CN110156876A - A kind of high-purity A40926B0 preparation method of suitable industrialized production - Google Patents
A kind of high-purity A40926B0 preparation method of suitable industrialized production Download PDFInfo
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- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
- C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
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Abstract
The invention belongs to bio-pharmaceutical separating and purifying technology fields, and in particular to the preparation method of the high purity product of glycopeptide antibiotics Dalbavancin precursor A40926 discloses a kind of high-purity A40926B0 preparation method of suitable industrialized production.A40926 fermentation liquid is pre-processed first, micro-filtration, ultrafiltration and nanofiltration are then carried out using film filtering, initial gross separation purifying and concentration are carried out to A40926 fermentation liquid；Sour precipitating is carried out again, is finally purified with preparative liquid chromatography preparation, and by being concentrated under reduced pressure and being lyophilized the high-purity A40926B0 for obtaining purity and being greater than 99%.
Description
Technical field
The invention belongs to bio-pharmaceutical separating and purifying technology fields, and in particular to glycopeptide antibiotics Dalbavancin precursor
The preparation method of the high purity product of A40926.
Background technique
Dalbavancin (Dalbavancin), also referred to as dalbavancin are before being obtained by microorganism Ye Ye village's actinomycete fermentation
Body object A40926 B0 is through chemically synthesized second generation of glycopeptide antibiotics.Dalbavancin is in May, 2014 by U.S. FDA batch
Quasi- listing, for treating acute bacterial caused by gram-positive bacteria (including methicillin-resistant staphylococcus aureus, MRSA)
Property skin and skin structure infection.Currently, domestic imitation medicine is not gone public also, sharing includes Zhongmei Huadong Pharmaceutical Co., Ltd. Hangzhou
The product of 4 medicines enterprise inside is in clinical stage.Dalbavancin is similar to the molecular structure of teicoplanin and physicochemical property, but
With wider array of antimicrobial spectrum and stronger antibacterial activity.A40926 is the precursor of Dalbavancin, the village You Yeye actinomycesNonomuraea spDalbavancin is made through esterification, amidation and hydrolysis after ATCC39727 fermentation obtains.But A40926
It is made of PA, PB, A, B0 and B1, wherein B0 is the precursor for synthesizing Dalbavancin, these structures of matter are similar with property, give
Isolating and purifying for B0 brings very big difficulty.Therefore preparation high-purity A40926 B0 component is the pass of large-scale production Dalbavancin
Key.Chinese invention patent CN107365357A discloses a kind of method for preparing purified of glycopeptide antibiotics intermediate A 40926,
Preparation method described in the patent includes alkalization, ceramic membrane filter, polyamide chromatography, isoelectric precipitation, redissolution and gel chromatography, pure
Degree reaches 95% or more.The preparation method step is more, and needs more chromatographic enrichment, and filler polyamide used and gel price are higher,
And polyamide filler is unstable to acid, is readily incorporated impurity, influences drug quality and safety.Chinese invention patent
CN101851277A discloses a kind of method for preparing purified of dalbavancin key intermediate A40926B0 component.The invention is related to
Method mainly for A40926B0 component crude product further purification, including gel chromatography, macroporous absorption chromatography and reversed-phase column
The B0 product purity of chromatography, acquisition is greater than 97%.The invention is needed through three kinds of normal pressure column chromatographies, and process is complicated, and mainly
A40926 B0 crude product is purified, the first purifying process of fermentation liquid is not directed to.
Summary of the invention
The main object of the present invention is to establish the high-purity A40926 B0 that suitable industrialized production needs and isolate and purify
Method solves the critical problem in existing Dalbavancin production, reduces production cost.
