CN1101396C - 用超滤法从发酵液中分离棒酸 - Google Patents
用超滤法从发酵液中分离棒酸 Download PDFInfo
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- CN1101396C CN1101396C CN96196471A CN96196471A CN1101396C CN 1101396 C CN1101396 C CN 1101396C CN 96196471 A CN96196471 A CN 96196471A CN 96196471 A CN96196471 A CN 96196471A CN 1101396 C CN1101396 C CN 1101396C
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- clavulanic acid
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- HZZVJAQRINQKSD-UHFFFAOYSA-N Clavulanic acid Natural products OC(=O)C1C(=CCO)OC2CC(=O)N21 HZZVJAQRINQKSD-UHFFFAOYSA-N 0.000 title claims abstract description 48
- HZZVJAQRINQKSD-PBFISZAISA-N clavulanic acid Chemical compound OC(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21 HZZVJAQRINQKSD-PBFISZAISA-N 0.000 title claims abstract description 48
- 229960003324 clavulanic acid Drugs 0.000 title claims abstract description 45
- 238000000108 ultra-filtration Methods 0.000 title claims abstract description 24
- 238000002955 isolation Methods 0.000 title claims description 6
- 238000000855 fermentation Methods 0.000 title abstract description 8
- 230000004151 fermentation Effects 0.000 title abstract description 8
- 239000000706 filtrate Substances 0.000 claims abstract description 25
- 150000003839 salts Chemical class 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 13
- 150000002148 esters Chemical class 0.000 claims abstract description 9
- 239000012528 membrane Substances 0.000 claims abstract description 7
- 238000001471 micro-filtration Methods 0.000 claims abstract description 7
- 239000007787 solid Substances 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 6
- 239000012466 permeate Substances 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 16
- 239000012530 fluid Substances 0.000 claims description 9
- 230000000149 penetrating effect Effects 0.000 claims description 9
- 239000012141 concentrate Substances 0.000 claims description 5
- 241000209094 Oryza Species 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 229920002492 poly(sulfone) Polymers 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 239000002904 solvent Substances 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000605 extraction Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- ABVRVIZBZKUTMK-JSYANWSFSA-M potassium clavulanate Chemical compound [K+].[O-]C(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21 ABVRVIZBZKUTMK-JSYANWSFSA-M 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- -1 alkali metal salt Chemical class 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 238000001223 reverse osmosis Methods 0.000 description 5
