CN111499669A - Method for refining spiramycin by adopting two DAC columns - Google Patents
Method for refining spiramycin by adopting two DAC columns Download PDFInfo
- Publication number
- CN111499669A CN111499669A CN202010354822.5A CN202010354822A CN111499669A CN 111499669 A CN111499669 A CN 111499669A CN 202010354822 A CN202010354822 A CN 202010354822A CN 111499669 A CN111499669 A CN 111499669A
- Authority
- CN
- China
- Prior art keywords
- spiramycin
- value
- solution
- acetone
- buffer salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- ACTOXUHEUCPTEW-BWHGAVFKSA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4r,5s,6r)-5-[(2s,4r,5s,6s)-4,5-dihydroxy-4,6-dimethyloxan-2-yl]oxy-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-10-[(2s,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-o Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)[C@@H]1CC[C@H](N(C)C)[C@@H](C)O1 ACTOXUHEUCPTEW-BWHGAVFKSA-N 0.000 title claims abstract description 89
- 239000004187 Spiramycin Substances 0.000 title claims abstract description 86
- 229930191512 spiramycin Natural products 0.000 title claims abstract description 86
- 229960001294 spiramycin Drugs 0.000 title claims abstract description 86
- 235000019372 spiramycin Nutrition 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000007670 refining Methods 0.000 title claims abstract description 14
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 172
- 239000000243 solution Substances 0.000 claims abstract description 98
- 238000000605 extraction Methods 0.000 claims abstract description 80
- 239000000337 buffer salt Substances 0.000 claims abstract description 63
- 238000011068 loading method Methods 0.000 claims abstract description 58
- 239000007788 liquid Substances 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000000843 powder Substances 0.000 claims abstract description 19
- 239000011259 mixed solution Substances 0.000 claims abstract description 15
- 238000001035 drying Methods 0.000 claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 10
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 69
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 66
- 238000003756 stirring Methods 0.000 claims description 33
- 238000005406 washing Methods 0.000 claims description 30
- 238000000746 purification Methods 0.000 claims description 24
- 238000000926 separation method Methods 0.000 claims description 24
- 235000006408 oxalic acid Nutrition 0.000 claims description 23
- 239000008346 aqueous phase Substances 0.000 claims description 22
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 21
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 21
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 21
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 19
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 239000003513 alkali Substances 0.000 claims description 8
- 239000012071 phase Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- 239000007791 liquid phase Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- SWXVUIWOUIDPGS-UHFFFAOYSA-N diacetone alcohol Chemical compound CC(=O)CC(C)(C)O SWXVUIWOUIDPGS-UHFFFAOYSA-N 0.000 claims description 6
- 238000011282 treatment Methods 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 4
- 238000004821 distillation Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 48
- 239000012488 sample solution Substances 0.000 abstract description 11
- 238000002386 leaching Methods 0.000 description 33
- 239000000523 sample Substances 0.000 description 33
- 239000000945 filler Substances 0.000 description 24
- 239000004480 active ingredient Substances 0.000 description 12
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 11
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 238000001514 detection method Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 238000001816 cooling Methods 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 201000007100 Pharyngitis Diseases 0.000 description 2
- HSZLKTCKAYXVBX-LYIMTGTFSA-N Spiramycin III Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@H]([C@@H]([C@@H](O[C@H]2[C@@H]([C@H]([C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@](C)(O)C3)[C@@H](C)O2)N(C)C)O)[C@@H](CC=O)C[C@H]1C)OC)OC(=O)CC)[C@H]1CC[C@H](N(C)C)[C@@H](C)O1 HSZLKTCKAYXVBX-LYIMTGTFSA-N 0.000 description 2
- ZPCCSZFPOXBNDL-LWXQEXJOSA-N Spiramycin-II Natural products CO[C@H]1[C@@H](CC(=O)O[C@H](C)CC=CC=C[C@H](O[C@H]2CC[C@@H]([C@@H](C)O2)N(C)C)[C@H](C)C[C@@H](CC=O)[C@@H]1O[C@@H]3O[C@H](C)[C@@H](O[C@H]4C[C@@](C)(O)[C@@H](O)[C@H](C)O4)[C@@H]([C@H]3O)N(C)C)OC(=O)C ZPCCSZFPOXBNDL-LWXQEXJOSA-N 0.000 description 2
- HSZLKTCKAYXVBX-VPIGJTHDSA-N Spiramycin-III Natural products CCC(=O)O[C@@H]1CC(=O)O[C@H](C)CC=CC=C[C@H](O[C@H]2CC[C@@H]([C@@H](C)O2)N(C)C)[C@H](C)C[C@H](CC=O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O[C@H]4C[C@@](C)(O)[C@@H](O)[C@H](C)O4)[C@@H]([C@H]3O)N(C)C)[C@H]1OC HSZLKTCKAYXVBX-VPIGJTHDSA-N 0.000 description 2
- 241000187758 Streptomyces ambofaciens Species 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000003120 macrolide antibiotic agent Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- ACTOXUHEUCPTEW-ZOTSFZJCSA-N spiramycin I Natural products CO[C@H]1[C@H](O)CC(=O)O[C@H](C)CC=CC=C[C@H](O[C@H]2CC[C@@H]([C@@H](C)O2)N(C)C)[C@H](C)C[C@H](CC=O)[C@@H]1O[C@@H]3O[C@H](C)[C@@H](O[C@H]4C[C@@](C)(O)[C@@H](O)[C@H](C)O4)[C@@H]([C@H]3O)N(C)C ACTOXUHEUCPTEW-ZOTSFZJCSA-N 0.000 description 2
- ZPCCSZFPOXBNDL-RSMXASMKSA-N spiramycin II Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@H]([C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)OC(C)=O)[C@H]1CC[C@H](N(C)C)[C@H](C)O1 ZPCCSZFPOXBNDL-RSMXASMKSA-N 0.000 description 2
- 229950001955 spiramycin i Drugs 0.000 description 2
- 229950006796 spiramycin ii Drugs 0.000 description 2
- 229950003659 spiramycin iii Drugs 0.000 description 2
- -1 3, 6-dideoxy-3-dimethylamino- β -D-glucopyranosyl Chemical group 0.000 description 1
- 206010001076 Acute sinusitis Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 201000005485 Toxoplasmosis Diseases 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 208000020029 respiratory tract infectious disease Diseases 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a method for refining spiramycin, which comprises the following steps: adjusting the pH value of a back extraction liquid obtained in the spiramycin production to 5.5-6.5, and passing the lower-layer water phase through activated carbon to be used as a loading solution; separating and purifying the sample solution by using a first DAC column, eluting, and dividing the obtained fraction into a first target fraction; adjusting the pH value of the first target fraction to 7.0-8.5, separating and purifying by using a second DAC column, eluting by using a mixed solution of buffer salt and acetone, and collecting a second target fraction; adjusting the pH value of the second target fraction to 7.0-8.0, and distilling under reduced pressure to remove acetone; adjusting the pH value to 8.5-10.5, and drying to obtain the spiramycin dry powder. The refining method can improve the yield of the spiramycin, improve the quality of the spiramycin product by reducing the contents of the impurities B, D and F in the spiramycin, and reduce the risk that the spiramycin product does not meet pharmacopoeia standards.