In order to achieve the above object, the present invention provides a kind of high-purity A40926 B0's for being suitable for industrialized production
Preparation method, this method comprises the following steps:
(1) microbial fermentation is terminated according to fermentation parameter, and ensures that A40926 fermentation liquid character is excellent, especially thallus is not
Self-dissolving can occur, 100-400 ppm macromolecular decolorizing flocculant is added, stand 1-2 h, the residue precipitated after mixing evenly
Thallus and supernatant.Fermented supernatant fluid is transferred in membrane filtration system fluid reservoir using press over system or self priming pump.Remove supernatant
The sodium hydroxide solution that 5-10% is added in the later remaining thallus of liquid adjusts pH value to 10-11, and remaining thallus volume 3- is then added
5 times of low-carbon alcohol solution stirs 30-90 minutes, obtains low-carbon alcohols soak.
(2) the low-carbon alcohols soak in step (1) is filtered by ceramic micro filter film, mycelium is removed, by degerming
Low-carbon alcohols soak afterwards is mixed with the supernatant in membrane filtration system storage tank, obtains mixed liquor, and mixed liquor progress ultrafiltration is gone
It is miscellaneous to obtain ultrafiltration mixed liquor；Ultrafiltration mixed liquor is subjected to nanofiltration removal of impurities again, obtains nanofiltration mixed liquor.Filter operation described in this step
Selecting filtering membrane material is metal film or ceramic membrane or natural macromolecule membrane or synthetic polymeric membrane, the filtering membrane material
The pore diameter range of matter is 50-200 nanometers, preferably 50-100 nanometers, 50-80 nanometers more preferable, and the control of film filtration temperature exists
35-45 DEG C, no more than 50 DEG C.Ultrafiltration membrane material selection metal film, ceramic membrane, natural macromolecule membrane or synthetic polymeric membrane,
It is preferred that natural macromolecule membrane or synthetic polymeric membrane, more preferable synthetic polymeric membrane.The molecular cut off of film successively uses 2 kinds of rule
Lattice are first filtered with 25-30KD filter membrane, then are filtered with the filter membrane that molecular cut off is 5-10KD, preferably 5-8KD, more
It is preferred that 5-6KD；Nanofiltration membrane material selection natural macromolecule membrane or synthetic polymeric membrane, preferably synthetic polymeric membrane are more preferably poly-
Styrene, Kynoar or teflon membrane filter.The molecular cut off of film is 300-1000, preferably 300-500.
(3) with pH to 3-5, the preferably 3.0-3.5 of the nanofiltration mixed liquor in dilute hydrochloric acid regulating step (2), 60-120 is stood
Minute, preferably 120 minutes.It is filtered under diminished pressure and is collected filter cake, filter cake is dissolved in sodium carbonate-bicarbonate buffer solution, is made
A40926 sample solution；The pH value of the sodium carbonate-bicarbonate buffer solution is 10-11 and the ethyl alcohol containing 5-15%.
(4) preparative liquid chromatography column is installed and fraction collection device, setting program is collected according to peak.Preparative liquid chromatography institute
It is that perhaps the ratio of the aqueous solution acetonitrile of methanol or methanol and water is 10:90-30:70 to acetonitrile with mobile phase.It will be in (3)
A40926 sample solution upper prop high pressure preparative liquid chromatography, first with the chromatographic column that balances each other is flowed, equilibrium volume is 3-5 times of column volume.
The elution of A40926 B0 component is carried out with gradient elution mode.Chromatographic column filler selects carbon 18 or carbon 8, preferably carbon 18.Chromatographic column
Length is 250-500 millimeters, preferably 500 millimeters.
(5) it collects the preparation chromatography efflux that (4) obtain and obtains high-purity A40926 B0 by being concentrated under reduced pressure and being lyophilized.
The temperature of the reduced pressure is controlled at 50-60 DEG C.
According to the domestic patent of invention and pertinent literature retrieved, it there are no and A40926 is carried out using preparative liquid chromatography
The report isolated and purified.It is to be isolated and purified using normal pressure column chromatography mostly, by reported by comparing existing document
The purity of separating and purifying technology method A40926 obtained is up to 97%.Multistage Membranes filtering and preparation according to the present invention
Liquid chromatography technology isolates and purifies A40926 purity obtained and reaches 99% or more, is above existing technical method.