- OBETXYAYXDNJHR-SSDOTTSWSA-M (2r)-2-ethylhexanoate Chemical compound CCCC[C@@H](CC)C([O-])=O OBETXYAYXDNJHR-SSDOTTSWSA-M 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 229910052783 alkali metal Inorganic materials 0.000 description 4
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 229940090805 clavulanate Drugs 0.000 description 3
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 235000011164 potassium chloride Nutrition 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 241000187180 Streptomyces sp. Species 0.000 description 2
- 241001655322 Streptomycetales Species 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium group Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- DKPFZGUDAPQIHT-UHFFFAOYSA-N butyl acetate Chemical compound CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 235000011056 potassium acetate Nutrition 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- YBRBMKDOPFTVDT-UHFFFAOYSA-N tert-butylamine Chemical class CC(C)(C)N YBRBMKDOPFTVDT-UHFFFAOYSA-N 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 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 1
- NKBWMBRPILTCRD-UHFFFAOYSA-N 2-Methylheptanoic acid Chemical compound CCCCCC(C)C(O)=O NKBWMBRPILTCRD-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 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 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 1
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical class O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000187433 Streptomyces clavuligerus Species 0.000 description 1
- 241000187215 Streptomyces jumonjinensis Species 0.000 description 1
- KXKVLQRXCPHEJC-UHFFFAOYSA-N acetic acid trimethyl ester Natural products COC(C)=O KXKVLQRXCPHEJC-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 150000004985 diamines Chemical class 0.000 description 1
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- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
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- 230000016615 flocculation Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- YKYONYBAUNKHLG-UHFFFAOYSA-N n-Propyl acetate Natural products CCCOC(C)=O YKYONYBAUNKHLG-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 235000010235 potassium benzoate Nutrition 0.000 description 1
- 239000004300 potassium benzoate Substances 0.000 description 1
- 229940103091 potassium benzoate Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 229940090181 propyl acetate Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
