CN101906126B - Method for separating and purifying citicoline by hydrophobic chromatography - Google Patents
Method for separating and purifying citicoline by hydrophobic chromatography Download PDFInfo
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- CN101906126B CN101906126B CN2010101078221A CN201010107822A CN101906126B CN 101906126 B CN101906126 B CN 101906126B CN 2010101078221 A CN2010101078221 A CN 2010101078221A CN 201010107822 A CN201010107822 A CN 201010107822A CN 101906126 B CN101906126 B CN 101906126B
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- cytidine diphosphate
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- 238000000034 method Methods 0.000 title claims abstract description 54
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 title abstract description 55
- 230000002209 hydrophobic effect Effects 0.000 title abstract description 19
- 238000004587 chromatography analysis Methods 0.000 title abstract description 14
- 229960001284 citicoline Drugs 0.000 title abstract description 8
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 238000000926 separation method Methods 0.000 claims abstract description 21
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000011347 resin Substances 0.000 claims abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 14
- 238000002425 crystallisation Methods 0.000 claims abstract description 13
- 230000008025 crystallization Effects 0.000 claims abstract description 11
- 238000001728 nano-filtration Methods 0.000 claims abstract description 11
- 238000010612 desalination reaction Methods 0.000 claims abstract description 9
- 239000012528 membrane Substances 0.000 claims description 41
- ZWIADYZPOWUWEW-XVFCMESISA-N CDP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-XVFCMESISA-N 0.000 claims description 38
- 238000010521 absorption reaction Methods 0.000 claims description 36
- 239000012530 fluid Substances 0.000 claims description 29
- 238000000746 purification Methods 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 10
- 239000010452 phosphate Substances 0.000 claims description 10
- 238000009987 spinning Methods 0.000 claims description 6
- DJHGAFSJWGLOIV-UHFFFAOYSA-N Arsenic acid Chemical compound O[As](O)(O)=O DJHGAFSJWGLOIV-UHFFFAOYSA-N 0.000 claims description 4
- 229940000488 arsenic acid Drugs 0.000 claims description 4
- 238000006068 polycondensation reaction Methods 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 150000003863 ammonium salts Chemical class 0.000 claims description 3
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 3
- 239000012266 salt solution Substances 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 2
- -1 phenolic aldehyde Chemical class 0.000 claims description 2
- 125000000024 selenono group Chemical group [H]O[Se](*)(=O)=O 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 239000012539 chromatography resin Substances 0.000 abstract description 5
- 238000005119 centrifugation Methods 0.000 abstract description 2
- 239000003480 eluent Substances 0.000 abstract 1
- 230000002779 inactivation Effects 0.000 abstract 1
- 230000008929 regeneration Effects 0.000 abstract 1
- 238000011069 regeneration method Methods 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 22
- 230000003197 catalytic effect Effects 0.000 description 13
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 10
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000004793 Polystyrene Substances 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 239000012535 impurity Substances 0.000 description 8
- 238000012545 processing Methods 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 7
- 239000013078 crystal Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 239000011591 potassium Substances 0.000 description 6
- 229910052700 potassium Inorganic materials 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 5
- 235000019270 ammonium chloride Nutrition 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 229960001231 choline Drugs 0.000 description 5
- 239000003456 ion exchange resin Substances 0.000 description 5
- 229920003303 ion-exchange polymer Polymers 0.000 description 5
- 235000010333 potassium nitrate Nutrition 0.000 description 5
- 239000004323 potassium nitrate Substances 0.000 description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 description 5