Description
Technical Field
The invention relates to the technical field of preparation of macrolide antibiotics, in particular to a method for refining spiramycin by adopting two DAC columns.
Background
Spiramycin, its english name is Spiramycin, its chemical name is (4R,5S,6S,7R,9R,10R,16R) - (11E,13E) -6- [ (0-2, 6-dideoxy-3-C-methyl- α -L-riboflavin-hexylpyran) - (1,4) - (3, 6-dideoxy-3-dimethylamino- β -D-glucopyranosyl) oxy ] -7-formyl-4-hydroxy-5-methoxy-9, 16-dimethyl-10- [ (2,3,4, 6-tetraethoxy-4-dimethylamino-D-erythro-hexylpyran) oxy ] -oxycyclohexanone-11, 13-dienyl-2-one insoluble, Spiramycin is a macrolide antibiotic obtained from the culture broth of streptomyces ambofaciens (streptomyces ambofaciens), is a white or yellowish powder, slightly odorous, slightly hygroscopic, in water, ethanol, acetone or petroleum ether.
Spiramycin is a very powerful bacteriostatic agent and exhibits bactericidal activity only at very high concentrations. Spiramycin has strong in vivo antibacterial action and antibacterial after effect (PAE), can enhance phagocytosis of phagocytes, is widely distributed in vivo, has higher concentration in tissue cells than erythromycin, and has less side effects than erythromycin. The spiramycin can be used for treating ear, nose, throat and respiratory tract infections caused by gram-positive bacteria and some gram-negative bacteria, is suitable for treating oral cavity and ear-nose-throat infection caused by sensitive bacteria, such as otitis media, periodontitis and acute sinusitis, and can also be used for treating toxoplasmosis.
Wherein, the structural formula of the spiramycin is as follows:
in the EP9.2 pharmacopoeia, spiramycin I is more than or equal to 80.0 percent; spiramycin II is less than or equal to 5.0 percent; spiramycin III is less than or equal to 10 percent; the sum of the spiramycin I, the spiramycin II and the spiramycin III is more than or equal to 90.0 percent. The impurity structural formula is as follows:
wherein the molecular formula of the impurity A is: c36H62N2O11The structural formula is as follows:
the molecular formula of impurity B is: c43H76N2O14The structural formula is as follows:
the molecular formula of impurity D is: c43H73N2O16The structural formula is as follows:
the molecular formula of impurity E is: c44H78N2O13The structural formula is as follows:
the molecular formula of the impurity F is spiramycin dimer, and the structural formula is as follows:
the molecular formula of impurity G is: c46H78N2O15The structural formula is as follows:
the molecular formula of impurity H is: c49H68N2O12The structural formula is as follows:
wherein, the impurity A, the impurity B, the impurity D, the impurity E, the impurity G and the impurity H are less than or equal to 2.0 percent, and the total amount of the impurities is less than or equal to 10 percent.
At present, in the production of spiramycin, spiramycin is extracted by adopting a butyl acetate extraction method, BA solution is washed by mixed solution of phosphate and oxalic acid, sodium chloride solution and purified water, and partial impurities in the BA solution are dissolved and removed by the washing solution. The method cannot specifically remove impurities B, D and F in the BA solution, so that the refining effect is poor.
The impurities except the impurity B, the impurity D and the impurity F are low in content, so that the impurities can be easily removed and meet the standard requirement through production extraction and back extraction steps. However, the contents of the impurities B, D and F in the production of spiramycin are high, and the impurities are not easy to remove through extraction and back extraction steps. B, D, F three impurities are intensively researched in the process so as to avoid the situation that the impurity B, the impurity D and the impurity F are high.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a spiramycin refining method which can improve the yield of spiramycin, improve the quality of spiramycin products by reducing the contents of impurities B, D and F in the spiramycin, and reduce the risk of the spiramycin products not meeting pharmacopoeia standards.
In order to achieve the purpose, the invention provides a method for refining spiramycin, which comprises the following steps: (1) adjusting the pH value of a back extraction liquid obtained in spiramycin production to 5.5-6.5, and after layering, removing butyl acetate from the back extraction liquid obtained in the lower water phase by using activated carbon to obtain a loading solution; (2) separating and purifying the sample loading solution by adopting a first DAC column according to the sample loading amount of 50-120%, eluting the first DAC column by using acetone, methanol or ethanol with the volume percentage concentration of 20-100% after separation and purification, and collecting fractions obtained in the separation and purification process of the first DAC column as first target fractions; (3) adjusting the pH value of the first target fraction to 7.0-8.5, then separating and purifying by adopting a second DAC column according to the sample loading amount of 6% -10%, eluting by adopting a mixed solution of buffer salt and acetone after separation and purification, and collecting the fraction obtained in the separation and purification process of the second DAC column as a second target fraction; and (4) adjusting the pH value of the second target fraction to 7.0-8.0 by using an alkali solution, and removing acetone by reduced pressure distillation; then dropwise adding an alkali solution to adjust the pH value to 8.5-10.5, simultaneously heating to 45-65 ℃, stirring, keeping the temperature, washing with the alkali solution after centrifugal filtration, and drying to obtain the spiramycin dry powder.
When the spiramycin product is transferred from an upper butyl acetate phase to a lower water phase within the range of pH value of 5.5-6.5; the second target fraction is easily crystallized when the pH is 8.5-10.5.
