evaporators RE202, REV202M, RE301, RE601, RE801, R-300, and RE201 manufactured by Yamato Scientific Co., Ltd. may be used.

[0064] The temperature for vacuum concentration is not particularly limited, but from the viewpoint of purification efficiency, it is preferably 4 to 80°C, more preferably 4 to 50°C, even more preferably 10 to 45°C, and most preferably 30 to 40°C. The pressure for vacuum concentration is not particularly limited, but from the viewpoint of purification efficiency, it is preferably 0.1 to 300 hPa, more preferably 1 to 200 hPa, even more preferably 10 to 100 hPa, and most preferably 50 to 80 hPa.

[0065] While there are no particular restrictions on the temperature, pressure, and other conditions during concentration as long as the target organic compound can be concentrated, a lower heat load is preferable. For example, when choline cytidinediphosphate is concentrated under reduced pressure, thermal decomposition occurs, producing 5'-cytidylic acid (5'-CMP) and choline uridinediphosphate (UDP-choline) as thermal decomposition products (structural formulas are shown below). By reducing the temperature and performing the concentration under reduced pressure for a short time, the heat load during the concentration process can be reduced, thereby suppressing the generation of decomposition impurities.

[0066] 5'-Cytidylic acid (5'-CMP)

[0067] Choline uridinediphosphate (UDP-choline)

[0068] The concentration ratio is not particularly limited as long as the target organic compound can be concentrated, but it may be carried out so that the volume after concentration is usually 1 or less, preferably 1 / 2 or less, more preferably 1 / 3 or less, and most preferably 1 / 4 compared to the volume before concentration. In another embodiment, the volume after concentration is preferably 1 to 1 / 50 times, more preferably 1 / 2 to 1 / 20 times, even more preferably 1 / 3 to 1 / 15 times, and most preferably 1 / 4 to 1 / 10 times compared to the volume before concentration.

[0069] The ratio of the mass of the eluent to the mass of the target organic compound in the concentrate of step (5) (eluent / target organic compound) is preferably 0.1 or less, more preferably 0.06 or less, and even more preferably 0.02 or less. In another embodiment, for those for which the mass ratio cannot be calculated, a value calculated by another analytical method such as HPLC area can be applied. The ratio of the area of ​​the eluent to the mass of the target organic compound in the concentrate of step (5) (eluent / target organic compound) is preferably 0.1 or less, more preferably 0.06 or less, and even more preferably 0.02 or less.

[0070] Membrane concentration is a method of concentrating organic compounds by selectively allowing low-molecular-weight substances such as solvents and salts to pass through separation membranes such as microfiltration membranes (MF), ultrafiltration membranes (UF), nanofiltration membranes (NF), reverse osmosis membranes (RO), and ion exchange membranes, thereby separating the target organic compound. Membrane concentration can be carried out by generally known methods, and any membrane filter may be used in such cases.

[0071] Typically, microfiltration is a pressure-driven membrane separation process in the liquid phase that blocks particles larger than 0.1 μm and dissolved polymers. Ultrafiltration is a pressure-driven membrane separation process in the liquid phase that blocks particles smaller than 0.1 μm and larger than 2 nm and dissolved polymers. Nanofiltration is a pressure-driven membrane separation process in the liquid phase that blocks particles smaller than 2 nm and dissolved molecules. Reverse osmosis is a pressure-driven separation process in the liquid phase where applied intermembrane pressure selectively moves the solvent against its osmotic pressure difference. Ion exchange is a process using a charged porous membrane that selectively moves ionized substances in the liquid phase by electrophoresis.

[0072] The materials for microfiltration membranes include cellulose acetate, polyvinyl alcohol, polyethylene, polypropylene, and polytetrafluoroethylene; the materials for ultrafiltration membranes include polyacrylonitrile, polysulfone, polyvinylidene fluoride, aromatic polyamide, and cellulose acetate; the materials for nanofiltration membranes include polyamide, cellulose acetate, polyethersulfone, polyester, polyimide, vinyl polymer, polyolefin, regenerated cellulose, polycarbonate, polysulfone, and polyacrylonitrile; the materials for reverse osmosis membranes include cellulose acetate, polyvinyl alcohol, aromatic polyamide, and polysulfone; and the materials for ion exchange membranes are organic polymers polymerized from styrene, chloromethylstyrene, dibenylbenzene, etc., molded into a sheet shape with immobilized ion exchange groups such as sulfonic acid and quaternary ammonium.

[0073] Depending on the size differences of the target organic compound and the eluent, the membrane used for membrane concentration can be selected. For example, if the target organic compound is a protein, an ultrafiltration membrane is suitable, while if the target organic compound is a low molecular weight compound such as cytidine diphosphate choline compounds, oxidized glutathione, flavin adenine dinucleotide, and pravastatin, a nanofiltration membrane is suitable. Concentration can also be performed using an ion exchange membrane, focusing on differences in charge state rather than differences in size of the target organic compound and the eluent.

[0074] The membrane can be in any shape, including flat membranes, spiral membranes, hollow fiber membranes, plate-like membranes, and tubular membranes.

[0075] Membrane concentration methods are classified into dead-end filtration (direct flow filtration) and cross-flow filtration (tangential flow filtration). Dead-end filtration attempts to filter the entire volume of solution perpendicular to the membrane, and as the target organic compound in the solution continues to accumulate on the membrane surface, the filterability decreases as filtration progresses. Cross-flow filtration filters the solution while it flows parallel to the membrane surface, and because the shear flow near the membrane surface suppresses the accumulation of the target organic compound on the membrane surface, filtration can be performed quickly and efficiently.

[0076] The pressure driving force may be either pressurization or depressurization. The pressure is not particularly limited, but from the viewpoint of purification efficiency, it is preferably 0.1 MPa to 10 MPa, more preferably 1 MPa to 8 MPa, even more preferably 1 to 6 MPa, and most preferably 1 to 3 MPa. The operating temperature is not particularly limited, but from the viewpoint of purification efficiency, it is preferably 0 to 100°C, more preferably 5 to 80°C, even more preferably 10 to 50°C, and most preferably 10 to 30°C.

[0077] The membrane filtration is preferably a nanofiltration (NF) membrane or a reverse osmosis (RO) membrane. Specifically, commercially available membranes such as SU600, SU-610, SU-620, SU-700, SU-710, SU-720 from Toray Industries, and NTR-7100, NTR-7400, NTR-7410, NTR-7430, NTR-7450, NTR-7250, NTR-729HF, and NTR-759HR from Nitto Denko Corporation can be used.

[0078] The target organic compound obtained by this disclosure may undergo purification steps such as activated carbon decolorization, membrane filtration, crystallization, and powdering. Known methods can be used for activated carbon decolorization, membrane filtration, crystallization, and powdering, and examples of powdering methods include freeze-drying and spray-drying. The order of activated carbon decolorization, membrane filtration, crystallization, and powdering is not restricted. Furthermore, the target organic compound obtained by this disclosure is useful, for example, as a component, raw material, or intermediate for products such as health foods, pharmaceuticals, or cosmetics, or for cell culture media.

[0079] The present disclosure will be specifically described below with reference to examples, but the present disclosure is not limited to the following examples unless it exceeds its gist. Unless otherwise specified, % refers to mass %. Oxidized glutathione, cytidine diphosphate choline, pravastatin, flavin adenine dinucleotide and their analogs, as well as acetic acid, formic acid, propionic acid and ammonia, were detected by liquid chromatography, and acetone, 2-butanol and ethyl acetate were detected by gas chromatography. Each detection method was performed according to the methods described in Analysis Examples 1 to 11 below. "Below the detection limit" for each eluent means less than the following values: Acetic acid: 0.947 mg / L Formic acid: 1.49 mg / L Propionic acid: 1.15 mg / L Ammonia: 0.08 mg / L Acetone: 0.10 mg / L 2-Butanol: 0.20 mg / L Ethyl acetate: 0.095 mg / L

[0080] [Test Example 1: Method for Producing a Solution High in Oxidized Glutathione (GSSG)] [Preparation Example 1] <Obtaining a fraction obtained by eluting GSSG using propionic acid as an eluent> As a solution containing GSSG, commercially available GSSG (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 16.3 L of an aqueous solution (referred to as Solution A) containing 9.1 g / L, and OH －Adsorption was carried out by passing GSSG through 972 mL of Marathon WBA (Dow Chemical) at SV = 0.93 and 24°C. Next, the column was washed with 0.44 L of deionized water at SV = 0.83 and 24°C. Subsequently, GSSG adsorbed on Marathon WBA was eluted by passing a 44 g / L aqueous solution of propionic acid through the column at SV = 0.83 and 24°C. The eluate was collected as a solution (referred to as Solution C) with an endpoint of 3.6 RV as shown in Figure 1, representing the first fraction where propionic acid was below the detection limit. The second fraction, where propionic acid was detected, was collected as a solution (referred to as Solution B) with an endpoint of 4.7 RV as shown in Figure 1, representing 1.1 L. During elution, the eluate was sampled at 0.05 RV intervals, and the eluate composition was analyzed. The concentrations of GSSG and propionic acid in the obtained solutions A, B, and C are shown in Table 1.

[0081]

[0082] The results of the analysis of the eluate composition are shown in Figure 1. In Figure 1, the horizontal axis shows the volume of liquid passed through the column as "volume of eluent passed through the column per unit volume of ion exchange resin, RV," and the vertical axis shows the concentrations of GSSG (left) and propionic acid (right).

[0083] [Comparative Example 1] <Obtaining a solution obtained by concentrating GSSG eluate under reduced pressure in one go> Solution B (446 mL) and solution C (676 mL) obtained by the method of Preparation Example 1 were mixed to make one solution, and a 1.1 L solution (referred to as solution D) containing 46.4 g / L of GSSG and 12.9 g / L of propionic acid was obtained. 301 mL of the 1.1 L solution D was placed in a 1000 mL round-bottom flask, and reduced-pressure concentration was started under conditions equivalent to an evaporation rate of 200 mL / h (internal flask temperature 37-40°C, vacuum setting 78 hPa; the same conditions as in Comparative Example 1 and Example 1). During concentration, the volume of solution D in the round-bottom flask was maintained at approximately 300 mL while supplying a volume of solution D equivalent to the amount of liquid evaporated to the round-bottom flask as needed. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the volume of liquid in the round-bottom flask reached 301 mL to obtain solution E. Next, to further remove propionic acid by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 200 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 300 mL. A total of 400 mL of deionized water was supplied to the round-bottom flask, and concentration was continued until the liquid volume in the flask reached 301 mL, yielding solution F.