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- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D503/00—Heterocyclic compounds containing 4-oxa-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. oxapenicillins, clavulanic acid derivatives; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
- C07D503/10—Heterocyclic compounds containing 4-oxa-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. oxapenicillins, clavulanic acid derivatives; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
- C07D503/12—Heterocyclic compounds containing 4-oxa-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. oxapenicillins, clavulanic acid derivatives; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 unsubstituted in position 6
- C07D503/14—Heterocyclic compounds containing 4-oxa-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. oxapenicillins, clavulanic acid derivatives; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 unsubstituted in position 6 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, other than a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, attached in position 3
- C07D503/16—Radicals substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical
- C07D503/18—Radicals substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical by oxygen atoms
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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Abstract
本发明揭示了棒酸或其药学上可接受的盐或酯的制备和/或纯化方法，包括以下步骤：用微量过滤法从含棒酸的发酵液中除去固体，形成第一滤液，用超滤法从第一滤液中进一步除去固体，形成第二滤液，通过除去水来浓缩第二滤液，处理经浓缩的第二滤液来分离棒酸或其药学上可接受的盐或酯，其特征在于用超滤膜过滤第一滤液，所述超滤膜的分子量截留值为10至30千道尔顿，渗透液流量为10至30升/(米2·小时)。
Description
技术领域
本发明涉及棒酸以及棒酸的药学上可接受的盐和酯的纯化和/或制备方法，特别是碱金属盐(尤其是棒酸钾)，但不仅限于此。
背景技术
棒酸是(2R，5R，Z)-30(2-羟基亚乙基)-7-氧代-4-氧杂-1-氮杂二环[3.2.0]庚烷-2-羧酸的常用名。棒酸及其碱金属盐和酯能有效地作为由一些革兰氏阳性和革兰氏阴性微生物所产生的β-内酰胺酶的抑制剂。除了抑制β-内酰胺酶以外，棒酸及其碱金属盐还与青霉素和头孢菌素类抗生素有协同作用。棒酸及其盐被用于药物制剂，以防止β-内酰胺抗生素的失活。商业上的制剂包括棒酸钾和三水合羟氨苄青霉素的复方。棒酸钾比游离酸或其它盐更稳定。
棒酸通过微生物的发酵来制备，这些微生物如链霉菌属的菌株，例如带小棒链霉菌(S.clavuligerus)NRRL 3585、S.jumonjinensis NRRL 5741和桂滨链霉菌(S.katsurahamanus)IFO 13716和Streptomyces sp.P6621 FERM P2804。如GB1508977中揭示，将发酵后得到的含水培养物根据常规的方法(如过滤和色谱纯化)进行纯化和浓缩，然后用有机溶剂对水溶液进行萃取，得到不纯的棒酸溶于溶剂的溶液。
GB1508977揭示了通过使发酵液流经阴离子交换树脂来进行过滤，得到棒酸盐的制备方法。这一方法可以得到可接受的产率，但是它需要使用复杂的色谱纯化方法，而且从商业角度考虑，使用树脂柱占了生产投资的重要部分。
GB1543563中揭示了一种发酵方法，其中培养基的pH值保持在6.3至6.7的范围内。通过由棒酸锂的再盐化(re-salting)来制备药学上可接受的盐，如棒酸钾。
EP-A-0026044中揭示了使用棒酸的叔丁胺盐作为中间体来纯化棒酸。BE-862211或DE2733230说明了所述的盐，据述该盐比棒酸的钠盐或钾盐更稳定。叔丁胺是一种有毒的化合物，而且很难将其从废水中除去，这导致了严重的污染问题。
EP-A-0562583揭示了使用棒酸和N，N′-单取代的对称1，2-乙二胺的盐(如二棒酸N，N′-二异丙基乙二铵)作为有用的中间体来从乙酸乙酯的萃取液中分离和制备纯的棒酸或棒酸的碱金属盐。
通常经过滤的含棒酸的发酵液含有10-20％的蛋白质，以棒酸的量计。这些蛋白质阻碍了接下去的棒酸的分离和纯化。用离子交换树脂或絮凝剂来处理经过滤的发酵液是耗时和昂贵的，并且会增加不稳定的产物的降解。
发明内容
根据本发明，棒酸或其药学上可接受的盐或酯的制备和/或纯化方法，包括以下步骤：