In a preferred embodiment, in step (1), the above-mentioned stripping solution is prepared by a method comprising: extracting spiramycin fermentation filtrate by butyl acetate to form BA solution; then, adding oxalic acid into dipotassium hydrogen phosphate, and adjusting the pH value to 2.5 +/-0.2 to obtain a primary back-extraction liquid; adding the primary back extraction liquid into the BA solution, stirring until the pH value is 6.4 +/-0.3, then adding oxalic acid to adjust the pH value to 4.8 +/-0.2, stirring uniformly, and standing for layering; recovering the lower aqueous phase, adding the primary back-extraction liquid into the upper BA liquid phase again, adjusting the pH value to 2.8 +/-0.3, stirring and mixing uniformly, standing for layering, and recovering the lower aqueous phase; the aqueous phases recovered twice are combined to obtain the final back-extraction solution.
After the pH of the BA solution is adjusted to 4.8 + -0.2, the pH still needs to be adjusted to be lower by adding the primary back-extraction liquid because: after the pH value of the BA solution is adjusted to 4.8 +/-0.2, the residual spiramycin is not extracted, and after the pH value of the BA solution is adjusted to 2.8 +/-0.3 from 4.8 +/-0.2, the residual spiramycin can be continuously extracted, so that the yield of the spiramycin is improved. However, the impurities are further extracted, so that two kinds of DAC columns are needed to remove the impurities to ensure the product quality.
In a preferred embodiment, in the step (2), the pH value of a back extraction liquid obtained in the spiramycin production is adjusted to 5.8-6.2; preferably, the pH value of a back extraction liquid obtained in the spiramycin production is adjusted to 6.0; and/or, the first DAC column is loaded with 80% of the loading amount.
In a preferred embodiment, in step (2), the first DAC column is eluted after loading with acetone at a concentration of 80% by volume.
20-100% of acetone is adopted to wash away impurities D in the DAC column 1 filler, ensure that the DAC column can be washed clean, the next batch of materials can be normally used, and simultaneously, the method also aims to protect the DAC column 1 filler and avoid the dry bed phenomenon.
In a preferred embodiment, in step (2), the loading time is 2-12min, preferably, the loading time is 6 min; and/or, the elution time is 2-10min, preferably, the elution time is 2 min.
In a preferred embodiment, in step (3), the pH of the first target fraction is adjusted to 7.4 to 8.5; preferably, the pH of the first target fraction is adjusted to 7.7; and/or, separating and purifying by using a second DAC column according to the sample loading amount of 8%; and/or, the step of eluting with buffer salt and acetone comprises: eluting with a first buffer salt, then eluting with a mixed solution of the first buffer salt and first acetone, and finally eluting with a mixed solution of a second buffer salt and second acetone; and/or said first acetone is present at a concentration of 15-25% by volume, most preferably said first acetone is present at a concentration of 20% by volume; and/or the concentration of the second diacetone is 25-40% by volume, and most preferably the concentration of the second diacetone is 30% by volume.
In a preferred embodiment, the first buffer salt is prepared from potassium dihydrogen phosphate and dipotassium hydrogen phosphate; and/or the second buffer salt is prepared by potassium dihydrogen phosphate and oxalic acid.
In a preferred embodiment, the elution time of the first buffer salt is 5-25min, preferably 12 min; the elution time of the mixed solution of the first buffer salt and the first acetone is 15-50min, and the preferable time is 24 min; the elution time of the mixed solution of the second buffer salt and the second diacetone is 10-30min, and the preferable time is 20 min.
In a preferred embodiment, in step (4), the pH of the second target fraction is adjusted to 7.0 to 8.0, and preferably, the pH of the second target fraction is adjusted to 7.5.
In a preferred embodiment, in the step (4), the alkali solution is a NaOH solution; and/or, the time of the heat preservation is 20-50min, preferably, the time of the heat preservation is 30 min.
Compared with the prior art, the invention has the following beneficial effects:
(1) the back extraction liquid obtained in spiramycin production is separated and purified through the first DAC column, the impurity D is adsorbed on the DAC column 1, liquid (first target fraction) which is rich in spiramycin active ingredients and is removed at the same time can be collected, and the content of the impurity D in a spiramycin finished product is reduced; and further separating and purifying the first fraction by a second DAC column, flushing the first fraction by using a mixed solution of acetone and buffer salt prepared from potassium dihydrogen phosphate and dipotassium hydrogen phosphate, passing the collected liquid (the first target fraction) of the column 1 through the second DAC column by using the elution capacity difference of the buffer salt and the acetone solution with different concentrations, adsorbing the spiramycin, the impurity B and the impurity F on the DAC column, after the sample loading is finished, balancing the filler of the DAC column by using the elution of the first buffer salt, removing the impurities (mainly the impurity B and the impurity F) on the DAC column by using the mixed solution of the first buffer salt and the acetone and the mixed solution of the second buffer salt and the acetone, and continuously eluting by using the second buffer salt and the acetone to ensure that the spiramycin is desorbed from the filler of the DAC column 2 to obtain the analytic liquid rich in the spiramycin.
In a word, the invention adopts two times of DAC columns for separation and purification, thereby realizing the reduction of the controllability of impurities B, D and F in the existing extraction production of spiramycin and leading the yield of the spiramycin to reach 20 percent; the method simplifies the original process of washing BA solution by buffer solution for multiple times, reduces water consumption, improves the back extraction yield and product quality, and improves the market competitiveness of spiramycin products.
(2) According to the method, the BA solution is subjected to back extraction twice to obtain the back extraction liquid, the spiramycin can be completely back extracted from the BA solution, and the yield of the spiramycin product is improved on the premise of ensuring the product quality.
Defining:
DAC column: dynamic axial compression column, DAC for short.
BA solution is butyl acetate solution containing spiramycin.
Drawings
FIG. 1 is a high performance liquid chromatogram of control 1 according to the present invention.
FIG. 2 is a high performance liquid chromatogram of example 1 according to the present invention.
FIG. 3 is a high performance liquid chromatogram of control 2 according to the present invention.
FIG. 4 is a high performance liquid chromatogram according to example 2 of the present invention.
FIG. 5 is a high performance liquid chromatogram of control group 3 according to the present invention.
FIG. 6 is a high performance liquid chromatogram of example 3 according to the present invention.
FIG. 7 is a high performance liquid chromatogram of control group 4 according to the present invention.
FIG. 8 is a high performance liquid chromatogram of example 4 according to the present invention.
FIG. 9 is a high performance liquid chromatogram of control group 5 according to the present invention.