[0084] [Example 1] <Obtaining a solution obtained by dividing the GSSG eluate and concentrating it under reduced pressure> Solution B (446 mL) and solution C (676 mL) were prepared by the method of Preparation Example 1. 301 mL of solution B was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 200 mL / h. During concentration, the volume of solution B in the round-bottom flask was maintained at approximately 300 mL by continuously supplying a volume of solution B equivalent to the amount of liquid evaporated. After the supply of 446 mL of solution B was completed, 676 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the volume of liquid in the round-bottom flask reached 301 mL to obtain solution G.

[0085] GSSG, propionic acid, and the decomposition impurity S-glutathionylcysteinylglycine (hereinafter referred to as GLT-CG) contained in solutions D to G obtained in Comparative Example 1 and Example 1 were measured by HPLC. The results are shown in Table 2.

[0086]

[0087] Solution E in Comparative Example 1 and Solution G in Example 1 were obtained by concentrating the same amount of solution in one go or in stages, respectively. Solution E, obtained by one go, had a propionic acid concentration of 16.1 g / L, which was higher than the propionic acid concentration of 5.0 g / L in Solution G, obtained by staged concentration. Solution F in Comparative Example 1 was obtained by continuing reduced-pressure concentration while adding deionized water to Solution E until the propionic acid concentration reached the same level as Solution G. The propionic acid concentration was at the same level in Solution F and Solution G, but in Solution F, the time required for concentration and the amount of solvent increased, and the amount of decomposed impurities also increased. The increase in the amount of decomposed impurities is presumed to be due to the increased time required for concentration. From these results, it became clear that by dividing the eluted fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first, efficient concentration can be achieved and the generation of decomposed impurities can be suppressed.

[0088] [Test Example 2: Method for Producing a Solution High in Cytidinedicholine Phosphate] [Preparation Example 2] <Obtaining a fraction in which cytidinedicholine eluted using formic acid as an eluent> As a solution containing cytidinedicholine phosphate, commercially available cytidinedicholine phosphate (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 25.8 L of an aqueous solution (referred to as Solution A) containing it at a concentration of 5.5 g / L, and OH －Adsorption was carried out by passing a 950 mL column of PA412M (manufactured by Mitsubishi Chemical Corporation) through a column at SV = 1.45 and 24°C. Next, the column was washed by passing 475 mL of deionized water through it at SV = 0.87 and 24°C. Subsequently, a 44.6 g / L formic acid aqueous solution was passed through the column at SV = 0.87 and 24°C to elute the cytidinediphosphate choline adsorbed on PA412M. From the eluate, 1.68 L was recovered as a solution with an endpoint of 2.7 RV (as shown in Figure 2) (referred to as solution C'), representing the first fraction where formic acid was below the detection limit. In addition, 0.67 L was recovered as a solution with an endpoint of 3.3 RV (as shown in Figure 2) (referred to as solution B'), representing the second fraction where formic acid was detected. Separately from the above, 950 mL of PA412M was prepared, and the same column elution test was performed to elute the adsorbed cytidinediphosphate choline. The eluate was collected as a solution (referred to as solution C'') with an endpoint of 2.8 RV as shown in Figure 3, representing the first fraction where formic acid was below the detection limit. Additionally, 0.37 L of the second fraction (referred to as solution B'') with an endpoint of 3.1 RV as shown in Figure 3, representing the second fraction where formic acid was detected, was collected. Solutions C' and C'' were mixed to form a single solution of 3.33 L (referred to as solution C). Similarly, solutions B' and B'' were mixed to form a single solution of 1.04 L (referred to as solution B). During elution, the eluate was sampled at 0.05 RV intervals, and the concentrations of cytidinediphosphate choline and formic acid in the eluate were analyzed. The concentrations of cytidinediphosphate choline and formic acid in the obtained solutions A, B, and C are shown in Table 3.

[0089]

[0090] The results of the analysis of the eluate composition are shown in Figures 2 and 3. In Figures 2 and 3, the horizontal axis represents the volume of liquid passed through the column, and the vertical axis represents the concentrations of cytidinediphosphate choline (left) and formic acid (right).

[0091] [Comparative Example 2] <Obtaining a solution obtained by concentrating the eluate of cytidinediphosphate choline in one go under reduced pressure> Solution B (276 mL) and solution C (887 mL) obtained by the method of Preparation Example 2 were mixed to make one solution, and a 1.16 L solution (referred to as solution D) containing 65.7 g / L of cytidinediphosphate choline and 5.5 g / L of formic acid was obtained. 189 mL of solution D was placed in a 500 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 100 mL / h (internal flask temperature 37-40°C, vacuum setting 78 hPa; the same conditions were used in Comparative Example 2 and Example 2). During concentration, solution D equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the round-bottom flask at approximately 189 mL. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask reached 189 mL, obtaining solution E. Next, to further remove formic acid by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 189 mL. A total of 600 mL of deionized water was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the flask reached 189 mL, obtaining solution F.

[0092] [Example 2] <Obtaining a solution obtained by dividing the eluate of cytidinediphosphate choline and concentrating it under reduced pressure> Solution B (276 mL) and solution C (887 mL) were prepared by the method of Preparation Example 2. 189 mL of solution B was placed in a 500 mL round-bottom flask, and concentration under reduced pressure was started under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, the volume of solution B in the round-bottom flask was maintained at approximately 189 mL by supplying a volume of solution B equivalent to the amount of liquid evaporated as needed. After the supply of 276 mL of solution B was completed, 887 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the volume of liquid in the round-bottom flask reached 189 mL, thereby obtaining solution G.

[0093] The cytidinediphosphate choline, formic acid, and decomposition impurities (5'-cytidylic acid (5'-CMP) and uridinediphosphate choline (UDP-choline)) contained in solutions D to G obtained in Comparative Example 2 and Example 2 were measured by HPLC. The results are shown in Table 4.

[0094]

[0095] Solution E in Comparative Example 2 and Solution G in Example 2 were obtained by concentrating the same amount of solution in one step or in stages, respectively. Solution E, obtained by one step, had a formic acid concentration of 14.5 g / L, which was higher than the formic acid concentration of 5.2 g / L in Solution G, obtained by stages. Solution F in Comparative Example 2 was obtained by continuing reduced-pressure concentration while adding deionized water to Solution E until the formic acid concentration reached the same level as Solution G. The formic acid concentration was at the same level in Solution F and Solution G, but in Solution F, the time required for concentration and the amount of solvent increased, and the ratio of 5'-cytidylic acid to cytidinediphosphate choline and the area ratio of uridinediphosphate choline, which are decomposed impurities, also increased. The increase in the amount of decomposed impurities is presumed to be due to the increased time required for concentration. From these results, it became clear that by dividing the eluted fraction based on the presence or absence of the eluent and concentrating the fraction in which the eluent was detected first, efficient concentration can be achieved and the generation of decomposed impurities can be suppressed.

[0096] [Test Example 3: Method for Producing a Solution High in Cytidinedicholine Phosphate] [Preparation Example 3] <Obtaining a fraction in which cytidinedicholine eluted using propionic acid as an eluent> As a solution containing cytidinedicholine phosphate, commercially available cytidinedicholine phosphate (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 64.4 L of an aqueous solution (referred to as Solution A) containing it at a concentration of 5.5 g / L, and OH －Adsorption was carried out by passing the solution through 2375 mL of PA412M (manufactured by Mitsubishi Chemical Corporation) at SV = 1.43 and 24°C. Next, 1.2 L of deionized water was passed through the column at SV = 0.84 and 24°C to wash the solution. Subsequently, a 36.3 g / L aqueous solution of propionic acid was passed through the column at SV = 0.84 and 24°C to eluate the cytidinediphosphate choline adsorbed on PA412M. 5.57 L of the eluate was collected as a solution (referred to as Solution C) with an endpoint of 3.9 RV as shown in Figure 4, representing the first fraction where propionic acid was below the detection limit. 1.72 L of the solution (referred to as Solution B) with an endpoint of 4.6 RV as shown in Figure 4, representing the second fraction where propionic acid was detected, was also collected. During elution, the eluate was sampled at 0.024 RV intervals, and the concentrations of cytidinediphosphate choline and propionic acid in the eluate were analyzed. Table 5 shows the concentrations of cytidinediphosphate choline and propionic acid in the obtained solutions A, B, and C.

[0097]

[0098] Figure 4 shows the results of the analysis of the eluate composition. In Figure 4, the horizontal axis represents the volume of eluent through the column, and the vertical axis represents the concentrations of cytidinediphosphate choline (left) and propionic acid (right).

[0099] [Comparative Example 3] <Obtaining a solution obtained by concentrating the eluate of cytidinediphosphate choline in one go under reduced pressure> Solution B (507 mL) and solution C (1641 mL) obtained by the method of Preparation Example 3 were mixed to make one solution, and a 2.15 L solution (solution D) containing 46.9 g / L of cytidinediphosphate choline and 5.8 g / L of propionic acid was obtained. 260 mL of solution D was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 150 mL / h (internal flask temperature 37-40°C, vacuum setting 78 hPa; the same conditions as in Comparative Example 3 and Example 3). During concentration, solution D in a volume equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the round-bottom flask at approximately 260 mL. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask reached 260 mL to obtain solution E. Next, to further remove propionic acid by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 150 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 260 mL. A total of 1000 mL of deionized water was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the flask reached 260 mL, obtaining solution F.

[0100] [Example 3] <Obtaining a solution obtained by dividing the eluate of cytidinediphosphate choline and concentrating it under reduced pressure> Solution B (507 mL) and solution C (1641 mL) were prepared by the method of Preparation Example 3. 260 mL of solution B was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 150 mL / h. During concentration, the volume of solution B in the round-bottom flask was maintained at approximately 260 mL by supplying a volume equivalent to the amount of liquid evaporated to the round-bottom flask as needed. After the supply of 507 mL of solution B was completed, 1641 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the volume of liquid in the round-bottom flask became 260 mL to obtain solution G.