- 235000011152 sodium sulphate Nutrition 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000010307 cell transformation Effects 0.000 description 3
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 3
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 description 3
- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 3
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 3
- 229930183912 Cytidylic acid Natural products 0.000 description 2
- 208000006558 Dental Calculus Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920005990 polystyrene resin Polymers 0.000 description 2
- 229920003987 resole Polymers 0.000 description 2
- 235000010344 sodium nitrate Nutrition 0.000 description 2
- 239000004317 sodium nitrate Substances 0.000 description 2
- 229940001516 sodium nitrate Drugs 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102000014461 Ataxins Human genes 0.000 description 1
- 108010078286 Ataxins Proteins 0.000 description 1
- 241000345998 Calamus manan Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 206010008025 Cerebellar ataxia Diseases 0.000 description 1
- 235000019743 Choline chloride Nutrition 0.000 description 1
- PCDQPRRSZKQHHS-CCXZUQQUSA-N Cytarabine Triphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-CCXZUQQUSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- IATUGZHPADVZPS-UHFFFAOYSA-N N1=CN=CC=C1.P(=O)(OCCC(=C)C)(O)O Chemical class N1=CN=CC=C1.P(=O)(OCCC(=C)C)(O)O IATUGZHPADVZPS-UHFFFAOYSA-N 0.000 description 1
- ALKWJXWZUTYERW-UHFFFAOYSA-L O.O.O.O.[Mn](=O)(Cl)Cl Chemical class O.O.O.O.[Mn](=O)(Cl)Cl ALKWJXWZUTYERW-UHFFFAOYSA-L 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 description 1
- 206010043118 Tardive Dyskinesia Diseases 0.000 description 1
- 208000009205 Tinnitus Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 201000004562 autosomal dominant cerebellar ataxia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 1
- 229960003178 choline chloride Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 208000024732 dysthymic disease Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 238000010438 heat treatment Methods 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- WCVHUIPWSPEOIG-UHFFFAOYSA-N n,n-dimethylheptadecan-1-amine Chemical compound CCCCCCCCCCCCCCCCCN(C)C WCVHUIPWSPEOIG-UHFFFAOYSA-N 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- 239000002718 pyrimidine nucleoside Substances 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
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- 235000011912 vitamin B7 Nutrition 0.000 description 1
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- 239000011701 zinc Substances 0.000 description 1
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Landscapes
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for separating and purifying citicoline by hydrophobic chromatography, which comprises the following steps: (1) carrying out enzyme inactivation, centrifugation and ultrafiltration pretreatment on the citicoline conversion solution; (2) adjusting the pH value of the pretreated citicoline conversion solution to 2.0-14.0, adding inorganic salt to prepare upper column liquid containing 0.01-5 mol/L of inorganic salt and 0.1-40 g/L of citicoline, adsorbing by hydrophobic chromatography resin, and eluting by pure water with the pH value of 2.0-14.0; (3) and (4) carrying out nanofiltration, desalination, concentration and crystallization on the eluent to obtain the citicoline. The method has simple separation process, low separation cost and easy crystallization of the product, and can obtain the CDP-choline product with high purity and high yield by regeneration treatment after the resin elution is finished.
Description
Technical field
The invention belongs to the bioseparation technology field, be specifically related to a kind of method of separating purified cytidine diphosphate choline by hydrophobic chromatography.
Background technology
Cytidine diphosphate (CDP-choline) is a nucleoside derivates (I), is the precursor substance of phosphatidylcholine, can increase the sympathin and the dopamine level of cns.Be widely used in the various cerebrovascular diseases of treatment clinically, senile dementia, dysthymia disorders, also to Parkinsonism, neural heariing loss and tinnitus, tardive dyskinesia, cerebellum and spinocerebellar ataxia have certain curative effect.