Detailed description of the invention
Fig. 1 is A40926 freeze-drying prods HPLC chromatogram in first embodiment of the invention.
Fig. 2 is A40926 freeze-drying prods HPLC chromatogram in second embodiment of the invention.
Fig. 3 is A40926 freeze-drying prods HPLC chromatogram in third embodiment of the invention.
Fig. 4 is A40926 freeze-drying prods HPLC chromatogram in four embodiment of the invention.
Fig. 5 is A40926 freeze-drying prods HPLC chromatogram in fifth embodiment of the invention.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
(1) when A40926 fermentation liquid proceed to 165 it is small when, mycelium and fermentation liquid characteristic index are evaluated, it is ensured that mycelia
Body does not have self-dissolving, and even dyeing is without vacuole, and in 385 mps, fermentation liquid total amount is 110 L for fermentation broth viscosity control.It will meet
It is transferred in pretreatment tank after stating the A40926 B0 fermentation liquid termination fermentation of fermentation parameter, according to volume mass than being added 200
The flocculant polyacrylamide of ppm, after mixing evenly stratification 60 minutes, the remaining thallus and supernatant precipitated.With
Afterwards, fermented supernatant fluid is transferred in membrane filtration system fluid reservoir using press over system, transfer volume is 78 L.The remaining bacterium of precipitating
The sodium hydroxide solution that 5-10% is added in body adjusts pH value to 10, and the methanol of remaining 3 times of volume of thallus is then added, stirs 30 points
Clock obtains 120L low-carbon alcohols soak.
(2) the low-carbon alcohols soak in step (1) is filtered by ceramic micro filter film, removes mycelium, filtering step
Suddenly the ceramic membrane aperture selected is 100 nanometers, when filtering out 50L clear liquid, is continuously added to the aqueous solution containing 10% ethyl alcohol, directly
Reach 240 L to supernatant volume is filtered out.Low-carbon alcohols soak after degerming is mixed with the supernatant in membrane filtration system storage tank
It closes, obtains 318L mixed liquor.The mixed liquor is subjected to ultrafiltration impurity elimination and concentration again, obtains ultrafiltration mixed liquor.Film filtration temperature control
System is at 35 DEG C.Ultrafiltration membrane material is polysulfones, carries out a ultrafiltration with the filter membrane that molecular cut off is 25-30KD first, then use 5-
The filter membrane of 10KD carries out second ultrafiltration.Ultrafiltration mixed liquor further progress nanofiltration removal of impurities and concentration are collected, nanofiltration mixed liquor is obtained,
Nanofiltration membrane material selection Kynoar, the molecular cut off of film are 300.
(3) by the nanofiltration mixed liquor obtained in (2) with the salt acid for adjusting pH value of 5-10% to 3.5, stand 60 minutes.Decompression
Filter cake is filtered and collects, with the sodium carbonate-bicarbonate buffer solution dissolving filter cake for containing 10% ethyl alcohol, A40926 content, which is made, is
10% sample solution.The pH value of the sodium carbonate-bicarbonate buffer solution is 10-11.
(4) preparative liquid chromatography column is installed and fraction collection device, setting program is collected according to peak.Chromatographic column filler is selected
Carbon 18.Chromatogram column length is 500 millimeters.Mobile phase A used in preparative liquid chromatography is ultrapure water (containing 0.5% ammonium formate), flowing
Phase B is pure methanol.First with the chromatographic column that balances each other is flowed, equilibrium volume is 3-5 times of column volume, then loading A40926 sample, i.e.,
(3) sample solution that the content obtained is 10%, flow control is in 5 ml/min.Gradient elution mode is 0-10min, B mobile phase from
5%-10%, 10-20min, B mobile phase are from 15%-30%, 20-25min, B mobile phase 40%.