[0101] The cytidinediphosphate choline, propionic acid, and decomposition impurities (5'-CMP and UDP-choline) contained in solutions D to G obtained in Comparative Example 3 and Example 3 were measured by HPLC. The results are shown in Table 6.

[0102]

[0103] Solution E in Comparative Example 3 and Solution G in Example 3 were obtained by concentrating the same amount of solution in one step or in stages, respectively. Solution E, obtained by one step, had a propionic acid concentration of 6.3 g / L, which was higher than the propionic acid concentration of 0.2 g / L in Solution G, obtained by stages. Solution F in Comparative Example 3 was obtained by continuing reduced-pressure concentration while adding deionized water to Solution E until the propionic acid concentration reached the same level as Solution G. The propionic acid concentration was at the same level in Solution F and Solution G, but in Solution F, the time required for concentration and the amount of solvent increased, and the amount of decomposed impurities also increased. The increase in the amount of decomposed impurities is presumed to be due to the increased time required for concentration. From these results, it became clear that by dividing the eluted fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first, efficient concentration can be achieved and the generation of decomposed impurities can be suppressed.

[0104] [Test Example 4: Method for Producing a Solution High in Cytidinediphosphate Choline] [Preparation Example 4] <Obtaining a fraction in which cytidinediphosphate choline has been eluted using acetic acid as an eluent> As a solution containing cytidinediphosphate choline and acetic acid, commercially available cytidinediphosphate choline (manufactured by Kyowa Hakko Bio Co., Ltd.) and acetic acid were dissolved in water to prepare 6.25 L of an aqueous solution (referred to as Solution A) containing cytidinediphosphate choline at a concentration of 56 g / L and acetic acid at a concentration of 8.0 g / L, and OH － The eluate was eluted through a 652 mL Marathon WBA (Dow Chemical) column at SV = 4.0 and 24°C. 4.55 L of the eluate was collected as the first fraction, where acetic acid was below the detection limit, with an endpoint of 8.9 RV (referred to as Solution C) as shown in Figure 5. Additionally, 1.96 L of the second fraction, where acetic acid was detected, was collected as the second fraction, with an endpoint of 11.9 RV (referred to as Solution B) as shown in Figure 5. During elution, the eluate was sampled at 0.1 RV intervals, and the concentrations of cytidinediphosphate choline and acetic acid in the eluate were analyzed. The concentrations of cytidinediphosphate choline and acetic acid in the obtained Solutions A, B, and C are shown in Table 7.

[0105]

[0106] Figure 5 shows the results of the analysis of the eluate composition. In Figure 5, the horizontal axis represents the volume of liquid passed through the column, and the vertical axis represents the concentrations of cytidinediphosphate choline (left) and acetic acid (right).

[0107] [Comparative Example 4] <Obtaining a solution obtained by concentrating the eluate of cytidinediphosphate choline in one go under reduced pressure> Solution B (602 mL) and solution C (1398 mL) obtained by the method of Preparation Example 4 were mixed to make one solution, and a 2.0 L solution (solution D) containing 51.6 g / L of cytidinediphosphate choline and 0.9 g / L of acetic acid was obtained. 260 mL of solution D was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 200 mL / h (internal flask temperature 37-40°C, vacuum setting 78 hPa; the same conditions as in Comparative Example 4 and Example 4). During concentration, solution D equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the round-bottom flask at approximately 260 mL. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask reached 260 mL to obtain solution E. Next, to further remove acetic acid by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 200 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was continuously supplied to the round-bottom flask, maintaining the liquid volume in the flask at approximately 260 mL. A total of 800 mL of deionized water was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the flask reached 260 mL, obtaining solution F.

[0108] [Example 4] <Obtaining a solution obtained by dividing the eluate of cytidinediphosphate choline and concentrating it under reduced pressure> Solution B (602 mL) and solution C (1398 mL) were prepared by the method of Preparation Example 4. 260 mL of solution B was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 200 mL / h. During concentration, the volume of solution B in the round-bottom flask was maintained at approximately 260 mL by continuously supplying a volume of solution B equivalent to the amount of liquid evaporated. After the supply of 602 mL of solution B was completed, 1398 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the volume of liquid in the round-bottom flask became 260 mL to obtain solution G.

[0109] The cytidinediphosphate choline, acetic acid, and decomposition impurities (5'-CMP and UDP-choline) contained in solutions D to G obtained in Comparative Example 4 and Example 4 were measured by HPLC. The results are shown in Table 8.

[0110]

[0111] Solution E in Comparative Example 4 and Solution G in Example 4 were obtained by concentrating the same amount of solution in one step or in stages, respectively. Solution E, obtained by one step, had an acetic acid concentration of 1.7 g / L, which was higher than the acetic acid concentration of Solution G, obtained by stages, which was 0.2 g / L. Solution F in Comparative Example 4 was obtained by continuing reduced-pressure concentration while adding deionized water to Solution E until the acetic acid concentration reached the same level as Solution G. The acetic acid concentration was at the same level in Solution F and Solution G, but in Solution F, the time required for concentration and the amount of solvent increased, and the amount of decomposed impurities also increased. The increase in the amount of decomposed impurities is presumed to be due to the increased time required for concentration. From these results, it became clear that by dividing the eluted fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first, efficient concentration can be achieved and the generation of decomposed impurities can be suppressed.

[0112] [Test Example 5: Method for Producing a Solution High in Cytidinediphosphate Choline] [Preparation Example 5] <Obtaining a fraction in which cytidinediphosphate choline has been eluted using acetic acid as an eluent> Cytidinediphosphate choline was produced by fermentation using the method described in Japanese Patent Publication No. 3369236, and the culture solution was inactivated and the pH was adjusted to 3.0 with sulfuric acid to obtain a culture solution containing cytidinediphosphate choline. Subsequently, the culture solution was heated to 50°C, and the bacterial cells were separated by cross-flow filter filtration (0.1 μm filter) to obtain a filtrate containing cytidinediphosphate choline. Next, the filtrate was H ＋ The filtrate was passed through a XUS40232-01 type (manufactured by Dow Chemical) column at SV = 0.38 and 5-10°C. Once the filtrate had passed through the column, deionized water cooled to 5-10°C was passed through at SV = 0.38, and the solution was collected when the elution of cytidinediphosphate choline began. 180 kL of solution A containing 7.2 g / L of cytidinediphosphate choline was obtained. Solution A was then OH －Adsorption was carried out by passing the sample through a PA412M (manufactured by Mitsubishi Chemical Corporation) column at SV = 1.5 and 5-10°C. Next, the sample was washed by passing deionized water through the column at SV = 0.84 and 40°C. Subsequently, a 31.8 g / L aqueous acetic acid solution was passed through the column at SV = 0.63 and 40°C to elute the cytidinediphosphate choline adsorbed on the PA412M. The eluate was collected as a solution with an endpoint of 5.3 RV (referred to as solution C') in Figure 6, representing the first fraction where acetic acid was below the detection limit. The second fraction, where acetic acid was detected, was collected as a solution with an endpoint of 6.8 RV (referred to as solution B') in Figure 6. During elution, the eluate was sampled at 0.3 RV intervals, and the concentrations of cytidinediphosphate choline and acetic acid in the eluate were analyzed. The concentrations of cytidinediphosphate choline and acetic acid in the obtained solutions A, B, and C are shown in Table 9.

[0113]

[0114] Figure 6 shows the results of the analysis of the eluate composition. In Figure 6, the horizontal axis represents the volume of liquid passed through the column, and the vertical axis represents the concentrations of cytidinediphosphate choline (left) and acetic acid (right).

[0115] [Comparative Example 5] <Obtaining a solution obtained by concentrating the eluate of cytidinediphosphate choline in one go under reduced pressure> Solutions B and C having the same cytidinediphosphate choline and acetic acid concentrations as solutions B' and C' obtained by the method of Preparation Example 5 were prepared. Solution B (800 mL) and solution C (1600 mL) were mixed to make one solution, and a 2.4 L solution (solution D) containing 64.7 g / L of cytidinediphosphate choline and 7.9 g / L of acetic acid was obtained. 450 mL of solution D was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started while maintaining the internal temperature of the flask at 37-40°C and the evaporation rate at approximately 100 mL / h. During concentration, a volume of solution D equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the round-bottom flask at approximately 450 mL. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask reached 450 mL to obtain solution E. Next, to further remove acetic acid by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 450 mL. A total of 2000 mL of deionized water was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the flask reached 450 mL, obtaining solution F.

[0116] [Example 5] <Obtaining a solution obtained by dividing the eluate of cytidinediphosphate choline and concentrating it under reduced pressure> Solutions B and C were prepared having the same cytidinediphosphate choline concentration and acetic acid concentration as solutions B' and C' obtained by the method of Preparation Example 5. Solution B (800 mL) and solution C (1600 mL) were prepared. 450 mL of solution B was placed in a 1000 mL round-bottom flask, and vacuum concentration was started under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, the volume of solution B in the round-bottom flask was maintained at approximately 450 mL by supplying a volume equivalent to the amount of liquid evaporated as needed. After the supply of 800 mL of solution B was completed, 1600 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the volume of liquid in the round-bottom flask became 450 mL to obtain solution G. Next, in order to further evaporate and remove acetic acid, vacuum concentration was restarted under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, the volume of deionized water equivalent to the amount of evaporated liquid was continuously supplied to the round-bottom flask, maintaining the liquid volume in the flask at approximately 450 mL. A total of 1400 mL of deionized water was supplied to the round-bottom flask, and the solution was concentrated until the liquid volume in the flask reached 450 mL, obtaining solution H.

[0117] The cytidinediphosphate choline, acetic acid, and decomposition impurities (5'-CMP and UDP-choline) contained in solutions D to G obtained in Comparative Example 5 and Example 5 were measured by HPLC. The results are shown in Table 10.