In the whole-cell catalytic conversion fluid of CDP-choline; There are similar compound of some physicochemical property and mesostate such as cytidine monophosphate, cytidine diphosphate(CDP) and cytidine triphosphate(CTP) etc.; And the substrate that has not been transformed such as inorganic phosphate and glucose etc.; The impurity such as the compounds such as albumen and nucleic acid that also have aqtocytolysis to produce in addition, separating difficulty is very big.Separation and purification about CDP-choline; Report is less both at home and abroad; Traditional separating technology ubiquity that technology is tediously long, the output yield is low, the shortcoming that separation costs is high, and ion exchange process adopts the type of elution of alcohol-water; Inflammable, explosive characteristic is arranged, bring potential safety hazard to production.The former good filial piety of rattan etc. (the special public clear 62-16497 of Japan) are to chemical method synthetic CDP-choline solution; Use strong-acid ion exchange resin RHPK-25.4～60 (H type) posts and separation and purification of weak-base ion-exchange resin RWA-30 (OH type) post two procedures; Can reach 98% through liquid chromatographic detection CDP-choline purity, yield is not reported.Zhang Jian etc. (CN101130797A) propose carbon post on the conversion fluid with pure water rinsing carbon post, is eluted CDP-choline with the ethanol alkaline solution again, and collect; Again the elutriant vaporization is concentrated, the macroporous ion exchange resin post is gone up in the liquid concentrator dilution, finish the after washing pillar; Wash-out and elutriant concentrated again; Then ultra-fine filter removal of impurities, decolouring are delivered in the CDP-choline solution heating of collecting, added ethanol and stir, and place freezer and spend the night; Again crystal solution is carried out spinning, vacuum-drying, oven dry back bagging and weighing hermetically drying is preserved.This technical process is loaded down with trivial details, and the product loss rate is high.(WO2007/018259A1.) such as village Tian Yingcheng with strongly-acid (H type) ion exchange column and active carbon column separation and purification cell transformation liquid, product yield is merely 80%.It is absorption carrier that Xu Renhua etc. (CN1944661A.) improve with the activated carbon on the basis of the above, with Cl
-Type ion exchange resin is carrier of separating separation and purification cell transformation liquid, and product yield slightly improves.
Though these methods have all realized the separation of CDP-choline, all to use twice resin or activated carbon column, cause production technique tediously long, the product loss rate is high, becomes the bottleneck of its development.Qiu Weiran etc. (CN101096380A.) have proposed employing ion exchange resin combination chromatography first; Separation of C DP-choline; And reclaiming the intact cytidylic acid of unreacted simultaneously, this method can reclaim a spot of unreacted cytidylic acid in the cell transformation liquid, but the yield of purge process CDP-choline is merely 80.9%; Failing equally, it is low to solve separation efficiency, the problem that product yield is low.
The CDP-choline molecule is made up of choline and isopentenyl monophosphate pyrimidine nucleotide; Measure through the X-ray diffraction, whole molecular formula height is rolled, and a plurality of molecule aggregations together; With 5 '-isopentenyl monophosphate pyrimidine nucleotide is core; Outside phosphoric acid and choline partly are exposed to,, present certain hydrophobicity with loose the combining of water molecules on every side.Utilize this characteristic, the present invention combines CDP-choline and hydrophobic medium, and 5 '-isopentenyl monophosphate pyrimidine nucleoside acid groups can drive phosphoric acid and choline partly has the trend that water gets into nonpolar phase of leaving, i.e. hydrophobicity absorption.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of hydrophobic chromatography separation and purification cytidine diphosphate; After, ultrafiltration etc. centrifugal through the CDP-choline conversion fluid is carried out handled; Carry out upper prop absorption, elution process again, can obtain high yield, highly purified CDP-choline solution.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
Hydrophobic chromatography separation and purification cytidine diphosphate comprises the steps:
(1) the cytidine diphosphate conversion fluid is through enzyme-deactivating, centrifugal and ultrafiltration pretreatment;
(2) with behind pretreated cytidine diphosphate conversion fluid accent pH2.0～14.0; Add inorganic salt and be mixed with the upper prop liquid that contains 0.01～5mol/L inorganic salt and 0.1～40g/L cytidine diphosphate; Through the hydrophobic chromatography resin absorption, again through the pure water wash-out of pH2.0～14.0;
(3) elutriant is obtained cytidine diphosphate through nanofiltration desalination and concentration, crystallization.
In the step (1), regulate cytidine diphosphate conversion fluid pH value to 2.0～3.0 and make enzyme-deactivating, through 6000～12000rpm spinning, 10～60min, getting supernatant is the ultra-filtration membrane ultrafiltration of 3000～8000Dolton through molecular weight cut-off again.Preferably, regulate cytidine diphosphate conversion fluid pH value to 2.0 and make enzyme-deactivating, through 8000～10000rpm spinning, 15～20min, getting supernatant is the ultra-filtration membrane ultrafiltration of 5000～6000Dolton through molecular weight cut-off again.Most preferably, regulate cytidine diphosphate conversion fluid pH value to 2.0 and make enzyme-deactivating, again through 8000rpm spinning 20min, getting supernatant is the ultra-filtration membrane ultrafiltration of 5000Dolton through molecular weight cut-off.The preferred hollow fiber ultrafiltration membrane of above-mentioned ultra-filtration membrane, mould material are PS membrane, and ultrafiltration pressure generally is controlled at 0.1～1.5MPa.Can thalline insolubles in the CDP-choline conversion fluid and macro-molecular protein etc. be removed through above-mentioned pre-treatment.