(5) part that A40926 B0 component chromatographic purity is greater than 99% is collected, merges efflux, is lyophilized after being concentrated under reduced pressure
Obtain high-purity A40926 B0.The temperature of reduced pressure is 50 DEG C, and vacuum degree is -0.09Mpa.Freeze-drying parameter is pre-freeze temperature
- 35 DEG C of degree, the pre-freeze time 1 hour, the control of cold trap temperature was at -45 DEG C, 25 DEG C of sublimation temperature, vacuum degree 10pa.
A40926 freeze-drying prods HPLC chromatogram is as shown in Figure 1 in embodiment 1.
A40926 freeze-drying prods HPLC purity testing result is as follows in embodiment 1
| Serial number | Retention time (min) | Peak area percent (%) | Theoretical cam curve | Separating degree |
| 1 | 2.017 | 0.23 | 12978 | 31.248 |
| 2 | 2.533 | 0.45 | 20479 | 3.950 |
| 3 | 4.333 | 0.21 | 41612 | 12.961 |
| 4 | 11.017 | 99.12 | 15491 | 15.379 |
| It amounts to | 100.00 |
Embodiment 2
(1) when A40926 fermentation liquid proceed to 180 it is small when, mycelium and fermentation liquid characteristic index are evaluated, it is ensured that mycelia
Body does not have self-dissolving, and even dyeing is without vacuole, and in 320 mps, fermentation liquid total amount is 90 L for fermentation broth viscosity control.It will meet above-mentioned
The A40926 B0 fermentation liquid of fermentation parameter is transferred in pretreatment tank after terminating fermentation, according to volume mass than being added 250
The flocculant polyacrylamide of ppm, after mixing evenly stratification 60 minutes.The remaining thallus and supernatant precipitated.With
Afterwards, fermented supernatant fluid is transferred in membrane filtration system fluid reservoir using press over system, transfer volume is 63 L.The remaining bacterium of precipitating
The sodium hydroxide solution that body is added 10% adjusts pH value to 10.8, and the ethyl alcohol of remaining 5 times of volume of thallus is then added, stirs 20 points
Clock obtains 163L low-carbon alcohols soak.
(2) the low-carbon alcohols soak in step (1) is filtered by ceramic micro filter film, removes mycelium, filtering step
Suddenly the ceramic membrane aperture selected is 50 nanometers.When filtering out 50L clear liquid, it is continuously added to the aqueous solution containing 10% ethyl alcohol, until
It filters out supernatant volume and reaches 320 L.Film filtration temperature is controlled at 35 DEG C.It is with film filtering by the low-carbon alcohols soak after degerming
Supernatant mixing in system storage tank, obtains 320L mixed liquor.The mixed liquor is subjected to ultrafiltration impurity elimination and concentration again, it is mixed to obtain ultrafiltration
Close liquid.Ultrafiltration membrane material is polysulfones, carries out a ultrafiltration with the filter membrane that molecular cut off is 25-30KD first, then use 5-10KD
Filter membrane carry out second ultrafiltration.Ultrafiltration mixed liquor further progress nanofiltration removal of impurities and concentration are collected, nanofiltration mixed liquor, nanofiltration are obtained
Membrane material selects Kynoar, and the molecular cut off of film is 300.
(3) the nanofiltration mixed liquor obtained in (2) dilute hydrochloric acid is adjusted into pH value to 3.5, stands 60 minutes.It is filtered under diminished pressure simultaneously
Collect filter cake, with contain 5% ethyl alcohol sodium carbonate-bicarbonate buffer solution dissolving filter cake, be made A40926 content be 10% it is upper
Sample liquid.The pH value of the sodium carbonate-bicarbonate buffer solution is 10-11.
