CN117500810A - Two-step resin purification process of ansamitocins - Google Patents
Two-step resin purification process of ansamitocins Download PDFInfo
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- 229920005989 resin Polymers 0.000 title claims abstract description 127
- 239000011347 resin Substances 0.000 title claims abstract description 127
- 238000000746 purification Methods 0.000 title claims abstract description 23
- 238000004458 analytical method Methods 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 90
- 238000010828 elution Methods 0.000 claims description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 238000011068 loading method Methods 0.000 claims description 16
- 238000004587 chromatography analysis Methods 0.000 claims description 15
- OPQNCARIZFLNLF-UHFFFAOYSA-N ansamitocin P-3 Natural products CN1C(=O)CC(OC(=O)C(C)C)C2(C)OC2C(C)C(OC(=O)N2)CC2(O)C(OC)C=CC=C(C)CC2=CC(OC)=C(Cl)C1=C2 OPQNCARIZFLNLF-UHFFFAOYSA-N 0.000 claims description 14
- OPQNCARIZFLNLF-JBHFWYGFSA-N ansamitocin P3 Chemical compound CO[C@@H]([C@@]1(O)C[C@H](OC(=O)N1)[C@@H](C)[C@@H]1O[C@@]1(C)[C@@H](OC(=O)C(C)C)CC(=O)N1C)\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 OPQNCARIZFLNLF-JBHFWYGFSA-N 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- PVNFMCBFDPTNQI-UIBOPQHZSA-N [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] acetate [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] 3-methylbutanoate [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] 2-methylpropanoate [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] propanoate Chemical compound CO[C@@H]1\C=C\C=C(C)\Cc2cc(OC)c(Cl)c(c2)N(C)C(=O)C[C@H](OC(C)=O)[C@]2(C)OC2[C@H](C)[C@@H]2C[C@@]1(O)NC(=O)O2.CCC(=O)O[C@H]1CC(=O)N(C)c2cc(C\C(C)=C\C=C\[C@@H](OC)[C@@]3(O)C[C@H](OC(=O)N3)[C@@H](C)C3O[C@@]13C)cc(OC)c2Cl.CO[C@@H]1\C=C\C=C(C)\Cc2cc(OC)c(Cl)c(c2)N(C)C(=O)C[C@H](OC(=O)C(C)C)[C@]2(C)OC2[C@H](C)[C@@H]2C[C@@]1(O)NC(=O)O2.CO[C@@H]1\C=C\C=C(C)\Cc2cc(OC)c(Cl)c(c2)N(C)C(=O)C[C@H](OC(=O)CC(C)C)[C@]2(C)OC2[C@H](C)[C@@H]2C[C@@]1(O)NC(=O)O2 PVNFMCBFDPTNQI-UIBOPQHZSA-N 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000003960 organic solvent Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000005086 pumping Methods 0.000 claims description 7
- 239000012074 organic phase Substances 0.000 claims description 5
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 229930126263 Maytansine Natural products 0.000 claims description 2
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 claims description 2
- 239000000047 product Substances 0.000 description 20
- 238000005406 washing Methods 0.000 description 20
- 238000000034 method Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000000706 filtrate Substances 0.000 description 10
- 239000000049 pigment Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 239000012535 impurity Substances 0.000 description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 239000000945 filler Substances 0.000 description 6
- 238000010898 silica gel chromatography Methods 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000009776 industrial production Methods 0.000 description 4
- QWPXBEHQFHACTK-KZVYIGENSA-N (10e,12e)-86-chloro-12,14,4-trihydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-15,16-dihydro-14h-7-aza-1(6,4)-oxazina-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-6-one Chemical compound CN1C(=O)CC(O)C2(C)OC2C(C)C(OC(=O)N2)CC2(O)C(OC)\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 QWPXBEHQFHACTK-KZVYIGENSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- QWPXBEHQFHACTK-UHFFFAOYSA-N Maytansinol Natural products CN1C(=O)CC(O)C2(C)OC2C(C)C(OC(=O)N2)CC2(O)C(OC)C=CC=C(C)CC2=CC(OC)=C(Cl)C1=C2 QWPXBEHQFHACTK-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
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- 238000004440 column chromatography Methods 0.000 description 2
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- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002148 esters Chemical group 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- -1 isobutyryl Chemical group 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 235000012239 silicon dioxide Nutrition 0.000 description 2
- 239000002910 solid waste Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 239000003242 anti bacterial agent Substances 0.000 description 1
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- 230000000903 blocking effect Effects 0.000 description 1
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- 230000030833 cell death Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
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- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/18—Bridged systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Saccharide Compounds (AREA)
Abstract
The two-step resin purification process of ansamitocins is provided, and the target product is obtained by carrying out multi-step resin extraction and analysis, merging and concentration post-treatment, wherein the resin extraction and analysis are carried out at least twice, the yield can reach more than 70%, and the purity can reach more than 85%.