用微量过滤法(microfiltration)从含棒酸的发酵液中除去固体，形成第一滤液，
用超滤法从第一滤液中进一步除去固体，形成第二滤液，
通过除去水来浓缩第二滤液，
处理经浓缩的第二滤液来分离棒酸或其药学上可接受的盐或酯，
其特征在于用超滤膜过滤第一滤液，所述超滤膜的分子量截留值(molecularweight cut-off)为10至30千道尔顿(kD)，渗透液(permeate)的流量为10至30升/(米2·小时)(1m-2h-1)。
使用本发明的超滤法，可以得到足够纯净的棒酸溶液，而不需要在转化成棒酸钾前形成中间体胺或二铵盐。较好的是，超滤是连续的，超滤膜包括聚砜。而且，第二滤液可以用反渗透进行浓缩，而不会将反渗透膜表面弄脏，或在其表面上形成不需要的凝胶层。可以提高流经反渗透装置的流量，而不需要经常性的清洗。
已发现，使用本发明能提高分离棒酸的效率，它的另一个优点是第二滤液经过超滤步骤后，基本上脱色了。
具体实施方式
发酵液的微量过滤法可以按我们的专利申请WO95/23870中所揭示的进行。根据所揭示的内容，较好的方法是将含有粗棒酸、菌丝体、蛋白质以及其它悬浮的固体物质的含水发酵液通过在pH为5.8至6.2之间，在约20至40℃的温度下进行微量过滤来纯化。
对第二滤液的进一步处理较好的包括通过反渗透来浓缩。将滤液浓缩至约原来体积的1/5，得到经浓缩的含水棒酸，该含水棒酸可用与水不溶混的溶剂(较好的为乙酸乙酯)经串联离心式逆流萃取进行萃取，例如按WO95/23870中所述的进行。
该萃取较好的在15至25℃的温度间，1至3的pH值间进行。然后，将萃取液干燥至含水量在0.1％(体积)以下，进一步通过蒸发进行浓缩，如果需要，用活性炭进行脱色。
萃取液中的棒酸可以与胺或二胺反应，然后分离棒酸的铵或二铵盐，再将该铵或二铵盐转化成药学上可接受的棒酸盐或酯。或者，浓缩液可以直接与金属供体反应，形成药学上可接受的盐，如钾盐。
金属供体可以是钾、钠、锂或镁的有机盐、碳酸盐、碳酸氢盐或氢氧化物。较好的是用有机盐，更好的是羧酸盐。考虑到棒酸钾的相对稳定性，较好的是使用钾盐。
羧酸可以选自乙酸、丙酸、己酸、苯甲酸，以及被以下基团所取代的苯甲酸：一个或多个C1-C10烷基(较好的是C1-C6烷基)；卤素；硝基；O、S或NR取代的杂烷基；被R、OR、SR或NR1R2中的一个基团所取代的C1-C10烷基，其中R、R1和R2各自为C1-C10烷基。
较好的金属供体包括2-乙基己酸钾、乙酸钾、2-乙基己酸锂和乙酸锂。
可以加入另一溶剂，包括C1-C10醇或其混合物。较好的是使用C1-C4醇。特别好的另一溶剂包括甲醇、乙醇、异丙醇和异丁醇，以及它们的混合物。特别好的是使用异丙醇。这些溶剂最好是干燥的，例如含有0％至4％的水。在加入到棒酸萃取液以前，金属供体溶解在这另一溶剂中。或者，金属供体可溶解在与棒酸萃取液相同的溶剂(如乙酸乙酯)中，这时另一溶剂应分开加入。
当2-乙基己酸钾溶解于异丙醇、乙酸钾溶解于甲醇或苯甲酸钾溶解于甲醇时，得到特别有利的结果。异丙醇中2-乙基己酸钾的浓度较好的为0.1摩尔/升(mol/l)至5摩尔/升，更好的为1摩尔/升至2.5摩尔/升，最好的为1.5摩尔/升至2摩尔/升，过量0.8至5摩尔(以棒酸的量计)，较好的是过量5至25％(摩尔)。
用于萃取发酵液的滤液的与水不溶混的溶剂较好的是选自乙酸乙酯、乙酸甲酯、乙酸丙酯、乙酸正丁酯、酮(如甲基乙基酮)、醇(如正丁醇、正戊醇)、或卤化溶剂(如二氯甲烷、氯仿)、或醚(如二乙醚)、或己烷，或者它们的混合物。更好的是使用乙酸乙酯。浓缩液使用活性炭来纯化，如果需要，使用硅胶柱。
在干燥的经浓缩的与水不溶混的溶剂(如乙酸乙酯)的萃取液中，粗棒酸的浓度在8克/升(g/l)至40克/升之间，较好的在20克/升至40克/升之间。
通过加入活性炭对棒酸盐浓缩液(较好的是在乙酸乙酯中)进行脱色。已发现，较合适的是每克棒酸用0.2至0.5克的活性炭，然而，根据需要也可以使用其它的用量。
本发明通过实施例作进一步的描述，但这些实施例并不意味着对本发明的限制。
实施例1
通过发酵Streptomyces sp.P6621 FERM P2804，并使用WO95/23870中所述的微量过滤法，得到经过滤的含水发酵液。微滤液渗透液(microfiltrate permeate)的棒酸含量为3.5克/升(gl-1)，蛋白质含量为0.56克/升，用1∶2的水稀释后在420纳米(nm)处的透光率为27.9％，将该微滤液渗透液连续地送入两步的过滤装置(型号为2NUF2000，产自IMP Promont Ljubljana，Slovenia)中。两个超滤洗涤组件(型号为GR62-3833/47P，产自Filmtech(Dow Chemical Co.的子公司))具有总膜面积为194.4米2(m2)的两个环路(loops)。安排这些装置，使得超滤组件并联或串联连接。膜的分子量截留值为20千道尔顿。经过滤的发酵液的温度为35℃，pH值在5.8至6.2之间，渗透液流量为12升/(米2·小时)。膜上的压力为5巴(Bar)，浓缩液和渗透液之间的纵向压差为1.4巴。通过第一步的超滤，得到流量为1600升/小时(1h-1)的渗透液和流量为400升/小时的浓缩液。将第一步超滤的浓缩液用于第二步超滤，并用软化水以800升/小时的流量进行洗涤。第二步超滤中渗透液的流量为800升/小时，并且将其与从第一环路出来的渗透液一起送入反渗透装置。
以2400升/小时的流量得到含棒酸的渗透液，它含有0.11克/升的蛋白质。透光率(420纳米)为45.1％，产率为95％，按棒酸检定法计算。
Claims (7)
1.棒酸或其药学上可接受的盐或酯的制备和/或纯化方法，包括以下步骤：
用微量过滤法从含棒酸的发酵液中除去固体，形成第一滤液，
用超滤法从第一滤液中进一步除去固体，形成第二滤液，
通过除去水来浓缩第二滤液，
处理经浓缩的第二滤液来分离棒酸或其药学上可接受的盐或酯，
其特征在于用超滤膜过滤第一滤液，所述超滤膜的分子量截留值为10至30千道尔顿，渗透液流量为10至30升/(米2·小时)。
2.如权利要求1所述的方法，其特征在于在超滤之前第一滤液的pH值为5.8至6.2。