In the step (2), described inorganic salt are inorganic sodium or inorganic potassium salt or inorganic ammonium salt, for example can make sodium-chlor, SODIUMNITRATE, sodium sulfate, ammonium chloride, ammonium sulfate, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, Repone K, saltpetre or vitriolate of tartar etc.
In the step (2); Described hydrophobic chromatography resin is for being the inertia skeleton with PS, ROHM or polycondensation phenolic aldehyde; With sulfonic group, phosphate, arsenic acid base or selenono resin, for example can be CAD-40, X-5, H103, ADS-17, AB-8, NKA-II or NKA-9 as functional group.Described hydrophobic chromatography resin needs through using behind 0.01～5mol/L inorganic salt solution immersion, 1～48h, and preferred 1～2mol/L inorganic salt solution soaks 2h.Described inorganic salt are inorganic sodium or inorganic potassium salt or inorganic ammonium salt, for example can make sodium-chlor, SODIUMNITRATE, sodium sulfate, ammonium chloride, ammonium sulfate, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, Repone K, saltpetre or vitriolate of tartar etc.The inorganic salt that soak resin can be identical or different with the inorganic salt that add in the upper prop liquid, preferably uses identical inorganic salt.
In the step (2); Preferred scheme is after pretreated cytidine diphosphate conversion fluid is transferred pH2.0～5.0; Add inorganic salt and be mixed with the upper prop liquid that contains 1～3mol/L inorganic salt and 5～15g/L cytidine diphosphate, through the hydrophobic chromatography resin absorption, again through the pure water wash-out of pH7.0～10.0.
In the step (2), the absorption flow velocity is 10～100mL/min, and elution flow rate is 5～50mL/min.Preferably, the absorption flow velocity is 30～50mL/min, and elution flow rate is 10～20mL/min.
In the step (2), the temperature of absorption and wash-out is 10～40 ℃.
In the step (3), described nanofiltration, its nf membrane molecular weight cut-off is 100～300Dolton, preferred molecular weight cut-off is 150Dolton.
The cytidine diphosphate conversion fluid its preparation method of mentioning among the present invention is the whole-cell catalytic synthesis method, specifically referring to " a kind of preparation method of cytidine diphosphate " (200810019854.9).
Crystallization method is not carried out any restriction among the present invention, the conventional method that can the cytidine diphosphate crystallization be separated out can, preferred method is referring to " saltouing-the dilution crystallization method of a kind of cytidine diphosphate " (200810019855.3).
Beneficial effect: the present invention combines membrane separation technique with the hydrophobic chromatography technology, and the CDP-choline conversion fluid is centrifugal, uf processing has been reduced sepn process greatly after one non-polar resin separates, the brief loss of each process step; The present invention adopts the pure water type of elution, has avoided the potential safety hazard that the alcohol-water formula is brought in the traditional technology.This work simplification technical process, lowered the product loss rate, improved separation efficiency and product yield.Above-mentioned separating technology is compared with traditional separating technology, has simple, the with low cost advantage of technical process.The purity of the CDP-choline solution that obtains through above-mentioned resin absorption sepn process surpasses 98%, and yield surpasses 90%.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The preparation of CDP-choline conversion fluid is operated as follows in following examples:
Yeast culture base (g/L): glucose 40, urea 2.0, potassium primary phosphate 1.5, bitter salt 0.5, Zinc vitriol 4.0 * 10
-3, Presfersul 3.0 * 10
-3, four hydration Manganous chloride tetrahydrates 0.3 * 10
-3, Calcium Chloride Powder Anhydrous 1.0 * 10
-3, vitamin H 0.05 * 10
-3Yeast saccharomyces cerevisiae inoculum size 10% was cultivated centrifugal 4000rpm, 20 minutes 24 hours in 30 ℃ of following 120rpm shaking tables.Get yeast slurry ,-7 ℃ of preservations are subsequent use.