[0118]

[0119] In comparative example solution F, the residual acetic acid was 0.0034 to choline cytidinediphosphate by mass ratio, and the concentration time was 40.5 hours. On the other hand, in example solution H, the residual acetic acid was 0.0028 to choline cytidinediphosphate by mass ratio, and the concentration time was 31.5 hours. The time required for concentration to sufficiently reduce the acetic acid was 9 hours longer in the comparative example, which resulted in more advanced generation of decomposition impurities in the comparative example. From these results, it became clear that by dividing the eluted fraction into those with and without the eluting agent and concentrating the fraction containing the eluting agent first, efficient concentration can be achieved and the generation of decomposition impurities can be suppressed. Furthermore, from these results, it was found that the production method of the present invention can also be applied to solutions containing choline cytidinediphosphate obtained by the culture method.

[0120] [Test Example 6: Method for Producing a Solution High in Flavin Adenine Dinucleotide] [Preparation Example 6] <Obtaining a fraction from which flavin adenine dinucleotide has been eluted using acetone as an eluent> As a solution containing flavin adenine dinucleotide, commercially available flavin adenine dinucleotide (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 7.5 L of an aqueous solution (referred to as Solution A) containing 10.0 g / L. This solution was then passed through 3500 mL of SP207 (manufactured by Mitsubishi Chemical Corporation) at SV = 0.83, 20°C, while shielding the resin column with aluminum foil to allow adsorption. Next, 7.0 L of deionized water was passed through the column at SV = 0.83, 20°C to wash the solution. Subsequently, a 20 wt% aqueous acetone solution was passed through the column at SV = 0.36, 20°C to elute the flavin adenine dinucleotide adsorbed on the SP207. The eluate was collected as a solution (referred to as Solution C) with an endpoint of 1.18 RV as shown in Figure 7, comprising the first fraction containing 1.2 g / L of acetone. Additionally, 793 mL of the second fraction (referred to as Solution B) was collected as a solution (referred to as Solution B) with an endpoint of 1.41 RV as shown in Figure 7, comprising the second fraction containing 45.2 g / L of acetone. During elution, the eluate was sampled at 0.017 RV intervals, and the eluate composition was analyzed. The concentrations of flavin adenine dinucleotide and acetone in the obtained solutions A, B, and C are shown in Table 11.

[0121]

[0122] The results of the analysis of the eluate composition are shown in Figure 7. In Figure 7, the horizontal axis shows the volume of eluent passed through the column as "volume of eluent passed through the column per unit volume of synthetic adsorbent resin, RV," and the vertical axis shows the concentrations of flavin adenine dinucleotide (left) and acetone (right).

[0123] [Comparative Example 6] <Obtaining a solution obtained by membrane-concentrating the eluate of flavin adenine dinucleotide in one step> Solution B (357 mL) and solution C (604 mL) obtained by the method of Preparation Example 6 were mixed to make one solution, and a 961 mL solution (solution D) containing 39.0 g / L of flavin adenine dinucleotide and 35.0 g / L of acetone was obtained. 250 mL of solution D was placed in a 1000 mL light-shielding round-bottom flask, and vacuum concentration was started under conditions equivalent to an evaporation rate of 100 mL / h (internal flask temperature 27-30°C, vacuum setting 48 hPa; the same conditions were used in Comparative Example 6 and Example 6). During concentration, a volume of solution D equivalent to the amount of liquid evaporated was supplied to the light-shielding round-bottom flask as needed, maintaining the liquid volume in the light-shielding round-bottom flask at approximately 250 mL. All of solution D was supplied to the light-shielding round-bottom flask, and concentration was carried out until the liquid volume in the light-shielding round-bottom flask reached 250 mL to obtain solution E. Next, to further remove acetone by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was continuously supplied to a light-shielding round-bottom flask, maintaining the liquid volume in the flask at approximately 250 mL. A total of 200 mL of deionized water was supplied to the light-shielding round-bottom flask, and concentration was carried out until the liquid volume in the light-shielding round-bottom flask reached 250 mL, obtaining solution F.

[0124] [Example 6] <Obtaining a concentrated solution by dividing the eluate of flavin adenine dinucleotide> Solution B (357 mL) and solution C (604 mL) obtained by the method of Preparation Example 6 were prepared. 250 mL of solution B was placed in a 1000 mL light-shielding round-bottom flask, and vacuum concentration was started under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, solution B in a volume equivalent to the amount of liquid evaporated was supplied to the light-shielding round-bottom flask as needed, maintaining the liquid volume in the light-shielding round-bottom flask at approximately 250 mL. After the supply of 357 mL of solution B was completed, 604 mL of solution C was similarly supplied to the light-shielding round-bottom flask, and concentration was carried out until the liquid volume in the light-shielding round-bottom flask was 250 mL to obtain solution G.

[0125] The flavin adenine dinucleotide, acetone, and impurities contained in solutions D to G obtained in Comparative Example 6 and Example 6 were measured by HPLC. The results are shown in Table 12.

[0126]

[0127] Solution E in Comparative Example 6 and Solution G in Example 6 were obtained by concentrating the same amount of solution in one go or in stages, respectively. Solution E, obtained by one go, had an acetone concentration of 0.63 g / L, which was higher than the acetone concentration of Solution G, obtained by staged concentration, which was 0.0011 g / L. Solution F in Comparative Example 6 was obtained by continuing the concentration of Solution E while adding deionized water until the acetone concentration reached the same level as Solution G. The acetone concentrations were at the same level in Solution F and Solution G, but the time required for concentration and the amount of solvent increased in Solution F. From these results, it became clear that concentration can be performed efficiently by dividing the eluent fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first.

[0128] [Test Example 7: Method for Producing a Solution High in Pravastatin] [Preparation Example 7] <Obtaining a fraction in which pravastatin has been eluted using ethyl acetate as an eluent> Commercially available pravastatin (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 4.2 L of an aqueous solution (referred to as Solution A) containing pravastatin at a concentration of 10.1 g / L. This solution was passed through 4220 mL of SP207 (manufactured by Mitsubishi Chemical Corporation) at SV = 1.0 and 54-62°C to allow adsorption. Next, 8.4 L of 8.5 g / L aqueous ammonia was passed through the column at SV = 1.0 and ambient temperature to wash, and then 4.2 L of deionized water was passed through the column at SV = 1.0 and ambient temperature to wash again. Subsequently, an 8.0 wt% aqueous ethyl acetate solution was passed through the column at SV = 1.0 and ambient temperature to elute the pravastatin adsorbed on the SP207. The eluate was collected as a solution (referred to as Solution C) with an endpoint of 2.67 RV as shown in Figure 8, representing the first fraction where ethyl acetate was below the detection limit. Additionally, 2.65 L of the solution (referred to as Solution B) with an endpoint of 3.36 RV as shown in Figure 8, representing the second fraction where ethyl acetate was detected, was collected. During elution, the eluate was sampled at 0.014 RV intervals, and the eluate composition was analyzed. The concentrations of pravastatin and ethyl acetate in the obtained solutions A, B, and C are shown in Table 13.

[0129]

[0130] The results of the analysis of the eluate composition are shown in Figure 8. In Figure 8, the horizontal axis shows the volume of liquid passed through the column as "volume of eluent passed through the column per unit volume of synthetic adsorbent resin, RV," and the vertical axis shows the concentrations of pravastatin (left) and ethyl acetate (right).

[0131] [Comparative Example 7] <Obtaining a solution obtained by concentrating the pravastatin eluate under reduced pressure in one go> Solution B (1000 mL) and solution C (1019 mL) obtained by the method of Preparation Example 7 were mixed to make one solution, and a 2019 mL solution (solution D) containing pravastatin 6.71 g / L and ethyl acetate 9.18 g / L was obtained. 200 mL of solution D was placed in a 1000 mL round-bottom flask, and reduced-pressure concentration was started under conditions equivalent to an evaporation rate of 150 mL / h (internal flask temperature 37-41°C, vacuum setting 75 hPa; the same conditions were used in Comparative Example 7 and Example 7). During concentration, solution D equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the round-bottom flask at approximately 200 mL. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask reached 200 mL to obtain solution E.

[0132] [Example 7] <Obtaining a solution obtained by dividing and concentrating the pravastatin eluate under reduced pressure> Solution B (1000 mL) and solution C (1019 mL) were prepared by the method of Preparation Example 7. 200 mL of solution B was placed in a 1000 mL round-bottom flask, and reduced pressure concentration was started under conditions equivalent to an evaporation rate of 150 mL / h. During concentration, solution B in a volume equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 200 mL. After the supply of 1000 mL of solution B was completed, the remaining 939 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask was 200 mL to obtain solution F.

[0133] The pravastatin, ethyl acetate, and decomposed impurities contained in solutions D to F obtained in Comparative Example 7 and Example 7 were measured by HPLC. The results are shown in Table 14.

[0134]

[0135] Solution E in Comparative Example 7 and Solution F in Example 7 were obtained by concentrating the same amount of solution in a single step or in a divided step, respectively. In Solution E of Comparative Example 7, obtained by single step concentration, the ethyl acetate concentration was 0.0020 g / L, while in Solution F, obtained by divided step concentration, no ethyl acetate was detected. In other words, although the time required for concentration, the amount of solvent, and the amount of decomposed impurities due to thermal load were at the same level for Solution E of Comparative Example 7 and Solution F of Example 7, the eluent concentration in Solution F was reduced to a level lower than that of Solution E. From these results, it became clear that concentration can be performed efficiently by dividing the elution fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first.

[0136] [Test Example 8: Method for Producing a Solution High in Flavin Adenine Dinucleotide] [Preparation Example 8] <Obtaining a fraction from which flavin adenine dinucleotide has been eluted using 2-butanol as an eluent> As a solution containing flavin adenine dinucleotide, commercially available flavin adenine dinucleotide (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 7.5 L of an aqueous solution (referred to as Solution A) containing it at a concentration of 10.1 g / L. This solution was passed through 3700 mL of SP207 (manufactured by Mitsubishi Chemical Corporation) at SV = 0.83, 20°C, while shielding the resin column with aluminum foil to allow adsorption. Next, 7.4 L of deionized water was passed through the column at SV = 0.83, 20°C to wash the solution. Subsequently, a 100 g / L aqueous solution of 2-butanol was passed through the column at SV = 0.36, 20°C to elute the flavin adenine dinucleotide adsorbed on the SP207. The eluate was collected as a solution (referred to as Solution C) in 2.73 L, with the first fraction containing 0.02 g / L of 2-butanol and ending at 1.87 RV as shown in Figure 9. The second fraction containing 48.4 g / L of 2-butanol was collected as a solution (referred to as Solution B) with the second fraction ending at 2.02 RV as shown in Figure 9. During elution, the eluate was sampled in 0.018 RV increments, and the eluate composition was analyzed. The concentrations of flavin adenine dinucleotide and 2-butanol in the obtained solutions A, B, and C are shown in Table 15.