(4) preparative liquid chromatography column is installed and fraction collection device, setting program is collected according to peak.Chromatographic column filler is selected
Carbon 18.Chromatogram column length is 500 millimeters.Mobile phase A used in preparative liquid chromatography is ultrapure water (containing 0.5% ammonium formate), flowing
Phase B is pure methanol.First with the chromatographic column that balances each other is flowed, equilibrium volume is 3-5 times of column volume, then loading A40926 sample, i.e.,
(3) sample solution that the content obtained is 10%.Flow control is in 5 ml/min.Gradient elution mode is 0-10min, B mobile phase from
5%-10%, 10-20min, B mobile phase are from 15%-30%, 20-25min, B mobile phase 40%.
(5) part that A40926 B0 component chromatographic purity is greater than 99% is collected, merges efflux, is lyophilized after being concentrated under reduced pressure
Obtain high-purity A40926 B0.The temperature of reduced pressure is 55 DEG C, and vacuum degree is -0.09Mpa.Freeze-drying parameter is pre-freeze temperature
- 35 DEG C of degree, the pre-freeze time 1 hour, the control of cold trap temperature was at -45 DEG C, 25 DEG C of sublimation temperature, vacuum degree 10pa.
A40926 freeze-drying prods HPLC chromatogram is as shown in Figure 2 in embodiment 2.
A40926 freeze-drying prods HPLC purity testing result is as follows in embodiment 2
| Serial number | Retention time (min) | Peak area percent (%) | Theoretical cam curve | Separating degree |
| 1 | 2.017 | 0.28 | 12978 | 30.481 |
| 2 | 2.533 | 0.33 | 20479 | 4.138 |
| 3 | 4.360 | 0.21 | 41932 | 13.689 |
| 4 | 11.000 | 99.19 | 154445 | 15.221 |
| It amounts to | 100.00 |
Embodiment 3
(1) when A40926 fermentation liquid proceed to 160-180 it is small when, mycelium and fermentation liquid characteristic index are evaluated, it is ensured that
Mycelium does not have self-dissolving, and even dyeing is without vacuole, and fermentation broth viscosity control, in 300-600 mps, fermentation liquid total amount is 110
L.It is transferred in pretreatment tank after the A40926 B0 fermentation liquid for meeting above-mentioned fermentation parameter is terminated fermentation, according to volume matter
The flocculant polyacrylamide than 200 ppm are added is measured, after mixing evenly stratification 120 minutes.The remaining bacterium precipitated
Body and supernatant.Then, fermented supernatant fluid is transferred in membrane filtration system fluid reservoir using press over system, transfer volume 78
L.The sodium hydroxide solution that the remaining thallus of precipitating is added 5% adjusts pH value to 10, and the third of remaining 4 times of volume of thallus is then added
Alcohol stirs 30 minutes, obtains 120 L low-carbon alcohols soaks.
(2) the low-carbon alcohols soak in step (1) is filtered by ceramic micro filter film, removes mycelium, filtering step
Suddenly the ceramic membrane aperture selected is 100 nanometers, when filtering out 50L clear liquid, is continuously added to the aqueous solution containing 10% ethyl alcohol, directly
Reach 240 L to supernatant volume is filtered out.This step film filtration temperature is controlled at 35 DEG C.By after degerming low-carbon alcohols soak with
Supernatant mixing in membrane filtration system storage tank, obtains 318L mixed liquor.The mixed liquor is subjected to ultrafiltration impurity elimination and concentration again, is obtained
To ultrafiltration mixed liquor.Ultrafiltration membrane material is polysulfones, carries out a ultrafiltration with the filter membrane that molecular cut off is 25-30KD first, then
Second ultrafiltration is carried out with the filter membrane of 5-10KD.It collects ultrafiltration mixed liquor and carries out nanofiltration removal of impurities and concentration, nanofiltration membrane material selection is poly-
Vinylidene, the molecular cut off of film are 300.