At capacity is in the reactive tank of 15L; The reaction solution 10L that 2800 gram yeast saccharomyces cerevisiae mud, hexadecyl trimethylamine brometo de amonio 10 grams and the water that modulation is cultivated by 60mM choline chloride 60,0.20M SODIUM PHOSPHATE, MONOBASIC, 30mM CMP, 0.3M glucose, 50mM sal epsom, 1mM Repone K, 2mM halfcystine, aforesaid method is formed; Transfer pH to 6.8 with sodium hydroxide, stirring at low speed reaction 10h under 37 ℃ of conditions is after reaction finishes; Centrifugation is carried out the cytidine diphosphate quantitative analysis to supernatant.
Embodiment 1:
According to above-mentioned whole-cell catalytic compound method, preparation CDP-choline conversion fluid 15L, wherein the concentration of CDP-choline is 9.47g/L.
Treatment process is following:
(1) the whole-cell catalytic conversion fluid is regulated pH to 2.0 enzyme that goes out with hydrochloric acid and live, through 8000rpm, the centrifugal back removal of 20min solid impurity.Get centrifuged supernatant through uf processing, collect ultrafiltration and see through liquid; Described ultra-filtration membrane is a PS membrane, and the ultra-filtration membrane molecular weight cut-off is 5000Dolton, and the ultra-filtration membrane WP is 1MPa.
(2) ultrafiltration sees through in the liquid and adds sodium-chlor, is mixed with the upper prop liquid that contains 2.5mol/L sodium-chlor, 5g/L CDP-choline, transfers pH to 4.0 with hydrochloric acid.The sulfonic group polystyrene resin soaks 1h through the sodium-chlor of 2.0mol/L in advance, refills and fills into post (aspect ratio is 10: 1, and following examples aspect ratio is identical).Upper prop liquid get into to be filled the hydrophobic chromatography post absorption of sulfonic group polystyrene resin, and absorption flow velocity is 80mL/min, when the concentration of CDP-choline in the absorption effluent reach sample introduction concentration 5% the time, think to stop the arrival breakthrough point adsorbing; Pure water with pH8.0 carries out wash-out, and elution flow rate is 40mL/min.Absorption and eluting temperature are 10 ℃, and it is 89.6% that wash-out finishes through the yield that records whole resin isolation process CDP-choline, and purity is 97.3%.
(3) elutriant is handled desalination through nanofiltration and is concentrated, and the nf membrane molecular weight cut-off is 100Dolton, again by the method crystallization of patent 200810019855.3, obtains purity and be 99.1% cytidine diphosphate sodium salt crystal.
Embodiment 2:
According to above-mentioned whole-cell catalytic compound method, preparation CDP-choline conversion fluid 15L, wherein the concentration of CDP-choline is 10.75g/L.
Treatment process is following:
(1) the whole-cell catalytic conversion fluid is regulated pH to 3.0 enzyme that goes out with hydrochloric acid and live, through 10000rpm, the centrifugal back removal of 20min solid impurity.Get centrifuged supernatant through uf processing, collect ultrafiltration and see through liquid; Described ultra-filtration membrane is a PS membrane, and the ultra-filtration membrane molecular weight cut-off is 3000Dolton, and the ultra-filtration membrane WP is 1.5MPa.
(2) ultrafiltration sees through in the liquid and adds ammonium sulfate, is mixed with the upper prop liquid that contains 0.01mol/L ammonium sulfate, 5g/L CDP-choline, transfers pH to 2.0 with hydrochloric acid, and the phosphate WL 140 soaks 48h through the ammonium sulfate of 0.03mol/L in advance, refills and fills into post.Upper prop liquid get into to be filled the hydrophobic chromatography post absorption of phosphate WL 140, and absorption flow velocity is 100mL/min, when the concentration of CDP-choline in the absorption effluent reach sample introduction concentration 5% the time, think to stop the arrival breakthrough point adsorbing; Pure water with pH7.0 carries out wash-out, and elution flow rate is 50mL/min.Absorption and eluting temperature are 10 ℃, and it is 87.5% that wash-out finishes through the yield that records whole resin isolation process CDP-choline, and purity is 96.3%.