[0137]

[0138] The results of the analysis of the eluate composition are shown in Figure 9. In Figure 9, the horizontal axis shows the volume of eluent passed through the tower as "volume of eluent passed through the tower per unit volume of synthetic adsorbent resin, RV," and the vertical axis shows the concentrations of flavin adenine dinucleotide (left) and 2-butanol (right).

[0139] [Comparative Example 8] <Obtaining a solution obtained by concentrating the eluate of flavin adenine dinucleotide under reduced pressure> Solution B (234 mL) and solution C (1229 mL) obtained by the method of Preparation Example 8 were mixed to make one solution, and a 1463 mL solution (solution D) containing 23.7 g / L of flavin adenine dinucleotide and 7.5 g / L of 2-butanol was obtained. 150 mL of solution D was placed in a 500 mL light-shielding round-bottom flask, and reduced-pressure concentration was started under conditions equivalent to an evaporation rate of 100 mL / h (internal flask temperature 30.8-31.3°C, vacuum setting 41 hPa; the same conditions as in Comparative Example 8 and Example 8). During concentration, a volume of solution D equivalent to the amount of liquid evaporated was supplied to the light-shielding round-bottom flask as needed, while maintaining the liquid volume in the light-shielding round-bottom flask to approximately 150 mL. Solution D was completely supplied to a light-shielding round-bottom flask, and concentrated until the volume of liquid in the flask reached 150 mL to obtain solution E. Next, in order to further remove 2-butanol by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, deionized water equivalent to the volume of liquid evaporated was continuously supplied to the light-shielding round-bottom flask, maintaining the volume of liquid in the flask at approximately 150 mL. A total of 100 mL of deionized water was supplied to the light-shielding round-bottom flask, and concentrated until the volume of liquid in the flask reached 150 mL to obtain solution F.

[0140] [Example 8] <Obtaining a solution obtained by dividing the eluate of flavin adenine dinucleotide and concentrating it under reduced pressure> Solution B (234 mL) and solution C (1229 mL) obtained by the method of Preparation Example 8 were prepared. 150 mL of solution B was placed in a 500 mL light-shielding round-bottom flask, and concentration under reduced pressure was started under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, solution B in a volume equivalent to the amount of liquid evaporated was supplied to the light-shielding round-bottom flask as needed, maintaining the liquid volume in the light-shielding round-bottom flask at approximately 150 mL. After the supply of 234 mL of solution B was completed, 1229 mL of solution C was similarly supplied to the light-shielding round-bottom flask, and concentration was carried out until the liquid volume in the light-shielding round-bottom flask reached 150 mL to obtain solution G.

[0141] The flavin adenine dinucleotide, 2-butanol, and impurities contained in solutions D to G obtained in Comparative Example 8 and Example 8 were measured by HPLC. The results are shown in Table 16.

[0142]

[0143] Solution E in Comparative Example 8 and Solution G in Example 8 were obtained by concentrating the same amount of solution in one step or in stages, respectively. Solution E, obtained by one step, had a 2-butanol concentration of 0.11 g / L, which was higher than Solution G, obtained by staged concentration, where the 2-butanol concentration was below the detection limit. Solution F in Comparative Example 8 was obtained by continuing the concentration of Solution E while adding deionized water until the 2-butanol concentration reached the same level as Solution G. The 2-butanol concentrations were at the same level in Solution F and Solution G, but the time required for concentration and the amount of solvent increased in Solution F. From these results, it became clear that concentration can be performed efficiently by dividing the eluent fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first.

[0144] [Test Example 9: Method for Producing a Solution High in Oxidized Glutathione (GSSG)] [Preparation Example 9] <Obtaining a fraction from which GSSG has been eluted using ammonia as an eluent> Commercially available GSSG (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in water to prepare 2.0 L of an aqueous solution (referred to as Solution A) containing 67.6 g / L of GSSG. This solution was passed through 1940 mL of XUS40232-01 (manufactured by Dow Chemical) at SV = 1.0 and 20 °C to allow adsorption. Next, 7.95 L of deionized water was passed through the column at SV = 1.0 and 20 °C to wash the solution. Subsequently, 9.37 g / L of ammonia water was passed through the column at SV = 1.0 and 20 °C to elute the GSSG adsorbed on XUS40232-01. The eluate was collected as a solution (referred to as Solution C) with an endpoint of 2.8 RV as shown in Figure 10, representing the first fraction where ammonia was below the detection limit. Additionally, 1.05 L of the solution (referred to as Solution B) was collected as a solution (referred to as Solution B) with an endpoint of 3.34 RV as shown in Figure 10, representing the second fraction where ammonia was detected. During elution, the eluate was sampled at 0.027 RV intervals, and the eluate composition was analyzed. The concentrations of GSSG and ammonia in the obtained solutions A, B, and C are shown in Table 17.

[0145]

[0146] The results of the analysis of the eluate composition are shown in Figure 10. In Figure 10, the horizontal axis shows the volume of liquid passed through the column as "volume of eluent passed through the column per unit volume of cation exchange resin, RV," and the vertical axis shows the concentrations of GSSG (left) and ammonia (right).

[0147] [Comparative Example 9] <Obtaining a solution obtained by concentrating GSSG eluate under reduced pressure in one go> Solution B (472.5 mL) and solution C (702 mL) obtained by the method of Preparation Example 9 were mixed to make one solution, and a 1174.5 mL solution (solution D) containing 44.9 g / L of GSSG and 2.85 g / L of ammonia was obtained. 250 mL of solution D was placed in a 1000 mL round-bottom flask, and reduced-pressure concentration was started under conditions equivalent to an evaporation rate of 100 mL / h (internal flask temperature 36.4-37.1°C, vacuum setting 60 hPa; the same conditions were used in Comparative Example 9 and Example 9). During concentration, solution D equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 250 mL. All of solution D was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the flask reached 250 mL to obtain solution E. Next, to further remove ammonia by evaporation, vacuum concentration was resumed under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, deionized water equivalent to the volume of evaporated liquid was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 250 mL. A total of 400 mL of deionized water was supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the flask reached 250 mL, obtaining solution F.

[0148] [Example 9] <Obtaining a solution obtained by dividing the GSSG eluate and concentrating it under reduced pressure> Solution B (472.5 mL) and solution C (702 mL) were prepared by the method of Preparation Example 9. 250 mL of solution B was placed in a 1000 mL round-bottom flask, and reduced-pressure concentration was started under conditions equivalent to an evaporation rate of 100 mL / h. During concentration, solution B in a volume equivalent to the amount of liquid evaporated was supplied to the round-bottom flask as needed, maintaining the liquid volume in the flask at approximately 250 mL. After the supply of 472.5 mL of solution B was completed, 702 mL of solution C was similarly supplied to the round-bottom flask, and concentration was carried out until the liquid volume in the round-bottom flask was 250 mL to obtain solution G.

[0149] GSSG, ammonia, and the decomposition impurity S-glutathionylcysteinylglycine (hereinafter referred to as GLT-CG) contained in solutions D to G obtained in Comparative Example 9 and Example 9 were measured by HPLC. The results are shown in Table 18.

[0150]

[0151] Solution E in Comparative Example 9 and Solution G in Example 9 are solutions obtained by concentrating the same amount of solution in one step or in stages, respectively. Solution E, obtained by one step, had an ammonia concentration of 12.05 g / L, which was higher than the ammonia concentration of 5.25 g / L in Solution G, obtained by stages. Solution F in Comparative Example 9 is an aqueous solution obtained by continuing the concentration of Solution E while adding 400 mL of deionized water. Despite performing hydroconcentration, the ammonia concentration of Solution F was 11.98 g / L, which is hardly a decrease from the ammonia concentration of Solution E (12.05 g / L). This is thought to be because ammonia hardly volatilizes in the low pH range as ammonium ions, so continuing hydroconcentration on a concentrated solution with a high concentration of GSSG, which is acidic when dissolved, cannot reduce the ammonia concentration. From these results, it has become clear that by dividing the eluent fraction based on the presence or absence of the eluent and concentrating the fraction containing the eluent first, the eluent, which is difficult to remove by one step, can be efficiently removed.

[0152] [Test Example 10: Method for Producing a Solution High in Cytidinediphosphate Choline] [Preparation Example 10] <Obtaining a fraction in which cytidinediphosphate choline has been eluted using acetic acid as an eluent> Cytidinediphosphate choline was produced by fermentation using the method described in Japanese Patent Publication No. 3369236, and the culture solution was inactivated and the pH was adjusted to 3.0 with sulfuric acid to obtain a culture solution containing cytidinediphosphate choline. Subsequently, the culture solution was heated to 50°C, and the bacterial cells were separated by cross-flow filtration (0.1 μm filter) to obtain a filtrate containing cytidinediphosphate choline. Next, the filtrate was H ＋ The filtrate was passed through a XUS40232-01 type (manufactured by Dow Chemical) column at SV = 0.31 and 5-10°C. After the filtrate had passed through the column, deionized water cooled to 5-10°C was passed through at SV = 0.31, and the filtrate was collected when the elution of cytidinediphosphate choline began. 295 kL of an aqueous solution (Solution A) containing cytidinediphosphate choline at a concentration of 5.3 g / L was obtained. Solution A was then OH －The material was passed through a PA412M (manufactured by Mitsubishi Chemical Corporation) column at SV = 1.5 and 3-10°C to adsorb to a load of 157 g / L - resin. Next, 4.0 RV of deionized water was passed through the column at SV = 1.3 and 40°C for washing. Subsequently, a 28.8 g / L aqueous acetic acid solution was passed through the column at SV = 0.90 and 40°C to elute the cytidinediphosphate choline adsorbed on the PA412M. The eluate was collected as a solution (referred to as Solution C) with an endpoint of 2.0 RV to 3.5 RV in Figure 11, representing the first fraction where acetic acid was below the detection limit. The second fraction (referred to as Solution B) with an endpoint of 3.5 RV to 3.9 RV in Figure 11, representing the second fraction where acetic acid was detected, was also collected. During elution, cytidinediphosphate choline was detected using the absorbance that increased in response to the elution as an indicator, and the collection of the first fraction was initiated. During elution, the eluate was sampled at approximately 0.2 RV intervals, and the absorbance and acetic acid concentration in the eluate were analyzed. The concentrations of cytidinediphosphate choline and acetic acid in the obtained solutions A, B, and C are shown in Table 19.