FIG. 10 is a high performance liquid chromatogram of example 5 according to the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
The difference of the following comparative examples is that (1) the back extraction solution of spiramycin is prepared without carrying out back extraction twice, but the spiramycin fermentation filtrate is extracted by butyl acetate to form BA solution, the back extraction solution (purified water: potassium dihydrogen phosphate 1L: 13g is prepared and pH is adjusted to 2.60 +/-0.20 by oxalic acid) is slowly added, the pH value is stirred to 6.2-6.3, the mixture is stirred for 20min and kept stand for 40min, the lower-layer aqueous phase is recovered to obtain the back extraction solution, and (2) the back extraction solution is obtained after washing and impurity removal by 0.02 mol/L buffer solution, saturated saline, purified water and the like after the spiramycin fermentation filtrate is extracted by butyl acetate to form BA solution without carrying out DAC column separation and purification twice;
all other aspects are the same.
Example 1
The method for refining the spiramycin specifically comprises the following steps:
(1) back extraction, namely performing butyl acetate extraction on spiramycin fermentation filtrate to form BA solution, adding oxalic acid into 0.1M/L potassium dihydrogen phosphate, adjusting the pH value to 2.5 to obtain primary back extraction solution, slowly adding the primary back extraction solution into the BA solution, stirring to ensure that the pH value is 6.34, adding oxalic acid to adjust the pH value to 4.85, stirring for 20min, standing for 40min, recovering a lower aqueous phase, adding the primary back extraction solution into the upper BA solution until the pH value of the lower back extraction solution is 2.87, performing back extraction, stirring for 20min, standing for 40min, recovering the lower aqueous phase, and combining the two aqueous phases to obtain back extraction solution;
(2) and (3) treatment, separation and purification of a stripping solution: adjusting the pH value of the back extraction liquid to 6.12, carrying out layering and organic membrane filtration to obtain a clarified sample solution, passing the back extraction liquid obtained from the lower water phase through an active carbon column to remove butyl acetate to obtain a first sample loading solution, and carrying out separation and purification on the first DAC column; wherein,
the first DAC column is a chromatographic column with the diameter of 20mm and filled with 40g of filler, the flow rate is 20m L/min, the balance is 10min of pure water, the loading of the effective components accounts for 80 percent (accounting for 32g of the filler), the washing is carried out by changing the sample and washing for 6min, the column is flushed by 80 percent acetone (80: 20) and the column is flushed by 80 percent acetone for 10 min;
the first DAC column collects a first target fraction: loading the sample for 6min, washing with water for 0-6min to 80% acetone for 0-2min (recovery section); impurity-containing fraction: washing with 80% acetone for 2-10min (high D impurity fraction, discarding);
(3) and (3) treating, separating and purifying the first target fraction: adjusting the pH value of the first target fraction recovered from the first DAC column to 7.82 by using a 3M NaOH solution to obtain a second sample solution, and performing separation and purification on the second DAC column; wherein,
a second DAC column, namely a chromatographic column, namely 50DAC (filled with 300g of filler), a flow rate of 50M L/min, a balance of pure water for 20min, a sample loading step of calculating a sample loading volume according to the concentration of the active ingredients of the second sample loading solution, wherein the sample loading amount of the active ingredients accounts for 8% (accounting for 24g of the mass of the filler), a first buffer salt leaching step of leaching for 12min, wherein the first buffer salt is 0.05M potassium dihydrogen phosphate +0.05M dipotassium hydrogen phosphate (pH value is 6.9), a 20% acetone (the first buffer salt is 80: 20 acetone) leaching step of leaching for 24min, and a 30% acetone (buffer salt 2: 70: 30) eluting step of leaching for 20min, wherein the second buffer salt is 0.1M potassium dihydrogen phosphate, and the pH value of oxalic acid is adjusted to 2.5;
the second DAC column collects a second target fraction:
column two collection section 1: leaching with 20% acetone (first buffer salt: 80: 20) for 0-27 min, wherein the pH value of the second column collection section 1 is greater than 7.3, and the contents of impurities F and B are high;
a second column collection section 2: leaching with 30% acetone (second buffer salt: acetone 70: 30) for 0-20 min, wherein the collection section is a second target fraction;
(4) and (3) drying: and (3) after the second target fraction is taken out, adjusting the pH value to 7.5 by using 3M NaOH, distilling under reduced pressure to remove acetone, cooling to room temperature, stirring, slowly dropwise adding NaOH to reach a pH value of 9.5, simultaneously heating to 55 ℃, stirring, keeping the temperature for 30 minutes, filtering, washing by using 3M NaOH to obtain spiramycin wet powder, and drying to obtain spiramycin dry powder.
The detection method of the spiramycin components and related substances is based on a high performance liquid phase detection method, and is shown in European pharmacopoeia EP 9.2. The contents of some related substances and components in the spiramycin dry powder obtained in example 1 are shown in Table 1, wherein the graph obtained in comparative example 1 is shown in figure 1, and the graph obtained in example 1 is shown in figure 2.