(3) elutriant is handled desalination through nanofiltration and is concentrated, and the nf membrane molecular weight cut-off is 150Dolton, again by the method crystallization of patent 200810019855.3, obtains purity and be 98.7% cytidine diphosphate sodium salt crystal.
Embodiment 3:
According to above-mentioned whole-cell catalytic compound method, preparation CDP-choline conversion fluid 15L, wherein the concentration of CDP-choline is 12.8g/L.
Treatment process is following:
(1) the whole-cell catalytic conversion fluid is regulated pH to 2.0 enzyme that goes out with hydrochloric acid and live, through 8000rpm, the centrifugal back removal of 20min solid impurity.Get centrifuged supernatant through uf processing, collect ultrafiltration and see through liquid; Described ultra-filtration membrane is a PS membrane, and the ultra-filtration membrane molecular weight cut-off is 8000Dolton, and the ultra-filtration membrane WP is 0.5MPa.
(2) ultrafiltration sees through liquid adding potassium primary phosphate, is mixed with the upper prop liquid that contains 1mol/L potassium primary phosphate, 10g/LCDP-choline, transfers pH to 4.0 with hydrochloric acid.Arsenic acid base polycondensation resol soaks 8h through the potassium primary phosphate of 1.5mol/L in advance, refills and fills into post.Upper prop liquid get into to be filled the hydrophobic chromatography post absorption of arsenic acid base polycondensation resol, and absorption flow velocity is 40mL/min, when the concentration of CDP-choline in the absorption effluent reach sample introduction concentration 5% the time, think to stop the arrival breakthrough point adsorbing; Pure water with pH10.0 carries out wash-out, and elution flow rate is 20mL/min.Absorption and eluting temperature are 25 ℃, and it is 92.7% that wash-out finishes through the yield that records whole resin isolation process CDP-choline, and purity is 99.1%.
(3) elutriant is handled desalination through nanofiltration and is concentrated, and the nf membrane molecular weight cut-off is 300Dolton, again by the method crystallization of patent 200810019855.3, obtains purity and be 99.4% cytidine diphosphate sodium salt crystal.
Embodiment 4:
According to above-mentioned whole-cell catalytic compound method, preparation CDP-choline conversion fluid 15L, wherein the concentration of CDP-choline is 10.2g/L.
Treatment process is following:
(1) the whole-cell catalytic conversion fluid is regulated pH to 3.0 enzyme that goes out with hydrochloric acid and live, through 8000rpm, the centrifugal back removal of 20min solid impurity.Get centrifuged supernatant through uf processing, collect ultrafiltration and see through liquid; Described ultra-filtration membrane is a PS membrane, and the ultra-filtration membrane molecular weight cut-off is 5000Dolton, and the ultra-filtration membrane WP is 1MPa.
(2) ultrafiltration sees through liquid adding ammonium chloride, is mixed with the upper prop liquid that contains 2.5mol/L ammonium chloride, 10g/L CDP-choline, transfers pH to 5.0 with hydrochloric acid.The sulfonic group WL 140 soaks 2h through the ammonium chloride of 2.5mol/L in advance, refills and fills into post.Upper prop liquid get into to be filled the hydrophobic chromatography post absorption of sulfonic group WL 140, and absorption flow velocity is 50mL/min, when the concentration of CDP-choline in the absorption effluent reach sample introduction concentration 5% the time, think to stop the arrival breakthrough point adsorbing; Pure water with pH7.0 carries out wash-out, and elution flow rate is 25mL/min.Absorption and eluting temperature are 25 ℃, and it is 91.6% that wash-out finishes through the yield that records whole resin isolation process CDP-choline, and purity is 98.4%.
(3) elutriant is handled desalination through nanofiltration and is concentrated, and the nf membrane molecular weight cut-off is 150Dolton, again by the method crystallization of patent 200810019855.3, obtains purity and be 99.0% cytidine diphosphate sodium salt crystal.