Description
The invention belongs to the field of biological pharmacy, relates to extraction and purification of ansamitocins of maytansinoids, and in particular relates to a method for purifying ansamitocins by two-step resin in fermentation culture.
Ansamitocins are maytansine antibiotics which are mainly produced by fermentation of microorganisms such as actinomycetes orange and have extremely strong pharmacological activities such as anti-tumor, anti-tubercle bacillus and antibacterial activities.
Ansericin P-3 is a major fermentation product that inhibits cell death by blocking microtubule formation thereby preventing cell mitosis, and has significant antitumor effects in vitro and in tumor-bearing animals.
The chemical structural formula of ansamitocins is:
the ansamitocins produced by the fermentation process are ansamitocins having different ester substituents at C-3, including P-3 (isobutyryl), P-3 '(n-isoacyl), P-2 (propionyl), P-4 (isovaleryl), P-4' (n-valeryl). These various ester substitutions can each be reduced to C-3 maytansinol (P-0), which is a precursor for the synthesis of maytansinol. In addition, some ansamitocins which cannot be reduced to maytansinol are produced by fermentation and need to be removed.
Most patents and literature currently describe fermentation formulations with little introduction to the purification process, and are primarily addressed by the following prior art.
CN108276427a discloses a method for purifying ansamitocins, which comprises the steps of obtaining fermentation broth, extracting with solvent (ethyl acetate, dichloromethane, acetonitrile, etc.), concentrating, neutral alumina column chromatography (ethyl acetate/petroleum ether elution), concentrating, and the final product purity is 80%.
CN104745655a discloses a method for purifying ansamitocins by chromatography on macroporous resins (pH 6-7, solvents water and organic solvents, the macroporous resins being selected from the group consisting of Amberlite XAD-4, XAD-16, diaion HP20JIP21 and Sepabeads sp825 s P850.S P70.S P700), silica gel/alumina column chromatography (ethyl acetate and hexane elution), poor solvent crystallization (heptane, hexane).
The remainder of the literature describes sporadically the use of silica gel chromatography and poor solvent crystallization (n-hexane, heptane, etc.) during the preparation.
The steps of separation and purification are complex, the method is not suitable for industrial production, most of normal phase filler is difficult to recycle and reuse for mixed solvent, and the purity of the separated target product is low or the yield is low. We need to develop a process with high yield, high purity and low cost suitable for industrial production.
Disclosure of Invention
The inventors found in the study that a high purity fraction containing P-3 can be obtained by using resin chromatography and high pressure chromatography through screening, based on the polarity difference between ansamitocin P-3 and its analogues and other impurities in the fermentation broth. And finally concentrating and filtering the fraction to obtain a crude product, and precipitating and drying again to obtain a product with higher purity. In addition, the purification process uses a single solvent, has low cost and is suitable for industrial production. The purification method uses resin chromatography and C8/C18 modified silica gel chromatography, wherein the C8/C18 modified silica gel chromatography can be recycled, and the problem of large solid waste caused by using a large amount of pure silica gel chromatography (pure silica gel chromatography can only be used once) in the conventional process can be solved. In addition, the C1-C5 organic solvent used in the purification method has higher boiling point and higher safety compared with heptane and the like used in the conventional process.
The invention provides a purification method of ansamitocin P-3, which specifically obtains a target product through multi-step resin extraction and analysis, merging and concentration post-treatment.
The ansamitocins according to the invention may be fermentatively synthesized or naturally occurring ansamitocins. The ansamitocin liquid is obtained by a conventional fermentation technology, and can be an ansamitocin fermentation liquid, an ansamitocin aqueous solution, an ansamitocin filtrate obtained by filtration, or an ansamitocin supernatant obtained by centrifugation.
The macroporous resin is used, the pore diameter of the macroporous resin is matched with the molecular weight of ansamitocins, and the adsorption efficiency can be ensured; and the resin can be used in a large scale, can be recycled, is beneficial to the large-scale production of the whole extraction process and saves the cost.