TABLE 1
| Categories | Impurity B% | Impurity D% | Impurity F% |
| Comparative example 1 | 1.0 | 1.4 | 1.5 |
| Example 1 | 0.8 | 0.7 | 1.1 |
Example 2
The method for refining the spiramycin specifically comprises the following steps:
(1) back extraction, namely, extracting spiramycin fermentation filtrate by butyl acetate to form BA solution, adding oxalic acid into 0.1M/L potassium dihydrogen phosphate, adjusting the pH value to 2.5 to obtain primary back extraction solution, slowly adding the primary back extraction solution into the BA solution, stirring to measure the pH value to 6.32, adding oxalic acid to adjust the pH value to 4.81, stirring for 20min, standing for 40min, recovering a lower aqueous phase, adding the primary back extraction solution into the upper BA solution until the pH value of the lower back extraction solution is 2.78, performing back extraction, stirring for 20min, standing for 40min, recovering the lower aqueous phase, and combining the two aqueous phases to obtain back extraction solution;
(2) and (3) treatment, separation and purification of a stripping solution: adjusting the pH value of the back extraction liquid to 6.05, carrying out layering and organic membrane filtration to obtain a clarified sample solution, passing the back extraction liquid obtained from the lower water phase through an active carbon column to remove butyl acetate to obtain a first sample solution, and carrying out separation and purification on the first DAC column; wherein,
the first DAC column is a chromatographic column with the diameter of 20mm and filled with 40g of filler, the flow rate is 20m L/min, the balance is 10min of pure water, the loading of the effective components is 100 percent (accounting for 40g of the filler), the washing is carried out after loading, the washing is carried out for 6min, the column flushing is carried out by 80 percent acetone (80: 20) and the column flushing is carried out by 80 percent acetone for 10 min;
the first DAC column collects a first target fraction: target fraction 1: loading the sample for 6min, washing with water for 0-6min to 80% acetone for 0-2 min; (recovery section); impurity-containing fraction: washing with 80% acetone for 2-10min (high D impurity fraction, discarding);
(3) and (3) treating, separating and purifying the first target fraction: adjusting the pH value of the first target fraction recovered from the first DAC column to 7.70 by using a 3M NaOH solution to obtain a second sample loading liquid, and performing separation and purification on the second DAC column; wherein,
a second DAC column, namely a chromatographic column, 50DAC (filled with 300g of filler), a flow rate of 50M L/min, balance of pure water for 20min, sample loading, namely, calculating the sample loading volume according to the concentration of the active ingredients of the second sample loading liquid, wherein the sample loading amount of the active ingredients is 6% (accounting for the mass of the filler, namely, 27g), leaching by using first buffer salt (the first buffer salt is 0.05M potassium dihydrogen phosphate +0.05M dipotassium hydrogen phosphate, and has a pH value of 6.9), leaching for 12min, leaching by using 20% acetone (the buffer salt is 1: 80: 20), leaching for 24min, leaching by using 30% acetone (the second buffer salt is 0.1M potassium dihydrogen phosphate, and the pH value of oxalic acid is adjusted to be 2.5), eluting by using the second buffer salt (the second buffer salt is 70: 30), and leaching for 20 min;
second DAC column collection fraction:
column two collection section 1: leaching with 20% acetone (first buffer salt: acetone 80: 20) for 0-27 min, wherein the pH value of the second column collection section 1 is greater than 7.3, and the content of the impurity F, B is high;
a second column collection section 2: leaching with 30% acetone (second buffer salt: acetone 70: 30) for 0-20 min, wherein the collection section is a second target fraction;
(4) and (3) drying: and (3) after the second target fraction is taken out, adjusting the pH value to 7.5 by using 3M NaOH, distilling under reduced pressure to remove acetone, cooling to room temperature, stirring, slowly dropwise adding NaOH to reach a pH value of 9.5, simultaneously heating to 55 ℃, stirring, keeping the temperature for 30 minutes, filtering, washing by using 3M NaOH to obtain spiramycin wet powder, and drying to obtain spiramycin dry powder.
The detection method of the spiramycin components and related substances is based on a high performance liquid phase detection method, and is shown in European pharmacopoeia EP 9.2. The contents of some related substances and components in the spiramycin dry powder obtained in example 2 are shown in Table 2, wherein the graph obtained in comparative example 2 is shown in figure 3, and the graph obtained in example 2 is shown in figure 4.
TABLE 2
| Categories | Impurity B% | Impurity D% | Impurity F% |
| Comparative example 2 | 1.0 | 1.4 | 1.5 |
| Example 2 | 0.8 | 0.7 | 1.1 |
Example 3
The method for refining the spiramycin specifically comprises the following steps:
(1) back extraction, namely performing butyl acetate extraction on spiramycin fermentation filtrate to form BA solution, adding oxalic acid into 0.1M/L potassium dihydrogen phosphate, adjusting the pH value to 2.3 to obtain primary back extraction solution, slowly adding the primary back extraction solution into the BA solution, stirring to measure the pH value to 6.29, adding oxalic acid to adjust the pH value to 4.77, stirring for 20min, standing for 40min, recovering a lower aqueous phase, adding the primary back extraction solution into the upper BA solution until the pH value of the lower back extraction solution is 2.69, performing back extraction, stirring for 20min, standing for 40min, recovering the lower aqueous phase, and combining the two aqueous phases to obtain back extraction solution;
(2) and (3) treatment, separation and purification of a stripping solution: adjusting the pH value of the back extraction liquid to 6.08, carrying out layering and organic membrane filtration to obtain a clarified sample solution, passing the back extraction liquid obtained from the lower water phase through an active carbon column to remove butyl acetate to obtain a first sample solution, and carrying out separation and purification on the first DAC column; wherein,
the first DAC column is a chromatographic column with the diameter of 20mm and filled with 40g of filler, the flow rate is 20m L/min, the balance is 10min of pure water, the loading of the effective components is 50 percent (accounting for 20g of the filler), the washing is carried out by changing the water after loading for 6min, the column washing is carried out by 80 percent acetone (80: 20) and the column washing is carried out by changing 80 percent acetone for 10 min;
the first DAC column collects a first target fraction: target fraction 1: loading sample for 6min, washing with water for 0-6min to 80% acetone for 0-2 min; (recovery section). Impurity-containing fraction: rinsing with 80% acetone for 2-10min (high D impurity fraction, discarding);
(3) and (3) treating, separating and purifying the first target fraction: regulating the pH value of the first target fraction recycled by the first DAC column to 7.86 by using a 3M NaOH solution to obtain a second sample loading liquid, and performing separation and purification on the second DAC column; wherein,
a second DAC column, namely a chromatographic column, namely 50DAC (filled with 300g of filler), a flow rate of 50M L/min, a balance of pure water for 20min, a sample loading step of calculating a sample loading volume according to the concentration of the active ingredients of the second sample loading solution, wherein the sample loading amount of the active ingredients is 6% (accounting for 18g of the mass of the filler), a first buffer salt leaching step of leaching for 12min, wherein the first buffer salt is 0.05M potassium dihydrogen phosphate +0.05M dipotassium hydrogen phosphate (pH value is 6.9), a 20% acetone (the first buffer salt is 80: 20 acetone) leaching step of leaching for 24min, and a 30% acetone (the second buffer salt is 70: 30) eluting step of leaching for 20min, wherein the second buffer salt is 0.1M potassium dihydrogen phosphate, and the pH value of oxalic acid is adjusted to 2.5;
second DAC column collection fraction:
column two collection section 1: leaching with 20% acetone (buffer salt 1: acetone 80: 20) for 0-27 min, wherein the pH value of the column II collection section 1 is greater than 7.3, and the contents of impurities F and B are high;
a second column collection section 2: a 30% acetone (buffer salt 2: acetone ═ 70: 30) leaching section for 0-20 min, and the collection section is a second target fraction;
(4) and (3) drying: and (3) after the second target fraction is taken out, adjusting the pH value to 7.5 by using 3M NaOH, distilling under reduced pressure to remove acetone, cooling to room temperature, stirring, slowly dropwise adding NaOH to reach the pH value of 9.5, simultaneously heating to 55 ℃, stirring, keeping the temperature for 30 minutes, filtering, washing by using 3M NaOH to obtain spiramycin wet powder, and drying to obtain spiramycin dry powder.