Embodiment 5:
According to above-mentioned whole-cell catalytic compound method, preparation CDP-choline conversion fluid 15L, wherein the concentration of CDP-choline is 16.6g/L.
Treatment process is following:
(1) the whole-cell catalytic conversion fluid is regulated pH to 2.0 enzyme that goes out with hydrochloric acid and live, through 8000rpm, the centrifugal back removal of 20min solid impurity.Get centrifuged supernatant through uf processing, collect ultrafiltration and see through liquid; Described ultra-filtration membrane is a PS membrane, and the ultra-filtration membrane molecular weight cut-off is 6000Dolton, and the ultra-filtration membrane WP is 0.5MPa.
(2) ultrafiltration sees through liquid adding sodium sulfate, is mixed with the upper prop liquid that contains 3mol/L sodium sulfate, 15g/L CDP-choline, transfers pH to 3.0.The sulfonic group WL 140 soaks 2h through the sodium sulfate of 3mol/L in advance, refills and fills into post.Upper prop liquid get into to be filled the hydrophobic chromatography post absorption of sulfonic group WL 140, and absorption flow velocity is 30mL/min, when the concentration of CDP-choline in the absorption effluent reach sample introduction concentration 5% the time, think to stop the arrival breakthrough point adsorbing; Pure water with pH9.0 carries out wash-out, and elution flow rate is 10mL/min.Absorption and eluting temperature are 40 ℃, and it is 91.9% that wash-out finishes through the yield that records whole resin isolation process CDP-choline, and purity is 99.2%.
(3) elutriant is handled desalination through nanofiltration and is concentrated, and the nf membrane molecular weight cut-off is 200Dolton, again by the method crystallization of patent 200810019855.3, obtains purity and be 99.4% cytidine diphosphate sodium salt crystal.
Embodiment 6:
According to above-mentioned whole-cell catalytic compound method, preparation CDP-choline conversion fluid 15L, wherein the concentration of CDP-choline is 15.3g/L.
Treatment process is following:
(1) the whole-cell catalytic conversion fluid is regulated pH to 2.0 enzyme that goes out with hydrochloric acid and live, through 8000rpm, the centrifugal back removal of 20min solid impurity.Get centrifuged supernatant through uf processing, collect ultrafiltration and see through liquid; Described ultra-filtration membrane is a PS membrane, and the ultra-filtration membrane molecular weight cut-off is 5000Dolton, and the ultra-filtration membrane WP is 1MPa.
(2) ultrafiltration sees through liquid adding saltpetre, is mixed with the upper prop liquid that contains 1.0mol/L saltpetre, 15g/L CDP-choline, transfers pH to 5.0 with hydrochloric acid.The sulfonic group WL 140 soaks 2h through the saltpetre of 3.0mol/L in advance, refills and fills into post.Upper prop liquid get into to be filled the hydrophobic chromatography post absorption of sulfonic group WL 140, and absorption flow velocity is 40mL/min, when the concentration of CDP-choline in the absorption effluent reach sample introduction concentration 5% the time, think to stop the arrival breakthrough point adsorbing; Pure water with pH7.0 carries out wash-out, and elution flow rate is 10mL/min.Absorption and eluting temperature are 40 ℃, and it is 92.6% that wash-out finishes through the yield that records whole resin isolation process CDP-choline, and purity is 98.7%.
(3) elutriant is handled desalination through nanofiltration and is concentrated, and the nf membrane molecular weight cut-off is 200Dolton, again by the method crystallization of patent 200810019855.3, obtains purity and be 99.1% cytidine diphosphate sodium salt crystal.
Claims (7)
1. the method for a separation and purification cytidine diphosphate is characterized in that this method comprises the steps:
(1) the cytidine diphosphate conversion fluid is through enzyme-deactivating, centrifugal and ultrafiltration pretreatment;
(2) in the step (2), pretreated cytidine diphosphate conversion fluid transferred pH2.0～5.0 after, add inorganic salt and be mixed with the upper prop liquid that contains 1～3mol/L inorganic salt and 5～15g/L cytidine diphosphate, through resin absorption, again through the pure water wash-out of pH7.0;
(3) elutriant is obtained cytidine diphosphate through nanofiltration desalination and concentration, crystallization;
Wherein, Described resin is for being the inertia skeleton with PS, ROHM or polycondensation phenolic aldehyde; With sulfonic group, phosphate, arsenic acid base or the selenono resin as functional group, described resin uses after soaking 1～48h through 0.01～5mol/L inorganic salt solution.