Aiming at the current situation that most of normal phase fillers are mixed solvents in the purification technology in the prior art, the recovery and the application are difficult, and the purity of the target product is low or the yield is low, the reverse phase fillers are used for preparing the high-pressure chromatography, and C18 fillers and the preparation method are selected from the fillers, so that the purification method has the advantages of good separation effect, high purity, safe operation and recoverable fillers.
The yield of the ansamitocin P-3 purified by the method provided by the invention can reach more than 70%, and the purity can reach more than 85% stably.
The invention provides a purification method of ansamitocin P-3, which comprises the steps of resin extraction, analysis, merging and concentration, and post-treatment to obtain a target product ansamitocin P-3.
As a specific embodiment, the resin is a macroporous resin.
As a specific embodiment, the resin extraction and resolution is a multi-step extraction and multi-step resolution, including at least two resin extractions and resolutions.
As a specific embodiment, the resin extraction and resolution is a two-step extraction and two-step resolution.
The invention further provides a purification method of ansamitocin P-3, which comprises the following steps:
a. the first step is resin extraction: carrying out resin adsorption on the ansamitocin fermentation liquor by using macroporous resin;
b. first step, resin analysis: c, carrying out resin analysis on the resin obtained in the step a by using an organic solvent, carrying out chromatographic elution, and collecting and combining chromatographic liquids;
c. and a second step of resin extraction: b, pumping the chromatographic liquid obtained in the step b into a resin column pre-filled with resin;
d. and a second step of resin analysis: c, carrying out resin analysis on the resin obtained in the step c by using an organic solvent, carrying out chromatographic elution, and collecting and combining chromatographic liquids;
e. post-treatment: and d, merging and concentrating the chromatographic liquid obtained in the step d, and performing post-treatment to obtain the target product ansamitocin P-3.
As a specific embodiment, the macroporous resin used in the step a is selected from LX-316, LX-392, LX-218, LX-30, LX-60, LX-T28, LX-T81, LX-T83, HZ-818, HZ-816, HZ-801, HZ-835.
As a specific embodiment, the macroporous resin used in step a is preferably LX-60 resin.
As a specific embodiment, the flow rate of the ansamitocin fermentation liquid in the step a into the resin column is 0.5-5 BV/h.
As a specific embodiment, the flow rate of the ansamitocin fermentation broth fed into the resin column in the step a is preferably 1-3 BV/h.
In a specific embodiment, the resin loading in the step a is 1 to 30g/L, preferably 10 to 20g/L.
In a specific embodiment, the resin loading in the step a is preferably 10 to 20g/L.
As a specific embodiment, the organic solvent in the step b is selected from any one or a mixture of two or more of ethanol, acetonitrile, methanol, acetone and ethyl acetate.
As a specific embodiment, the organic solvent in the step b is preferably ethanol.
As a specific embodiment, the flow rate of the resin analysis in the step b is 0.5-5 BV/h.
As a specific embodiment, the flow rate of the resin analysis in the step b is preferably 1-3 BV/h.
As a specific embodiment, the resin chromatographic elution in the step b is isocratic elution or gradient elution, wherein the elution range of the organic phase is 10-100%.
In a specific embodiment, the resin chromatography in the step b is preferably performed by eluting with 20% ethanol for 2 to 4CV and then with 100% ethanol for 2 to 4CV.
As a specific embodiment, the macroporous resin used in step c is selected from the group consisting of LX-316, LX-392, LX-218, LX-30, LX-60, LX-T28, LX-T81, LX-T83, HZ-818, HZ-816, HZ-801, HZ-835.
As a specific embodiment, the macroporous resin used in step c is preferably HZ-818 resin.
In a specific embodiment, the flow rate of the eluent in the step c into the resin column is 0.5-5 BV/h.
As a specific embodiment, the flow rate of the eluent in the step c into the resin column is preferably 2BV/h.
As a specific embodiment, the resin chromatography loading amount in the step c is 5g/L to 10g/L.
As a specific embodiment, the organic solvent in the step d is selected from any one or a mixture of two or more of ethanol, acetonitrile, methanol, acetone and ethyl acetate.
As a specific embodiment, the organic solvent in the step d is preferably ethanol.
As a specific embodiment, the flow rate of the resin analysis in the step d is 0.5-5 BV/h.
As a specific embodiment, the flow rate of the resin analysis in the step d is preferably 1-3 BV/h.
As a specific embodiment, the resin chromatographic elution in the step d is isocratic elution or gradient elution, wherein the elution range of the organic phase is 10-100%.