[0153]

[0154] The results of the analysis of the eluate composition are shown in Figure 11. In Figure 11, the horizontal axis shows the volume of liquid passed through the column as "volume of eluent passed through the column per unit volume of ion exchange resin, RV," and the vertical axis shows the absorbance (= cytidinediphosphate choline concentration) (left) and the concentration of acetic acid (right).

[0155] [Comparative Example 10] <Obtaining a solution obtained by membrane-concentrating the eluate of cytidinediphosphate choline in one go> 64.6 mL of solution B obtained by the method of Preparation Example 10 and 185.4 mL of solution C were mixed to make one solution, and a 250 mL solution (referred to as solution D) containing 73.8 g / L of cytidinediphosphate choline and 4.7 g / L of acetic acid was obtained. Solution D was then filtered in a stirred cell type cross-flow membrane filter (effective membrane area 38 cm²). ２Using a Nitto Denko NTR-7250 membrane, membrane concentration was started at room temperature so that the raw water pressure in the cell reached 2 MPa. During membrane concentration, a volume of solution D equivalent to the amount of permeated treated liquid was continuously supplied to the filter to maintain a constant volume of circulating liquid on the raw water side of the filter. The point at which the supply of solution D ended was recorded as solution E. Next, in order to further remove acetic acid, a stirred cell type cross-flow membrane filter (effective membrane area 38 cm²) was used. ２ Using a Nitto Denko NTR-7250 membrane, membrane concentration was restarted so that the raw water pressure in the cell reached 2 MPa. During membrane concentration, an amount of deionized water equivalent to the amount of permeated treated liquid was supplied to the filter as needed, maintaining a constant volume of circulating liquid on the raw water side of the filter. The point at which a total of 50 mL of deionized water had been supplied was recorded as solution F.

[0156] [Example 10] <Obtaining a membrane-concentrated solution by dividing the eluate of cytidinediphosphate choline> 64.6 mL of solution B and 185.4 mL of solution C were prepared by the method of Preparation Example 10. Solution B was placed in a stirring cell type cross-flow membrane filter (effective membrane area 38 cm²). ２ Using a Nitto Denko NTR-7250 membrane, membrane concentration was started at room temperature so that the raw water pressure in the cell reached 2 MPa. During membrane concentration, a volume of solution B equivalent to the amount of treated liquid permeated was continuously supplied to the filter to maintain a constant volume of circulating liquid on the raw water side of the filter. After the supply of solution B was completed, solution C was similarly supplied to a stirred cell-type cross-flow membrane filter (effective membrane area 38 cm²). ２ The solution was supplied to the membrane (Nitto Denko NTR-7250), and the amount of circulating liquid on the raw water side of the filter was maintained at a constant level. The point at which the supply of solution C was completed was recorded as solution G.

[0157] The cytidine diphosphate choline, acetic acid, and decomposition impurities (5'-cytidylic acid (5'-CMP) and uridine diphosphate choline (UDP-choline)) contained in solutions D to G obtained in Comparative Example 10 and Example 10 were measured by HPLC. The results are shown in Table 20.

[0158]

[0159] Solution E in Comparative Example 10 and Solution G in Example 10 are solutions obtained by concentrating the same amount of solution in one step or in stages, respectively. Solution E, obtained by one step, had an acetic acid concentration of 4.3 g / L, which was higher than the acetic acid concentration of Solution G, obtained by stage concentration, which was 2.0 g / L. Solution F in Comparative Example 10 was obtained by continuing membrane concentration while adding deionized water to Solution E until the acetic acid concentration reached the same level as Solution G. The acetic acid concentration was at the same level in Solution F and Solution G, but the time required for concentration and the amount of solvent increased in Solution F. From these results, it became clear that concentration can be performed efficiently by dividing the eluted fraction into those with and without the eluent and concentrating the fraction containing the eluent first. Unlike vacuum concentration, membrane concentration does not require heating, so it is thought that decomposition of cytidinediphosphate choline due to heating does not occur, and this is supported by the data shown in Table 20.

[0160] The analysis of each component in the above experiment was performed according to the following method. <Analysis Example 1: Concentration Measurement of GSSG and GLT-CG> A 0.17 g / L GSSG standard solution (standard) and sample solutions were prepared in buffer so that the GSSG content was approximately 0.1 to 0.3 g / L. Analysis was performed under the following conditions to detect the GSSG peak (retention time: 5.3 mins), and GSSG was quantified using the one-check scale method from the peak area value of the standard. Sample solutions were prepared in buffer so that the GSSG concentrations were 0.5 g / L and 0.01 g / L, and analysis was performed under the following conditions to detect GLT-CG (retention time 8.1 mins). The peak area value of GLT-CG in the sample solution with a GSSG concentration of 0.5 g / L and the peak area value of GSSG in the sample solution with a GSSG concentration of 0.01 g / L were compared to calculate the area ratio of GLT-CG.

[0161] (Analysis conditions for GSSG and GLT-CG) GSSG and GLT-CG were analyzed by HPLC-UV under the following conditions: Column: Hibar LiChrosorb RP-18 (5 μm) 4 mmφ × 250 mm (manufactured by Kanto Chemical Co., Ltd.) Column temperature: 40°C Injection volume: 20 μL Mobile phase: 1.1 g of sodium 1-heptanesulfonate was dissolved in 100 mL of methanol and 0.05 mol / L phosphate buffer to make a 1000 mL solution. (For the 0.05 mol / L phosphate buffer solution, 7.1 g of disodium hydrogen phosphate (dodecahydrate) was dissolved in 900 mL of water, adjusted to pH 3 with phosphoric acid, and then diluted with water to 1000 mL.) Mobile phase flow rate: 0.85 mL / min Detector: UV-Vis absorption detector (SPD-20AV, Shimadzu Corporation) Detection wavelength: 220 nm GSSG detection limit: 0.036 mg / L GLT-CG detection limit: 0.024 mg / L

[0162] (Calculation of GSSG and GLT-CG concentrations) The peak area value of the GSSG standard solution was defined as A, the peak area value of GSSG in a sample solution with a concentration of approximately 0.1 to 0.3 g / L as B, the peak area value of GLT-CG in a sample solution with a concentration of 0.5 g / L as C, and the peak area value of GSSG in a sample solution with a concentration of 0.01 g / L as D. The concentrations of GSSG and GLT-CG in the sample solution were calculated using the following formulas (1a) and (1b).

[0163] GSSG concentration in sample solution (g / L) = 0.17 × (B / A) ... Equation (1a) GLT-CG concentration in sample solution (area ratio) = (C / D) ÷ 50 ... Equation (1b)

[0164] <Analysis Example 2: Measurement of Cytidinediphosphate Choline Concentration> A 0.02 g / L cytidinediphosphate choline standard solution (standard product) and a sample solution were prepared with distilled water to achieve a cytidinediphosphate choline concentration of approximately 0.01 to 0.03 g / L. Analysis was performed under the following conditions, and the cytidinediphosphate choline peak (retention time: 7.5 minutes) was detected. Cytidinediphosphate choline was quantified using the one-point measurement curve method from the peak area value of the standard product.

[0165] (Analysis conditions for cytidinediphosphate choline) Cytidinediphosphate choline was analyzed by HPLC-UV under the following conditions: Column: Shodex Asahipak NH2P-50 φ4.6×250 mm (manufactured by Resonaq Corporation) Column temperature: 40°C Injection volume: 10 μL Mobile phase: 0.03 mol / L potassium dihydrogen phosphate aqueous solution adjusted to pH 3.5 with phosphoric acid Mobile phase flow rate: 0.5 mL / min Detector: UV-Vis absorption detector (SPD-20A, manufactured by Shimadzu Corporation) Detection wavelength: 254 nm Detection limit: 0.00749 mg / L

[0166] (Calculation of cytidinediphosphate choline concentration) The peak area value of the cytidinediphosphate choline standard solution was set to A, and the peak area value of cytidinediphosphate choline in the sample solution was set to B. The concentration of cytidinediphosphate choline in the sample solution was calculated using the following formula (2).

[0167] The concentration of cytidinediphosphate choline in the sample solution (g / L) = 0.02 × (B / A) ... Equation (2)

[0168] <Analysis Example 3: Concentration Measurement of UDP-Choline and 5'-CMP> Sample solutions were prepared with distilled water to achieve concentrations of 1 g / L and 0.005 g / L of 5'-CMP standard solution (prescription) and choline cytidinediphosphate, respectively. Analysis was performed under the following conditions, and peaks for choline cytidinediphosphate (retention time: 22.9 min), UDP-Choline (retention time: 29.5 min), and 5'-CMP (retention time: 15 min) were detected. 5'-CMP was quantified using the one-point scale method from the peak area value of the 5'-CMP standard, and the area ratio of UDP-Choline was calculated by comparing it with the peak area value of the 0.005 g / L choline cytidinediphosphate sample solution.