2. the method for separation and purification cytidine diphosphate according to claim 1; It is characterized in that in the step (1); Regulate cytidine diphosphate conversion fluid pH value to 2.0～3.0 and make enzyme-deactivating; Through 6000～12000rpm spinning, 10～60min, getting supernatant is the ultra-filtration membrane ultrafiltration of 3000～8000Dolton through molecular weight cut-off again.
3. the method for separation and purification cytidine diphosphate according to claim 2; It is characterized in that in the step (1); Regulate cytidine diphosphate conversion fluid pH value to 2.0 and make enzyme-deactivating; Through 8000～10000rpm spinning, 15～20min, getting supernatant is the ultra-filtration membrane ultrafiltration of 5000～6000Dolton through molecular weight cut-off again.
4. the method for separation and purification cytidine diphosphate according to claim 1 is characterized in that in the step (2) that described inorganic salt are inorganic sodium or inorganic potassium salt or inorganic ammonium salt.
5. the method for separation and purification cytidine diphosphate according to claim 1 is characterized in that in the step (2), the absorption flow velocity is 10～100mL/min, and elution flow rate is 5～50mL/min.
6. the method for separation and purification cytidine diphosphate according to claim 5 is characterized in that in the step (2), the absorption flow velocity is 30～50mL/min, and elution flow rate is 10～20mL/min.
7. the method for separation and purification cytidine diphosphate according to claim 1 is characterized in that in the step (3), described nanofiltration, and its nf membrane molecular weight cut-off is 100～300 Dolton.
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| CN106432411A (en) * | 2016-09-23 | 2017-02-22 | 精晶药业股份有限公司 | Method for extracting alanyl-glutamine from enzymatic conversion liquid |
| CN109836468A (en) * | 2017-11-24 | 2019-06-04 | 苏州华赛生物工程技术有限公司 | A method of the purifying citicoline sodium from microbial fermentation solution |
| CN107827943B (en) * | 2017-11-28 | 2022-06-10 | 绍兴厚普生物科技有限责任公司 | Method for extracting cytosine nucleoside from fermentation liquor |
| CN110714212B (en) * | 2019-10-12 | 2021-04-30 | 常州大学 | Method for preparing super-hydrophobic nickel film in aqueous solution system by nickel chloride one-step method |
| CN111487337A (en) * | 2020-04-16 | 2020-08-04 | 南通秋之友生物科技有限公司 | Online control method for citicoline sodium crystallization process |
| CN113769794B (en) * | 2021-07-06 | 2024-04-05 | 沁浩膜技术(厦门)有限公司 | Ion exchange system and method for continuously removing impurities in citicoline sodium |
| CN114262726A (en) * | 2022-01-11 | 2022-04-01 | 深圳华酶生物科技有限公司 | Method for synthesizing citicoline sodium by using cytidine enzymatic method |
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| WO2007018259A1 (en) * | 2005-08-10 | 2007-02-15 | Kyowa Hakko Kogyo Co., Ltd. | Method for purification of cytidinediphosphoric choline |
| CN101096380A (en) * | 2007-05-10 | 2008-01-02 | 南通秋之友生物科技有限公司 | Method for purifying citicoline from biotransformation or multienzyme reaction liquid |
| CN101130797A (en) * | 2007-08-01 | 2008-02-27 | 张剑 | Ubelin manufacturing technique |
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| WO2007018259A1 (en) * | 2005-08-10 | 2007-02-15 | Kyowa Hakko Kogyo Co., Ltd. | Method for purification of cytidinediphosphoric choline |
| CN101096380A (en) * | 2007-05-10 | 2008-01-02 | 南通秋之友生物科技有限公司 | Method for purifying citicoline from biotransformation or multienzyme reaction liquid |
| CN101130797A (en) * | 2007-08-01 | 2008-02-27 | 张剑 | Ubelin manufacturing technique |
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