In a specific embodiment, the resin chromatography in the step d is preferably performed by eluting with 30% ethanol for 2-4 CV and then with 80% ethanol for 2-10 CV.
As a specific embodiment, the resin chromatography in the step d is more preferably performed by eluting 3CV with 30% ethanol and then 6CV with 80% ethanol.
As a specific embodiment, the elution is gradient elution, wherein the elution gradient of the organic phase is 10-100%, and the aqueous phase is selected from aqueous solutions of acetic acid, phosphoric acid and trifluoroacetic acid.
As a specific embodiment, the acid concentration in the aqueous phase of the gradient elution ranges from 0 to 5%; as a specific embodiment, the acid concentration in the aqueous phase of the gradient elution is preferably in the range of 0.05 to 0.2%.
The beneficial effects of the invention are as follows:
1. the invention uses two steps of resin extraction, and can finish the purification of more than 85% purity products by using two different macroporous resins, and has the outstanding advantages of low price of resin raw materials, low equipment requirement, and product quality control meeting GMP standard, and can be applied to small-scale laboratories and industrial production. Compared with the purification level about 80% recorded in the prior art, the method provided by the invention has the advantages that recrystallization is not used yet, the steps are simple and convenient, the recovery is easy, the steps of alumina or silica gel chromatography and the like are not needed to be introduced, and the solid waste pollution is avoided.
2. The solvent in the process is single, the solvent adaptation among the steps minimizes the possibility of introducing impurities from the solvent, the cost is low, the operation is simple and convenient, the environment is friendly, the purity of the purified ansamitocin P-3 can be ensured to be more than 85% on the basis that the recovery rate of fermentation liquor reaches 70%, and the purity of the P series homolog can be ensured to be more than 95%.
FIG. 1-HPLC diagram of fermentation broth;
FIG. 2-HPLC chart of first step resin analysis solution;
FIG. 3-HPLC chart of second step resin analysis solution;
FIG. 4-HPLC plot of the final product after drying.
The experimental method without specific conditions noted in the embodiments of the present invention is generally conventional conditions or conditions suggested by the manufacturer of the raw materials or goods; reagents of unspecified origin are typically conventional reagents commercially available or may be prepared from known reagents by conventional methods.
The present invention will be described in further detail with reference to specific examples.
Example 1
After the ansamitocin fermentation liquid is obtained by a biological fermentation method, solid-liquid separation is carried out by using a plate frame, and the normalized peak area percentage of the product in the fermentation liquid is 42.81%. The plate and frame filtrate was pumped into a first resin column (the resin column was pre-packed with LX-60 resin) and the column loading was 2BV/h with a loading of 10g/L. After the column is finished, washing 3BV with 20% edible ethanol, washing away partial pigment and impurity protein, resolving with edible ethanol, collecting resolving solution 0.5BV, directly pumping into a preparation tank, collecting 2BV, and collecting the product with normalized peak area percentage of 55.74%;
the resin chromatography combined solution obtained in the last step is added with equal volume of water and stirred for 10 minutes. Then the mixture was injected into a resin chromatographic column (the resin column was pre-packed with HZ-818 resin) in the second step at a flow rate of 2BV/h and a loading of 10g/L. After the column is finished, washing 3BV with 30% edible ethanol, washing off residual pigment and foreign protein, resolving with 80% ethanol, collecting after resolving 0.5BV, detecting, and main peak purity 85.18%.
All resin chromatograms were combined, concentrated to dryness in vacuo, dissolved in ethanol, then precipitated with water and dried in vacuo (25 ℃ C., 12 h). The target product ansamitocin P-3 is obtained, and the normalized peak area percentage of the main peak is 91.82 percent.
Example 2
Plate and frame filtrate was obtained as in example 1, and the plate and frame filtrate was fed into a first resin column (the resin column was pre-packed with HZ-801 resin) at a flow rate of 2BV/h on the column with a loading of 10g/L. After the column is finished, washing 3BV with 20% edible ethanol, washing away partial pigment and impurity protein, resolving with edible ethanol, collecting resolving solution 0.5BV, directly pumping into a preparation tank, collecting 2BV, and collecting the product with normalized peak area percentage of 50.10%;
the resin chromatography combined solution obtained in the last step is added with equal volume of water and stirred for 10 minutes. Then the mixture was injected into a resin chromatographic column (the resin column was pre-packed with HZ-835 resin) in the second step at a flow rate of 2BV/h and a loading of 10g/L. After the column is finished, washing 3BV with 30% edible ethanol, washing off residual pigment and foreign protein, resolving with 80% ethanol, collecting after resolving 0.5BV, detecting, and main peak purity 65.46%.