[0169] (Analysis conditions for UDP-choline and 5'-CMP) UDP-choline and 5'-CMP were analyzed by HPLC-UV under the following conditions: Column: Partisil 10SAX 4.0 × 250 mm, 10 μm, 2 in series (Avantor) Column temperature: 30°C Injection volume: 10 μL Mobile phase: 0.06 mol / L potassium dihydrogen phosphate aqueous solution adjusted to pH 3.5 with phosphoric acid Mobile phase flow rate: 0.45 mL / min Detector: UV-Vis absorption detector (SPD-20AV, Shimadzu Corporation) Detection wavelength: 254 nm UDP-choline detection limit: 0.051 mg / L 5'-CMP detection limit: 0.043 mg / L

[0170] (Calculation of UDP-choline and 5'-CMP concentrations) The peak area value of the 5'-CMP standard solution was set to A, and the peak area value of the 5'-CMP in the sample solution was set to B. The concentration of 5'-CMP in the sample solution was calculated using the following formula (3a). In addition, the peak area value of the 0.005 g / L cytidinediphosphate choline sample solution was set to C, and the peak area value of UDP-choline in the sample solution was set to D. The area ratio of UDP-choline in the sample solution was calculated using the following formula (3b).

[0171] The 5'-CMP concentration in the sample solution (g / L) = 0.011 × (B / A) ... Equation (3a) The UDP-cholinerial ratio in the sample solution = (D / C) ÷ 200 ... Equation (3b)

[0172] <Analysis Example 4: Measurement of the Concentrations of Acetic Acid, Formic Acid, and Propionic Acid> Standard solutions (preparations) of 0.23 g / L acetic acid, 0.13 g / L formic acid, and 0.13 g / L propionic acid were prepared, and sample solutions were prepared with deionized water so that the content of these organic acids was approximately 0.1 to 0.4 g / L. Analysis was performed under the following conditions, and each peak (acetic acid (retention time: 17 min), formic acid (retention time: 55 min), propionic acid (retention time: 27.8 min)) was detected. These organic acids were quantified using the one-point scale method from the peak area values ​​of the preparations.

[0173] (Analytical conditions for acetic acid, formic acid, and propionic acid) These organic acids were quantified by HPLC-UV under the following conditions: Column: Shodex Asahipak NH2P-50 φ4.6 × 250 mm (manufactured by Resonaq Corporation) Column temperature: 50°C Injection volume: 10 μL Mobile phase: 0.03 mol / L potassium dihydrogen phosphate aqueous solution adjusted to pH 3.5 with phosphoric acid Mobile phase flow rate: 0.40 mL / min Detector: UV-Vis absorption detector (SPD-20A, manufactured by Shimadzu Corporation) Detection wavelength: 216 nm Detection limit for acetic acid: 0.947 mg / L Detection limit for formic acid: 1.49 mg / L Detection limit for propionic acid: 1.15 mg / L

[0174] (Calculation of acetic acid, formic acid, and propionic acid content) The peak area value of the standard solution of the organic acid was set to A, and the peak area value of the sample solution was set to B. The concentrations of the organic acids in the sample solution were calculated using the following formulas (4a), (4b), and (4c), respectively.

[0175] Acetic acid content in sample solution (g / L) = 0.23 × (B / A) ... Equation (4a) Formic acid content in sample solution (g / L) = 0.13 × (B / A) ... Equation (4b) Propionic acid content in sample solution (g / L) = 0.13 × (B / A) ... Equation (4c)

[0176] <Analysis Example 5: Measurement of Pravastatin Concentration> A pravastatin standard solution (standard sample) with a concentration of 0.11 g / L was prepared by dissolving pravastatin (purity: 99.8%) manufactured by Kyowa Hakko Bio Co., Ltd. in a water / methanol mixture (11:9). A sample solution was prepared with a water / methanol mixture (11:9) to a pravastatin concentration of approximately 0.1 g / L, and analysis was performed under the following conditions. The pravastatin peak (retention time: 27 minutes) was detected, and the amount of pravastatin was quantified using the one-point measurement curve method from the peak area value of the standard sample.

[0177] (Analysis conditions for pravastatin) Pravastatin was analyzed by HPLC-UV under the following conditions: Column: Inertsil ODS-2 (5 μm) 4.6 mmΦ × 150 mm (GL Sciences) Column temperature: 35°C Injection volume: 10 μL Mobile phase: Solution of water: methanol: triethylamine: acetic acid mixed in a ratio of 550:450:1:1 Mobile phase flow rate: 1.2 mL / min Detector: UV-Vis absorption detector (SPD-20A, Shimadzu Corporation) Detection wavelength: 238 nm Detection limit: 0.057 mg / L

[0178] (Calculation of pravastatin concentration) The peak area value of the pravastatin standard solution was denoted as A, and the peak area value of pravastatin in the sample solution was denoted as B. The concentration of pravastatin in the sample solution was calculated using the following formula (5a).

[0179] Pravastatin concentration in sample solution (g / L) = 0.11 × (B / A) ... Equation (5a)

[0180] <Analysis Example 6: Concentration Measurement of RT56.4 (Pravastatin Analogue)> A sample solution was prepared with a water / methanol mixture (11:9) to achieve a pravastatin concentration of approximately 1.0 g / L. Analysis was performed under the following conditions, and the peaks for pravastatin (retention time: 27 minutes) and RT56.4 (retention time: 56.4 minutes) were detected. The area ratio of the RT56.4 peak was calculated by comparing it with the peak area value of the 1.0 g / L pravastatin sample solution.

[0181] (Analysis conditions for pravastatin analogues) Pravastatin analogues were analyzed by HPLC-UV under the following conditions: Column: Inertsil ODS-2 (5 μm) 4.6 mmΦ × 150 mm (GL Sciences) Column temperature: 35°C Injection volume: 10 μL Mobile phase: Solution of water, methanol, triethylamine, and acetic acid mixed in a ratio of 550:450:1:1 Mobile phase flow rate: 1.2 mL / min Detector: Ultraviolet-Visible absorption detector (SPD-20A, Shimadzu Corporation) Detection wavelength: 238 nm

[0182] (Calculation of RT56.4 (Pravastatin analogue) concentration) The peak area value of the 0.005 g / L pravastatin sample solution was denoted as A, and the peak area value of RT56.4 in the 1.0 g / L sample solution was denoted as B. The area ratio of RT56.4 in the sample solutions was calculated using the following formula (6a).

[0183] RT56.4 area ratio in sample solution = B / A ... Equation (6a)

[0184] <Analysis Example 7: Concentration Measurement of Flavin Adenine Dinucleotide> A standard solution of flavin adenine dinucleotide (standard sample) with a concentration of approximately 0.2 g / L was prepared by dissolving flavin adenine dinucleotide (purity: 96.2%) manufactured by Kyowa Hakko Bio Co., Ltd. in purified water. A sample solution was prepared with purified water to achieve a concentration of approximately 0.2 g / L of flavin adenine dinucleotide, and the peak of flavin adenine dinucleotide (retention time: 10 minutes) was detected. The amount of flavin adenine dinucleotide was quantified using the one-check curve method from the peak area value of the standard sample.

[0185] (Analysis conditions for flavin adenine dinucleotide) Flavin adenine dinucleotide was analyzed by HPLC-UV under the following conditions: Column: Inertsil ODS-2 (5 μm) 4.6 mmΦ × 150 mm (GL Sciences) Column temperature: 35°C Injection volume: 5 μL Mobile phase: Potassium dihydrogen phosphate solution prepared by dissolving 1 g of potassium dihydrogen phosphate in 500 mL of purified water, mixed with methanol in a 4:1 ratio Mobile phase flow rate: Adjusted to approximately 0.8-0.9 mL / min so that the retention time of flavin adenine dinucleotide is approximately 10 minutes Detector: Ultraviolet-Visible absorption detector (SPD-20AV, Shimadzu Corporation) Detection wavelength: 260 nm Detection limit: 0.026 mg / L

[0186] (Calculation of flavin adenine dinucleotide concentration) The peak area value of the flavin adenine dinucleotide standard solution was denoted as A, and the peak area value of the flavin adenine dinucleotide in the sample solution was denoted as B. The concentration of flavin adenine dinucleotide in the sample solution was calculated using the following formula (7a).

[0187] The concentration of flavin adenine dinucleotide in the sample solution (g / L) = 0.2 × (B / A) ... Equation (7a)

[0188] <Analysis Example 8: Concentration Measurement of Flavin Adenine Dinucleotide> Flavin adenine dinucleotide (purity: 96.2%) manufactured by Kyowa Hakko Bio Co., Ltd. was adjusted to the concentrations shown in Table 21, and the flavin adenine dinucleotide solution was analyzed using the method shown in Analysis Example 7 to quantify the flavin adenine dinucleotide. The flavin adenine dinucleotide solution was also measured for absorbance at a wavelength of 430 nm, and a calibration curve was determined from the obtained concentration and absorbance data. The flavin adenine dinucleotide solution obtained during the example was adjusted with purified water so that the absorbance at a wavelength of 430 nm was within the calibration curve range, and the absorbance of the sample solution was measured under the following conditions, and the flavin adenine dinucleotide was quantified from the calibration curve.

[0189]

[0190] When the absorbance of the sample solution is A, the resulting calibration curve is shown in the following equation (8a).

[0191] The concentration of flavin adenine dinucleotide in the sample solution (g / L) = 0.0887 × A - 0.00011... Equation (8a)

[0192] (Analysis conditions for absorbance) Measuring instrument: Spectrophotometer (U3900H, Hitachi, Ltd.) Wavelength: 430 nm

[0193] <Analysis Example 9: Concentration Measurement of Flavin Adenine Dinucleotide Related Substances> A sample solution prepared with purified water to a flavin adenine dinucleotide concentration of approximately 0.5 g / L was analyzed under the following conditions. Peaks for flavin adenine dinucleotide (retention time: 10 minutes), RT14 (retention time: 14 minutes), and RT15 (retention time: 15 minutes) were detected, and the area ratios of the RT14 and RT15 peaks were calculated by comparing them with the peak area values ​​of the 0.5 g / L flavin adenine dinucleotide sample solution.