3.如权利要求1或2所述的方法，其特征在于超滤是连续的。
4.如权利要求3所述的方法，其特征在于使用两步的装置进行连续超滤。
5.如以上权利要求中任一项所述的方法，其特征在于超滤膜包括聚砜。
6.如以上权利要求中任一项所述的方法，其特征在于渗透液的流量为12升/(米2·小时)。
7.如以上权利要求中任一项所述的方法，其特征在于分子量截留值为20千道尔顿。
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| SIP9500265 | 1995-08-28 | ||
| SIP-9500265 | 1995-08-28 | ||
| SI9500265A SI9500265A1 (en) | 1995-08-28 | 1995-08-28 | Process for purification of the aqueous fermented broth filtrate of streptomyces sp. p 6621 ferm p 2804 by ultrafiltration |
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| AT400033B (de) | 1992-03-10 | 1995-09-25 | Biochemie Gmbh | Neues verfahren zur isolierung und reinigung von clavulansäure und zur herstellung von pharmakologisch verträglichen salzen derselben |
| SI9500074A (en) * | 1995-03-10 | 1996-10-31 | Lek Tovarna Farmacevtskih | Process for preparation of alkani salts of clavulanic acid. |
| AT403375B (de) * | 1995-11-15 | 1998-01-26 | Biochemie Gmbh | Verfahren zur fällung von alkalisalzen der clavulansäure |
| ES2105988B1 (es) * | 1996-03-15 | 1998-07-01 | Antibioticos Sa | Procedimiento para purificar 7-sustituido-amino-desacetoxi-cefalosporinas mediante el empleo de membranas de filtracion. |
| GB0022841D0 (en) | 2000-09-18 | 2000-11-01 | Smithkline Beecham Plc | Process |
| JP2004094465A (ja) * | 2002-08-30 | 2004-03-25 | Fujitsu Ltd | 解析支援装置 |
| CN102933717A (zh) * | 2007-04-27 | 2013-02-13 | 科学与工业研究委员会 | 在固态发酵中采用棒状链霉菌mtcc 1142制备棒酸的方法 |
| CN102838624B (zh) * | 2012-09-07 | 2014-10-29 | 安徽普朗膜技术有限公司 | 一种从发酵液中纯化克拉维酸的方法 |
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| Publication number | Publication date |
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| CZ31898A3 (cs) | 1998-07-15 |
| US6274575B1 (en) | 2001-08-14 |
| UA59343C2 (uk) | 2003-09-15 |
| RU2169151C2 (ru) | 2001-06-20 |
| HUP9802532A2 (hu) | 1999-03-29 |
| BG102253A (en) | 1998-08-31 |
| EP0859780A1 (en) | 1998-08-26 |
| HU221353B1 (en) | 2002-09-28 |
| AU6829796A (en) | 1997-03-19 |
| CA2226683C (en) | 2002-04-30 |
| NZ316136A (en) | 1998-09-24 |
| HUP9802532A3 (en) | 1999-04-28 |
| WO1997008175A1 (en) | 1997-03-06 |
| SK10398A3 (en) | 1998-07-08 |
| RO116813B1 (ro) | 2001-06-29 |
| ZA967273B (en) | 1997-03-04 |
| SI9500265A1 (en) | 1997-02-28 |
| CN1193975A (zh) | 1998-09-23 |
| EE03567B1 (et) | 2001-12-17 |
| PL324855A1 (en) | 1998-06-22 |
| EE9800060A (et) | 1998-08-17 |
| BG63086B1 (bg) | 2001-03-30 |
| JP2000512125A (ja) | 2000-09-19 |
| AU714892B2 (en) | 2000-01-13 |
| KR19990035966A (ko) | 1999-05-25 |
| CA2226683A1 (en) | 1997-03-06 |
| US6127358A (en) | 2000-10-03 |
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