All resin chromatograms were combined, concentrated to dryness in vacuo, dissolved in ethanol, then precipitated with water, dried (vacuum dried, 25 ℃ C., 12 h). The target product ansamitocin P-3 is obtained, and the normalized peak area percentage of the main peak is 75.54 percent.
Example 3
Plate and frame filtrate was obtained as in example 1, and the plate and frame filtrate was fed into a first resin column (the resin column was pre-packed with LX-60 resin) at a flow rate of 2BV/h on the column with a loading of 20g/L. After the column is finished, washing 3BV with 20% edible ethanol, washing away partial pigment and impurity protein, resolving with edible ethanol, collecting resolving solution 0.5BV, directly pumping into a preparation tank, collecting 2BV, and collecting the product with normalized peak area percentage of 44.48%;
the resin chromatography combined solution obtained in the last step is added with equal volume of water and stirred for 10 minutes. Then the mixture is injected into a resin chromatographic column (the resin column is pre-filled with HZ-818 resin) in the second step, the flow rate is 2BV/h, and the loading is 20g/L. After the column is finished, washing 3BV with 30% edible ethanol, washing off residual pigment and foreign protein, resolving with 80% ethanol, collecting after resolving 0.5BV, and detecting, wherein the main peak purity is 55.13%.
All resin chromatograms were combined, concentrated to dryness in vacuo, dissolved in ethanol, then precipitated with water, dried (vacuum dried, 25 ℃ C., 12 h). The target product ansamitocin P-3 is obtained, and the normalized peak area percentage of the main peak is 65.75 percent.
Example 4
Plate and frame filtrate was obtained as in example 1, and the plate and frame filtrate was fed into a first resin column (the resin column was pre-packed with LX-60 resin) at a flow rate of 3BV/h and a loading of 10g/L. After the column is finished, washing 3BV with 20% edible ethanol, washing away partial pigment and impurity protein, resolving with edible ethanol, collecting resolving solution 0.5BV, directly pumping into a preparation tank, collecting 2BV, and collecting the product with normalized peak area percentage of 52.90%;
the resin chromatography combined solution obtained in the last step is added with equal volume of water and stirred for 10 minutes. Then the mixture was injected into a resin chromatographic column (the resin column was pre-packed with HZ-818 resin) in the second step at a flow rate of 3BV/h and a loading of 10g/L. After the column is finished, washing 3BV with 30% edible ethanol, washing off residual pigment and foreign protein, resolving with 80% ethanol, collecting after resolving 0.5BV, detecting, and main peak purity 68.87%.
All resin chromatograms were combined, concentrated to dryness in vacuo, dissolved in ethanol, then precipitated with water, dried (vacuum dried, 25 ℃ C., 12 h). The target product ansamitocin P-3 is obtained, and the normalized peak area percentage of the main peak is 82.25 percent.
Example 5
Plate and frame filtrate was obtained as in example 1, and the plate and frame filtrate was fed into a first resin column (the resin column was pre-packed with LX-60 resin) at a flow rate of 2BV/h on the column with a loading of 10g/L. After the column is finished, washing 3BV with 15% edible ethanol, washing away partial pigment and impurity protein, resolving with 80% acetonitrile, collecting resolving solution 0.5BV, directly pumping into a preparation tank, collecting 2BV, and collecting the product with normalized peak area percentage of 54.13%;
the resin chromatography combined solution obtained in the last step is added with equal volume of water and stirred for 10 minutes. Then the mixture was injected into a resin chromatographic column (the resin column was pre-packed with HZ-818 resin) in the second step at a flow rate of 2BV/h and a loading of 10g/L. After the column is finished, washing 3BV with 20% edible ethanol, washing off residual pigment and impurity protein, resolving with 70% acetonitrile, collecting after resolving 0.5BV, detecting, and the main peak purity is 80.45%.
All resin chromatograms were combined, concentrated to dryness in vacuo, dissolved in ethanol, then precipitated with water, dried (vacuum dried, 25 ℃ C., 12 h). The target product ansamitocin P-3 is obtained, and the normalized peak area percentage of the main peak is 88.45 percent.
The above embodiments are only for understanding the method and core idea of the present invention, and do not limit the scope of the present invention. It will be apparent to those skilled in the art that any possible variations or substitutions can be made without departing from the spirit of the invention.