(3) the nanofiltration mixed liquor obtained in (2) dilute hydrochloric acid is adjusted into pH value to 5, stands 100 minutes.It is filtered under diminished pressure simultaneously
Collect filter cake, and with the sodium carbonate-bicarbonate buffer solution dissolving filter cake for containing 8% ethyl alcohol, it is 10% that A40926 content, which is made,
Sample solution.The pH value of the sodium carbonate-bicarbonate buffer solution is 10-11.The temperature of reduced pressure is controlled at 50-60 DEG C.
(4) preparative liquid chromatography column is installed and fraction collection device, setting program is collected according to peak.Chromatographic column filler is selected
Carbon 8,5-10 μm of partial size.20-30 millimeters of chromatography column internal diameter, chromatogram column length is 300 millimeters.Preparative liquid chromatography stream used
Dynamic phase A is ultrapure water, and Mobile phase B is acetonitrile, and the ratio of acetonitrile and water is 10:90-30:70, and Detection wavelength is 210-230 nm,
Chromatography peak detection is carried out using diode array detector.First with the chromatographic column that balances each other is flowed, equilibrium volume is 3-5 times of cylinder
It accumulates, then loading A40926 sample, flow control is in 5 ml/min.Gradient elution mode is 0-10min, and B mobile phase is from 5%-
10%, 10-20min, B mobile phase are from 15%-30%, 20-25min, B mobile phase 40%.
(5) part that A40926 B0 component chromatographic purity is greater than 99% is collected, merges efflux, is lyophilized after being concentrated under reduced pressure
Obtain high-purity A40926 B0.The temperature of reduced pressure is 60 DEG C, and vacuum degree is -0.09Mpa.Freeze-drying parameter is pre-freeze temperature
- 35 DEG C, pre-freeze time 1h of degree, the control of cold trap temperature is at -45 DEG C, 25 DEG C of sublimation temperature, vacuum degree 10pa.
A40926 freeze-drying prods HPLC chromatogram is as shown in Figure 3 in embodiment 3.
A40926 freeze-drying prods HPLC purity testing result is as follows in 3 embodiment 3 of table
| Serial number | Retention time (min) | Peak area percent (%) | Theoretical cam curve | Separating degree |
| 1 | 2.017 | 0.57 | 9012 | 27.837 |
| 2 | 2.533 | 0.17 | 56887 | 4.528 |
| 3 | 4.350 | 0.25 | 60382 | 15.116 |
| 4 | 11.083 | 99.01 | 17013 | 16.053 |
| It amounts to | 100.00 |
Embodiment 4
(1) when A40926 fermentation liquid proceeds to 160-180 hours, mycelium and fermentation liquid characteristic index are evaluated, really
Protecting mycelium does not have self-dissolving, and even dyeing is without vacuole, and fermentation broth viscosity control, in 300-600 mps, fermentation liquid total amount is
110 L.It is transferred in pretreatment tank after the A40926 B0 fermentation liquid for meeting above-mentioned fermentation parameter is terminated fermentation, according to body
The flocculant polyacrylamide of 200 ppm is added in product mass ratio, after mixing evenly stratification 80 minutes.The residue precipitated
Thallus and supernatant.Then, fermented supernatant fluid is transferred in membrane filtration system fluid reservoir using press over system, transfer volume 78
L.The sodium hydroxide solution that the remaining thallus of precipitating is added 8% adjusts pH value to 11, and the low of remaining 3 times of volume of thallus is then added
Carbon alcohol, the low-carbon alcohols are the mixture of methanol and ethyl alcohol, stir 30 minutes, obtain 120 L low-carbon alcohols soaks.