The detection method of the spiramycin components and related substances is based on a high performance liquid phase detection method, and is shown in European pharmacopoeia EP 9.2. The contents of some related substances and components in the spiramycin dry powder obtained in example 3 are shown in Table 3, wherein the graph obtained in comparative example 1 is shown in figure 5, and the graph obtained in example 3 is shown in figure 6.
TABLE 3
| Categories | Impurity B% | Impurity D% | Impurity F% |
| Comparative example 3 | 1.0 | 1.4 | 1.5 |
| Example 3 | 0.8 | 0.7 | 1.2 |
Example 4
The method for refining the spiramycin specifically comprises the following steps:
(1) back extraction, namely performing butyl acetate extraction on spiramycin fermentation filtrate to form BA solution, adding oxalic acid into 0.1M/L potassium dihydrogen phosphate, adjusting the pH value to 2.7 to obtain primary back extraction solution, slowly adding the primary back extraction solution into the BA solution, stirring to measure the pH value to 6.03, adding oxalic acid to adjust the pH value to 4.72, stirring for 20min, standing for 40min, recovering a lower aqueous phase, adding the primary back extraction solution into the upper BA solution until the pH value of the lower back extraction solution is 2.96, performing back extraction, stirring for 20min, standing for 40min, recovering the lower aqueous phase, and combining the two aqueous phases to obtain back extraction solution;
(2) and (3) treatment, separation and purification of a stripping solution: adjusting the pH value of the back extraction liquid to 5.87, layering, filtering by an organic membrane to obtain a clarified sample solution, passing the back extraction liquid obtained from the lower water phase through an active carbon column to remove butyl acetate to obtain a first sample solution, and separating and purifying the first sample solution by a first DAC column; wherein,
the first DAC column is a chromatographic column with the diameter of 20mm and filled with 40g of filler, the flow rate is 20m L/min, the balance is 10min of pure water, the loading amount of active ingredients is 120 percent (accounting for 48g of the filler), the washing is carried out after loading, the washing is carried out for 6min, the column flushing is carried out by 80 percent acetone (80: 20) and the column flushing is carried out by 80 percent acetone for 10 min;
the first DAC column collects a first target fraction: target fraction 1: loading sample for 6min, washing with water for 0-6min to 80% acetone for 0-2 min; (recovery section). Impurity-containing fraction: rinsing with 80% acetone for 2-10min (high D impurity fraction, discarding);
(3) and (3) treating, separating and purifying the first target fraction: regulating the pH value of the first target fraction recycled by the first DAC column to 7.96 by using a 3M NaOH solution in a subsection to obtain a second sample loading liquid, and performing separation and purification on the second DAC column; wherein,
a second DAC column, namely a chromatographic column, namely 50DAC (filled with 300g of filler), a flow rate of 50M L/min, a balance of pure water for 20min, a sample loading step of calculating a sample loading volume according to the concentration of the active ingredients of the second sample loading solution, wherein the sample loading amount of the active ingredients is 10% (accounting for 30g of the mass of the filler), a first buffer salt leaching step of leaching for 12min, wherein the first buffer salt is 0.05M potassium dihydrogen phosphate +0.05M dipotassium hydrogen phosphate (pH value is 6.9), a 20% acetone (the first buffer salt is 80: 20 acetone) leaching step of leaching for 24min, and a 30% acetone (the second buffer salt is 70: 30) eluting step of leaching for 20min, the second buffer salt is 0.1M potassium dihydrogen phosphate, and the pH value of oxalic acid is adjusted to 2.5;
second DAC column collection fraction:
column two collection section 1: leaching with 20% acetone (buffer salt 1: acetone 80: 20) for 0-27 min, wherein the pH value of the column II collection section 1 is greater than 7.3, and the contents of impurities F and B are high;
a second column collection section 2: a 30% acetone (buffer salt 2: acetone ═ 70: 30) leaching section for 0-20 min, and the collection section is a second target fraction;
(4) and (3) drying: and (3) after the second target fraction is taken out, adjusting the pH value to 7.5 by using 3M NaOH, distilling under reduced pressure to remove acetone, cooling to room temperature, stirring, slowly dropwise adding NaOH to reach a pH value of 9.5, simultaneously heating to 55 ℃, stirring, keeping the temperature for 30 minutes, filtering, washing by using 3M NaOH to obtain spiramycin wet powder, and drying to obtain spiramycin dry powder.
The detection method of the spiramycin components and related substances is based on a high performance liquid phase detection method, and is shown in European pharmacopoeia EP 9.2. The contents of some related substances and components in the spiramycin dry powder obtained in example 4 are shown in Table 4, wherein the graph obtained in comparative example 4 is shown in figure 7, and the graph obtained in example 4 is shown in figure 8.