Claims (7)
- A resin purification process of ansamitocin comprises the steps of resin extraction and analysis, merging and concentrating, wherein the resin extraction and analysis are multi-step extraction and multi-step analysis, and the resin extraction and analysis are performed at least twice.
- A purification process as claimed in claim 1, comprising the steps of:a. the first step is resin extraction: carrying out resin adsorption on the ansamitocin fermentation liquor by using macroporous resin;b. first step, resin analysis: c, carrying out resin analysis on the resin obtained in the step a by using an organic solvent, carrying out chromatographic elution, and collecting and combining chromatographic liquids;c. and a second step of resin extraction: b, pumping the chromatographic liquid obtained in the step b into a second resin column which is preloaded with resin;d. and a second step of resin analysis: c, carrying out resin analysis on the resin obtained in the step c by using an organic solvent, carrying out chromatographic elution, and collecting and combining chromatographic liquids;e. post-treatment: and d, merging and concentrating the chromatographic liquid obtained in the step d, and performing post-treatment to obtain the target product ansamitocin P-3.
- A purification process as claimed in claim 2, wherein the macroporous resin used in step a is selected from LX-316, LX-392, LX-218, LX-30, LX-60, LX-T28, LX-T81, LX-T83, HZ-818, HZ-816, HZ-801, HZ-835, preferably LX-60 resins;and/or, the flow rate of the ansamitocin fermentation liquor in the step a, which is injected into the resin column, is 0.5-5 BV/h, preferably 1-3 BV/h;and/or the resin loading in the step a is 1-30 g/L, preferably 10-20 g/L.
- A purification process as claimed in claim 2, wherein the organic solvent in step b is selected from any one or a mixture of two or more of ethanol, acetonitrile, methanol, acetone, ethyl acetate, preferably ethanol;and/or the flow rate of the resin analysis in the step b is 0.5-5 BV/h, preferably 1-3 BV/h.And/or, the resin chromatographic elution in the step b is isocratic elution or gradient elution, wherein the elution range of the organic phase is 10-100%, preferably, 20% ethanol is used for eluting 2-4 CV, and then 100% ethanol is used for eluting 2-4 CV.
- A purification process as claimed in claim 2, wherein the macroporous resin used in step c is selected from LX-316, LX-392, LX-218, LX-30, LX-60, LX-T28, LX-T81, LX-T83, HZ-818, HZ-816, HZ-801, HZ-835, preferably HZ-818 resin;and/or, the flow rate of the eluent in the step c entering the resin column is 0.5-5 BV/h, preferably 2BV/h;and/or, the loading amount of the resin chromatography in the step c is 5g/L to 10g/L.
- A purification process as claimed in claim 2, wherein the organic solvent in step d is selected from any one or a mixture of two or more of ethanol, acetonitrile, methanol, acetone, ethyl acetate, preferably ethanol;and/or the flow rate of the resin analysis in the step d is 0.5-5 BV/h, preferably 1-3 BV/h;and/or, the resin chromatographic elution in the step d is isocratic elution or gradient elution, wherein the elution range of the organic phase is 10-100%, preferably, 30% ethanol is used for eluting 2-4 CV, and then 80% ethanol is used for eluting 2-10 CV; more preferably, 3CV is eluted with 30% ethanol followed by 6CV with 80% ethanol.
- Use of the ansamitocin purification process according to any one of claims 1 to 6 for the preparation of maytansine.
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| CN202110678428 | 2021-06-18 | ||
| CN2021106784281 | 2021-06-18 | ||
| PCT/CN2022/096846 WO2022262590A1 (en) | 2021-06-18 | 2022-06-02 | Two-step resin purification process of ansamitocin |
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| WO2007067698A2 (en) * | 2005-12-08 | 2007-06-14 | Millennium Pharmaceuticals, Inc. | Isolation of ansamitocins |
| CN105713069B (en) * | 2016-03-11 | 2019-01-15 | 浙江大学 | A kind of purification process of bacilysin |
| CN110051705B (en) * | 2019-05-08 | 2021-07-30 | 山西省人民医院 | Method for extracting and purifying inonotus obliquus polyphenol |
| CN110590883B (en) * | 2019-09-09 | 2020-11-13 | 艾美科健（中国）生物医药有限公司 | Method for extracting and separating spinosad from saccharopolyspora spinosa fermentation liquor by adopting resin |
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