(2) the low-carbon alcohols soak in step (1) is filtered by ceramic micro filter film, removes mycelium, filtering step
Suddenly the ceramic membrane aperture selected is 100 nanometers, when filtering out 50L clear liquid, is continuously added to the aqueous solution containing 10% ethyl alcohol, directly
Reach 240 L to supernatant volume is filtered out.This step film filtration temperature is controlled at 35 DEG C.By after degerming low-carbon alcohols soak with
Supernatant mixing in membrane filtration system storage tank, obtains 318L mixed liquor.The mixed liquor is subjected to ultrafiltration impurity elimination and concentration again, is obtained
To ultrafiltration mixed liquor.Ultrafiltration membrane material is polysulfones, carries out a ultrafiltration with the filter membrane that molecular cut off is 25-30KD first, then
Second ultrafiltration is carried out with the filter membrane of 5-10KD.It collects ultrafiltration mixed liquor and carries out nanofiltration removal of impurities and concentration, nanofiltration membrane material selection is poly-
Vinylidene, the molecular cut off of film are 600.
(3) the nanofiltration mixed liquor obtained in (2) dilute hydrochloric acid is adjusted into pH value to 4, stands 60 minutes.It is filtered under diminished pressure and receives
Collect filter cake, with the sodium carbonate-bicarbonate buffer solution dissolving filter cake for containing 10% ethyl alcohol, the loading that A40926 content is 10% is made
Liquid.The pH value of the sodium carbonate-bicarbonate buffer solution is 10.
(4) preparative liquid chromatography column is installed and fraction collection device, setting program is collected according to peak.Chromatographic column filler selects carbon
18.Chromatogram column length is 500 millimeters.Mobile phase A used in preparative liquid chromatography is ultrapure water (containing 0.5% ammonium formate), Mobile phase B
For pure methanol, the ratio of methanol and water is that 10:90-30:70 Detection wavelength is 210-230 nm, uses diode array detector
Carry out chromatography peak detection.First with the chromatographic column that balances each other is flowed, equilibrium volume is 3-5 times of column volume, then loading A40926 sample,
Flow control is in 5 ml/min.Gradient elution mode is 0-10min, and B mobile phase is from 5%-10%, 10-20min, B mobile phase from
15%-30%, 20-25min, B mobile phase 40%.
(5) part that A40926 B0 component chromatographic purity is greater than 99% is collected, merges efflux, is lyophilized after being concentrated under reduced pressure
Obtain high-purity A40926 B0 component.The temperature of reduced pressure is 58 DEG C, and vacuum degree is -0.09Mpa.It is pre- that parameter, which is lyophilized,
Freeze -35 DEG C of temperature, the pre-freeze time 1 hour, the control of cold trap temperature was at -45 DEG C, 25 DEG C of sublimation temperature, vacuum degree 10pa.
A40926 freeze-drying prods HPLC chromatogram is as shown in Figure 4 in embodiment 4.
A40926 freeze-drying prods HPLC purity testing result is as follows in embodiment 4
| Serial number | Retention time (min) | Peak area percent (%) | Theoretical cam curve | Separating degree |
| 1 | 2.517 | 0.77 | 20211 | 35.144 |
| 2 | 4.333 | 0.21 | 30572 | 10.720 |
| 3 | 11.017 | 99.02 | 16809 | 14.779 |
| It amounts to | 100.00 |
Embodiment 5
(1) when A40926 fermentation liquid proceed to 160-180 it is small when, mycelium and fermentation liquid characteristic index are evaluated, it is ensured that
Mycelium does not have self-dissolving, and even dyeing is without vacuole, and fermentation broth viscosity control, in 300-600 mps, fermentation liquid total amount is 110
L.Flocculant polyacrylamide according to volume mass than 200 ppm are added, after mixing evenly stratification 60 minutes.It is sunk
The remaining thallus and supernatant in shallow lake.Then, fermented supernatant fluid is transferred in membrane filtration system fluid reservoir using press over system, is shifted
Volume is 78 L.The remaining thallus of precipitating is added sodium hydroxide solution and adjusts pH value to 10, and remaining 3 times of volume of thallus is then added
Methanol, stir 30 minutes, obtain 120 L low-carbon alcohols soaks.