TABLE 4
| Categories | Impurity B% | Impurity D% | Impurity F% |
| Comparative example 4 | 1.0 | 1.4 | 1.5 |
| Example 4 | 0.8 | 0.7 | 1.2 |
Example 5
The method for refining the spiramycin specifically comprises the following steps:
(1) back extraction, namely performing butyl acetate extraction on spiramycin fermentation filtrate to form BA solution, adding oxalic acid into 0.1M/L potassium dihydrogen phosphate, adjusting the pH value to 2.3 to obtain primary back extraction solution, slowly adding the primary back extraction solution into the BA solution, stirring to measure the pH value to 5.71, adding oxalic acid to adjust the pH value to 4.69, stirring for 20min, standing for 40min, recovering a lower aqueous phase, adding the primary back extraction solution into the upper BA solution until the pH value of the lower back extraction solution is 2.92, performing back extraction, stirring for 20min, standing for 40min, recovering the lower aqueous phase, and combining the two aqueous phases to obtain back extraction solution;
(2) and (3) treatment, separation and purification of a stripping solution: adjusting the pH value of the back extraction liquid to 6.09, filtering by an organic membrane to obtain a clarified sample solution, blowing air, removing an organic solvent butyl acetate to obtain a first sample loading solution, and performing separation and purification by a first DAC column; wherein,
the first DAC column is a chromatographic column with the diameter of 20mm and filled with 40g of filler, the flow rate is 20m L/min, the balance is 10min of pure water, the loading of the effective components accounts for 80 percent (accounting for 32g of the filler), the washing is carried out by changing the water after loading for 6min, the column washing is carried out by 80 percent acetone (80: 20) and the column washing is carried out by changing 80 percent of acetone for 10 min;
the first DAC column collects a first target fraction: loading the sample for 6min, washing with water for 0-6min to 80% acetone for 0-2min (recovery section); impurity-containing fraction: washing with 80% acetone for 2-10min (high D impurity fraction, discarding);
(3) and (3) treating, separating and purifying the first target fraction: adjusting the pH value of the first target fraction recovered from the first DAC column to 7.79 by using a 3M NaOH solution to obtain a second sample loading liquid, and performing separation and purification on the second DAC column; wherein,
the second DAC column is a chromatographic column, 50DAC (filled with 300g of filler), the flow rate is 50M L/min, the balance is pure water for 20min, the loading volume is calculated according to the concentration of the active ingredients of the second loading liquid, the loading amount of the active ingredients accounts for 8 percent (accounting for the mass of the filler, namely 24g), the first buffer salt is leached (the first buffer salt is 0.05M potassium dihydrogen phosphate and 0.05M dipotassium hydrogen phosphate, the pH value is 6.9), the leaching is carried out for 12min, the 20 percent acetone (the first buffer salt is 80: 20) is leached, the leaching is carried out for 24min, the second buffer salt is 30 percent acetone (the second buffer salt is 0.1M potassium dihydrogen phosphate, the pH value of oxalic acid is adjusted to be 2.5), the acetone is eluted, the second DAC column is leached, the elution is carried out for 20min, and the second DAC column collection fraction:
column two collection section 1: leaching with 20% acetone (first buffer salt: 80: 20) for 0-27 min, wherein the pH value of the second column collection section 1 is greater than 7.3, and the contents of impurities F and B are high;
a second column collection section 2: leaching with 30% acetone (second buffer salt: acetone 70: 30) for 0-20 min, wherein the collection section is a second target fraction;
(4) and (3) drying: and (3) after the second target fraction is taken out, adjusting the pH value to 7.5 by using 3M NaOH, distilling under reduced pressure to remove acetone, cooling to room temperature, stirring, slowly dropwise adding NaOH to reach a pH value of 9.5, simultaneously heating to 55 ℃, stirring, keeping the temperature for 30 minutes, filtering, washing by using 3M NaOH to obtain spiramycin wet powder, and drying to obtain spiramycin dry powder.
The detection method of the spiramycin components and related substances is based on a high performance liquid phase detection method, and is shown in European pharmacopoeia EP 9.2. The contents of some related substances and components in the spiramycin dry powder obtained in example 5 are shown in the following table 5, wherein the graph obtained in the comparative example 5 is shown in the attached figure 9, and the graph obtained in example 5 is shown in the attached figure 10.
TABLE 5
| Categories | Impurity B% | Impurity D% | Impurity F% |
| Comparative example 5 | 1.0 | 1.4 | 1.5 |
| Example 5 | 0.7 | 0.8 | 1.1 |
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A method for refining spiramycin is characterized by comprising the following steps:
(1) adjusting the pH value of a back extraction liquid obtained in spiramycin production to 5.5-6.5, and after layering, removing butyl acetate from the back extraction liquid obtained in the lower water phase by using activated carbon to obtain a loading solution;
(2) separating and purifying the sample loading solution by adopting a first DAC column according to the sample loading amount of 50-120%, eluting the first DAC column by using acetone, methanol or ethanol with the volume percentage concentration of 20-100% after separation and purification, and collecting fractions obtained in the separation and purification process of the first DAC column as first target fractions;
(3) adjusting the pH value of the first target fraction to 7.0-8.5, then separating and purifying by adopting a second DAC column according to the sample loading amount of 6% -10%, eluting by adopting a mixed solution of buffer salt and acetone after separation and purification, and collecting the fraction obtained in the separation and purification process of the second DAC column as a second target fraction; and
(4) adjusting the pH value of the second target fraction to 7.0-8.0 by using an alkali solution, and removing acetone by reduced pressure distillation; then dropwise adding an alkali solution to adjust the pH value to 8.5-10.5, simultaneously heating to 45-65 ℃, stirring, keeping the temperature, washing with the alkali solution after centrifugal filtration, and drying to obtain the spiramycin dry powder.
2. The process according to claim 1, characterized in that, in step (1), the stripping solution is prepared by a method comprising: extracting spiramycin fermentation filtrate by butyl acetate to form BA solution; then, adding oxalic acid into dipotassium hydrogen phosphate, and adjusting the pH value to 2.5 +/-0.2 to obtain a primary back-extraction liquid; adding the primary back extraction liquid into the BA solution, stirring until the pH value is 6.4 +/-0.3, then adding oxalic acid to adjust the pH value to 4.8 +/-0.2, stirring uniformly, and standing for layering; recovering the lower aqueous phase, adding the primary back-extraction liquid into the upper BA liquid phase again, adjusting the pH value to 2.8 +/-0.3, stirring and mixing uniformly, standing for layering, and recovering the lower aqueous phase; the aqueous phases recovered twice are combined to obtain the final back-extraction solution.
3. The process according to claim 1, wherein in the step (2), the pH of the back-extract obtained in the spiramycin production is adjusted to 5.8-6.2; preferably, the pH value of a back extraction liquid obtained in the spiramycin production is adjusted to 6.0;
and/or, the first DAC column is loaded with 80% of the loading amount.
4. The process of claim 1, wherein in step (2), the first DAC column is eluted with acetone at a concentration of 80% by volume after loading.
5. The process according to claim 1, characterized in that in step (2), the loading time is 2-12min, preferably 6 min;
and/or, the time of elution is 2-10min, preferably, the time of elution is 2 min.
6. The process according to claim 1, characterized in that, in step (3), the first target fraction is adjusted to a pH value of 7.4 to 8.5; preferably, the pH of the first target fraction is adjusted to 7.7;
and/or, separating and purifying by using a second DAC column according to the sample loading amount of 8%;
and/or, the step of eluting with buffer salt and acetone comprises: eluting with a first buffer salt, then eluting with a mixed solution of the first buffer salt and first acetone, and finally eluting with a mixed solution of a second buffer salt and second acetone;
and/or the first acetone has a concentration of 15-25% by volume, most preferably the first acetone has a concentration of 20% by volume;
and/or the volume percentage concentration of the second diacetone is 25-40%, and most preferably, the volume percentage concentration of the second diacetone is 30%.