[0194] (Analysis conditions for RT14 and RT15 (flavin adenine dinucleotide analogs)) Column: Inertsil ODS-2 (5 μm) 4.6 mmΦ × 150 mm (GL Sciences) Column temperature: 35°C Injection volume: 5 μL Mobile phase: Potassium dihydrogen phosphate solution prepared by dissolving 1 g of potassium dihydrogen phosphate in 500 mL of purified water, mixed with methanol in a 4:1 ratio Mobile phase flow rate: Adjusted to approximately 0.8-0.9 mL / min so that the retention time of flavin adenine dinucleotide is approximately 10 minutes Detector: Ultraviolet-Visible absorption detector (SPD-20AV, Shimadzu Corporation) Detection wavelength: 216 nm

[0195] (Calculation of concentrations of RT14 and RT15 (flavin adenine dinucleotide analogs)) The peak area value of the 0.5 g / L flavin adenine dinucleotide sample solution was defined as A, the peak area value of the 0.5 g / L sample solution after a retention time of 14 minutes as B, and the peak area value of the 0.5 g / L sample solution after a retention time of 15 minutes as C. The area ratio of RT14 and RT15 in the sample solution was calculated using the following formulas (9a, 9b).

[0196] Area ratio of RT14 in the sample solution = B / A ... Equation (9a) Area ratio of RT15 in the sample solution = C / A ... Equation (9b)

[0197] <Analysis Example 10: Measurement of Ammonia Concentration> A standard ammonia solution (reference sample) with a concentration of 0.041 g / L was prepared by dissolving ammonium chloride manufactured by Kanto Chemical Co., Ltd. in 0.02 mol / L hydrochloric acid. Sample solutions were prepared with 0.02 mol / L hydrochloric acid so that the ammonia concentration was approximately 0.02 to 0.06 g / L, and analysis was performed under the following conditions. The ammonia peak (retention time: 11 minutes) was detected, and the ammonia was quantified using the one-point measurement curve method from the peak area value of the reference sample.

[0198] (Analysis conditions for ammonia) Ammonia was analyzed by HPLC-UV under the following conditions. Column: YMC-Pack ODS-AQ 6mmΦ×150mm (manufactured by YMC) Column temperature: 40℃ Sample injection volume: 20μL Mobile phase: 14.7g of sodium citrate dihydrate and 7.1g of anhydrous sodium sulfate were dissolved in purified water to prepare 4000mL. After adjusting the pH to 3.8 with sulfuric acid, 15g of sodium lauryl sulfate and 600mL of 1-propanol were added and mixed, and then the volume was adjusted to 5000mL with purified water. Reaction solution: 37g of boric acid, 22g of sodium hydroxide, 8.2mL of Briji-35, 1.2g of α-phthalaldehyde, and 9.2g of acetylcysteine ​​were dissolved in purified water to prepare 2L. Flow rate: Mobile phase 1.2 mL / min, reaction solution 0.4 mL / min; Detector: Spectrofluorescence detector (RF-20A XS, Shimadzu Corporation); Wavelength: Excitation wavelength 355 nm, fluorescence wavelength 455 nm; Detection limit: 0.08 mg / L

[0199] (Calculation of ammonia concentration) The peak area value of the ammonia standard solution was denoted as A, and the peak area value of the ammonia in the sample solution was denoted as B. The ammonia concentration of the sample solution was calculated using the following formula (10a).

[0200] Ammonia concentration in sample solution (g / L) = 0.041 × (B / A) ... Equation (10a)

[0201] <Analysis Example 11: Concentration Measurement of Acetone, 2-Butanol, and Ethyl Acetate> Acetone standard solution (standard) prepared by adjusting acetone (purity: 99.5%) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. to 0.052 g / L, 2-butanol standard solution (standard) prepared by adjusting 2-butanol (purity: 99% or higher) manufactured by Kanto Chemical Co., Ltd. to 0.010 g / L, and ethyl acetate standard solution (standard) prepared by adjusting ethyl acetate (purity: 99.8%) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. to 0.12 g / L. Sample solutions were prepared with purified water so that the content of these organic solvents was approximately 0.1 to 120 mg / L for acetone, approximately 0.20 to 100 mg / L for 2-butanol, and approximately 0.09 to 60 mg / L for ethyl acetate. Analysis was performed under the following conditions, and each peak (acetone (retention time: 0.8 min), 2-butanol (retention time: 3.0 min), ethyl acetate (retention time: 1.2 min)) was detected. These organic solvents were quantified using the one-check curve method from the peak area values ​​of the standard.

[0202] (Analytical conditions for acetone, 2-butanol, and ethyl acetate) Acetone, 2-butanol, and ethyl acetate were analyzed by gas chromatography under the following conditions: Column: DB-WAX (Serial No. USR573331H) Column temperature: 35°C Equilibrium time: 3.0 min Sample injection volume: 1 μL Injection mode: Split Split ratio: -1.0 Carrier gas: Helium Total carrier gas flow rate: 50.0 mL / min Carrier gas column flow rate: 27.4 mL / min Pressure: 100.0 kPa Detector: DFID Detector temperature: 230.0°C Detection limit (acetone): 0.10 mg / L Detection limit (2-butanol): 0.20 mg / L Detection limit (ethyl acetate): 0.095 mg / L

[0203] (Calculation of Acetone, 2-Butanol, and Ethyl Acetate Content) The peak area value of the standard solution of the organic solvent was denoted as A, and the peak area value of the sample solution as B. The concentrations of the organic solvents in the sample solution were calculated using the following formulas (11a), (11b), and (11c), respectively.

[0204] Acetone concentration in sample solution (g / L) = 0.052 × (B / A) ... Equation (11a) 2-butanol concentration in sample solution (g / L) = 0.010 × (B / A) ... Equation (11b) Ethyl acetate concentration in sample solution (g / L) = 0.12 × (B / A) ... Equation (11c)

Claims

(1) A step of adsorbing the target organic compound onto a stationary carrier, (2) A step of passing an eluent through a stationary carrier on which the target organic compound has been adsorbed, (3) The eluted fraction containing the target organic compound is (a) A fraction containing the eluent at a first concentration, and (b) Fractions in which the concentration of the eluent is less than the first concentration. The process of dividing into (4) A step of concentrating the fraction in (a) above to obtain a concentrate, (5) A step of adding the fraction (b) to the concentrate and concentrating it. A method for producing a target organic compound, including [the specified compound]. 　 The method according to claim 1, wherein the concentration in steps (4) and (5) includes vacuum concentration. 　 The method according to claim 2, wherein the eluent comprises at least one of a volatile acid, a volatile base, and a volatile organic solvent. 　 The method according to claim 2, wherein the target organic compound is a compound having a solubility of 10 to 800 g / L at 20 ± 5°C, and the solvent of the eluent is water. 　 The method according to claim 4, wherein the solubility is solubility in water. 　 The method according to claim 2, wherein the temperature at which the concentration is performed under reduced pressure in steps (4) and (5) is 80°C or lower. 　 The method according to claim 2, wherein the target organic compound is a peptide, a nucleotide or derivative thereof, a fatty acid, a statin, a vitamin, a sugar, or a terpenoid. 　 The method according to claim 2, wherein the target organic compound is a cytidine diphosphate choline compound, oxidized glutathione, flavin adenine dinucleotide, or pravastatin. 　 The method according to claim 2, wherein the immobilizing carrier is an ion exchange resin, a synthetic adsorption resin, activated carbon, or silica gel. 　 The method according to claim 9, wherein the ion exchange resin is a strongly basic anion exchange resin, a weakly basic anion exchange resin, a strongly acidic cation exchange resin, or a weakly acidic cation exchange resin. 　 The method according to claim 3, wherein the volatile acid is an inorganic acid or an organic acid having 1 to 3 carbon atoms and a carboxyl group. 　 The method according to claim 11, wherein the volatile acid is hydrochloric acid, nitric acid, formic acid, acetic acid, or propionic acid. 　 The method according to claim 11, wherein the volatile acid is formic acid, acetic acid, or propionic acid. 　 The method according to claim 1, wherein the concentration in steps (4) and (5) includes membrane concentration. 　 The method according to claim 14, wherein the eluent comprises at least one of an acid, a base, and an organic solvent. 　 The method according to claim 14, wherein the target organic compound is a compound having a solubility of 10 to 800 g / L at 20 ± 5°C, and the solvent of the eluent is water. 　 The method according to claim 16, wherein the solubility is solubility in water. 　 The method according to claim 14, wherein the temperature at which the membrane is concentrated in steps (4) and (5) is 80°C or lower. 　 The method according to claim 14, wherein the target organic compound is a peptide, a nucleotide or derivative thereof, a fatty acid, a statin, a vitamin, a sugar, or a terpenoid. 　 The method according to claim 14, wherein the target organic compound is a cytidine diphosphate choline compound, oxidized glutathione, flavin adenine dinucleotide, or pravastatin. 　 The method according to claim 14, wherein the immobilizing carrier is an ion exchange resin, a synthetic adsorption resin, activated carbon, or silica gel. 　 The method according to claim 21, wherein the ion exchange resin is a strongly basic anion exchange resin, a weakly basic anion exchange resin, a strongly acidic cation exchange resin, or a weakly acidic cation exchange resin. 　 The method according to claim 15, wherein the acid is an inorganic acid or an organic acid having 1 to 3 carbon atoms and a carboxyl group. 　 The method according to claim 23, wherein the acid is hydrochloric acid, nitric acid, formic acid, acetic acid, or propionic acid. 　 The method according to claim 23, wherein the acid is formic acid, acetic acid, or propionic acid. 　 The method according to claim 1, wherein the concentration of the eluent in the fraction (a) separated in step (3) is 100 g / L or less, and the fraction contains the target organic compound. 　 The method according to claim 1, wherein the concentration of the eluent in the fraction (a) separated in step (3) is 100 g / L or less, and the fraction contains 10 g / L or more of the target organic compound. 　 The method according to claim 1, wherein the ratio of the mass of the eluent to the mass of the target organic compound in the fraction (a) separated in step (3) (eluent / target organic compound) is 0.06 or more. 　 The method according to claim 1, wherein the ratio of the mass of the eluent to the mass of the target organic compound in the fraction (b) separated in step (3) is less than 0.

06. The method according to claim 1, wherein the ratio of the mass of the eluent to the mass of the target organic compound in the solution obtained after concentration in step (5) is less than 0.06 (eluent / target organic compound). 　 The method according to claim 1, wherein the concentration of the eluent in the (b) fraction separated in step (3) is 1 / 2 or less of the concentration in the (a) fraction.

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