7. The treatment method according to claim 6, wherein the first buffer salt is formulated with potassium dihydrogen phosphate and dipotassium hydrogen phosphate;
and/or the second buffer salt is prepared by potassium dihydrogen phosphate and oxalic acid.
8. The process according to claim 6, characterized in that the first buffer salt is eluted for a time of 5-25min, preferably for a time of 12 min; the elution time of the mixed solution of the first buffer salt and the first acetone is 15-50min, and the preferable time is 24 min; the elution time of the mixed solution of the second buffer salt and the second diacetone is 10-30min, and the preferable time is 20 min.
9. The process according to claim 1, characterized in that in step (4), the second target fraction is adjusted to a pH value of 7.0-8.0, preferably the second target fraction is adjusted to a pH value of 7.5.
10. The process of claim 1, wherein in step (4), the alkali solution is NaOH solution;
and/or the time for heat preservation is 20-50min, preferably, the time for heat preservation is 30 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010354822.5A CN111499669A (en) | 2020-04-29 | 2020-04-29 | Method for refining spiramycin by adopting two DAC columns |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010354822.5A CN111499669A (en) | 2020-04-29 | 2020-04-29 | Method for refining spiramycin by adopting two DAC columns |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111499669A true CN111499669A (en) | 2020-08-07 |
Family
ID=71868185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010354822.5A Pending CN111499669A (en) | 2020-04-29 | 2020-04-29 | Method for refining spiramycin by adopting two DAC columns |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111499669A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112694508A (en) * | 2020-12-11 | 2021-04-23 | 无锡福祈制药有限公司 | Method for preparing high-purity spiramycin |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5990288A (en) * | 1997-10-21 | 1999-11-23 | Vitro Diagnostics, Inc. | Method for purifying FSH |
| CN103087124A (en) * | 2012-11-21 | 2013-05-08 | 浙江海正药业股份有限公司 | Method for preparing high-purity adriamycin |
| CN105669789A (en) * | 2016-02-23 | 2016-06-15 | 华北制药集团新药研究开发有限责任公司 | Preparation method for norvancomycin |
| US20170174737A1 (en) * | 2014-07-21 | 2017-06-22 | Merck Sharp & Dohme Corp. | Chromatography process for purification of insulin and insulin analogs |
| CN108864229A (en) * | 2018-07-13 | 2018-11-23 | 广东东阳光药业有限公司 | The method for separating the method for berythromycin and preparing berythromycin reference substance |
| CN110746473A (en) * | 2018-07-10 | 2020-02-04 | 浙江华谱新创科技有限公司 | Purification process for reducing content of lincomycin B component |
-
2020
- 2020-04-29 CN CN202010354822.5A patent/CN111499669A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5990288A (en) * | 1997-10-21 | 1999-11-23 | Vitro Diagnostics, Inc. | Method for purifying FSH |
| CN103087124A (en) * | 2012-11-21 | 2013-05-08 | 浙江海正药业股份有限公司 | Method for preparing high-purity adriamycin |
| WO2014079300A1 (en) * | 2012-11-21 | 2014-05-30 | 浙江海正药业股份有限公司 | Method for preparing highly pure doxorubicin |
| US20150299241A1 (en) * | 2012-11-21 | 2015-10-22 | Zhejiang Hisun Pharmaceutical Co., Ltd. | Method for preparing highly pure doxorubicin |
| US20170174737A1 (en) * | 2014-07-21 | 2017-06-22 | Merck Sharp & Dohme Corp. | Chromatography process for purification of insulin and insulin analogs |
| CN105669789A (en) * | 2016-02-23 | 2016-06-15 | 华北制药集团新药研究开发有限责任公司 | Preparation method for norvancomycin |
| CN110746473A (en) * | 2018-07-10 | 2020-02-04 | 浙江华谱新创科技有限公司 | Purification process for reducing content of lincomycin B component |
| CN108864229A (en) * | 2018-07-13 | 2018-11-23 | 广东东阳光药业有限公司 | The method for separating the method for berythromycin and preparing berythromycin reference substance |
Non-Patent Citations (1)
| Title |
|---|
| 李志强 等: "一种阿霉素提取工艺的研究", 《化工管理》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112694508A (en) * | 2020-12-11 | 2021-04-23 | 无锡福祈制药有限公司 | Method for preparing high-purity spiramycin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1074791C (en) | Process for producing calcium D-pantothenate | |
| CN106928323B (en) | A kind of preparation method of high-purity oritavancin key intermediate A82846B | |
| CN101613390A (en) | A method for separating and purifying high-purity cordycepin | |
| CN110183519B (en) | Separation and purification method of dalbavancin key intermediate A40926 | |
| CN106188188A (en) | A kind of preparation method of avilamycin | |
| CN111499669A (en) | Method for refining spiramycin by adopting two DAC columns | |
| CN104846043B (en) | A kind of technique for being separated from zymotic fluid and purifying feldamycin | |
| CN111171096A (en) | Extraction method of pleocidin | |
| CN107043431B (en) | Purification method of bacterial capsular polysaccharide | |
| WO2016004848A1 (en) | Fidaxomicin purification method | |
| CN102311486A (en) | Method for separating and extracting enramycin by using macroporous weakly-acidic cationic resin | |
| CN113717236B (en) | Separation and purification method of hyaluronic acid | |
| CN104610282A (en) | Method for purifying cefazolin acid | |
| CN103936846B (en) | A kind of purification process of protamine sulfate | |
| CN112079941A (en) | Method for preparing heparin sodium and heparan sulfate sodium from crude heparin sodium | |
| CN112409426B (en) | Preparation method of sisomicin sulfate | |
| CN113801174B (en) | Process for recovering breviscapine from breviscapine acid precipitation waste liquid | |
| CN101838315A (en) | Method for separating Ramoplanin | |
| CN107778357A (en) | A kind of extraction of Pneumocandin B0, purification process | |
| CN105819444A (en) | Composite activated carbon and its application in purifying tacrolimus | |
| CN113045611A (en) | Preparation method of high-purity lincomycin hydrochloride | |
| CN112480127A (en) | Novel method for producing mitomycin | |
| CN101450960B (en) | Abomacetin extraction by salting-out method | |
| CN113416223B (en) | Method for separating and purifying salidroside and product thereof | |
| CN120699072A (en) | A method for purifying tylvalosin tartrate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200807 |
|
| RJ01 | Rejection of invention patent application after publication |