(2) the low-carbon alcohols soak in step (1) is filtered by ceramic micro filter film, removes mycelium, filtering step
Suddenly the ceramic membrane aperture selected is that 100 nm are continuously added to the aqueous solution containing 10% ethyl alcohol when filtering out 50L clear liquid, up to
It filters out supernatant volume and reaches 240 L.Film filtration temperature is controlled at 35 DEG C.It is with film filtering by the low-carbon alcohols soak after degerming
Supernatant mixing in system storage tank obtains 318L mixed liquor, then the mixed liquor is carried out ultrafiltration impurity elimination and concentration, and it is mixed to obtain ultrafiltration
Close liquid.Ultrafiltration membrane material is polysulfones, carries out a ultrafiltration with the filter membrane that molecular cut off is 25-30KD first, then use 5-10KD
Filter membrane carry out second ultrafiltration.The progress nanofiltration removal of impurities of collection ultrafiltration mixed liquor and concentration, nanofiltration membrane material selection Kynoar,
The molecular cut off of film is 1000.
(3) the nanofiltration mixed liquor obtained in (2) dilute hydrochloric acid is adjusted into pH value to 3.8, stands 110 minutes.It is filtered under diminished pressure
And filter cake is collected, with the sodium carbonate-bicarbonate buffer solution dissolving filter cake for containing 10% ethyl alcohol, it is 10% that A40926 content, which is made,
Sample solution.The pH value of the sodium carbonate-bicarbonate buffer solution is 10.5.
(4) preparative liquid chromatography column is installed and fraction collection device, setting program is collected according to peak.Chromatographic column filler is selected
C18.Chromatogram column length is 500 mm.Mobile phase A used in preparative liquid chromatography is ultrapure water (containing 0.5% ammonium formate), Mobile phase B
For pure methanol.First with the chromatographic column that balances each other is flowed, equilibrium volume is 3-5 times of column volume, then loading A40926 sample, flow velocity control
System is in 5 ml/min.Gradient elution mode is 0-10min, and B mobile phase is from 5%-10%, 10-20min, B mobile phase from 15%-30%,
20-25min, B mobile phase 40%.
(5) part that A40926 B0 component chromatographic purity is greater than 99% is collected, merges efflux, is lyophilized after being concentrated under reduced pressure
Obtain high-purity A40926 B0.The temperature of reduced pressure is 55 DEG C, and vacuum degree is -0.09Mpa.Freeze-drying parameter is pre-freeze temperature
- 35 DEG C of degree, the pre-freeze time 1 hour, the control of cold trap temperature was at -45 DEG C, 25 DEG C of sublimation temperature, vacuum degree 10pa.
A40926 freeze-drying prods HPLC chromatogram is as shown in Figure 5 in embodiment 5.
A40926 freeze-drying prods HPLC purity testing result is as follows in embodiment 5
| Serial number | Retention time (min) | Peak area percent (%) | Theoretical cam curve | Separating degree |
| 1 | 2.017 | 0.71 | 12978 | 29.394 |
| 2 | 2.533 | 0.26 | 31999 | 4.150 |
| 3 | 11.167 | 99.03 | 22557 | 23.429 |
| It amounts to | 100.00 |
Claims (6)
1. a kind of preparation method of the high-purity A40926B0 of suitable industrialized production, it is characterised in that the following steps are included:
(1) it is transferred in pretreatment tank after the A40926B0 fermentation liquid for meeting fermentation parameter being terminated fermentation, macromolecule is added
Decolorization flocculation agent is stood after mixing evenly, and supernatant is transferred to membrane filtration system by the remaining thallus and supernatant precipitated
In storage tank, remaining thallus sodium hydroxide solution adjusts pH value to 10-11, and the 50- of remaining 2-5 times of volume of thallus is then added
75% low-carbon alcohol solution stir process 30-90 minutes, obtains low-carbon alcohols soak；
(2) by the low-carbon alcohols soak filtering in (1), removal mycelium, it is with film filtering by the low-carbon alcohols soak after degerming
Supernatant mixing in system storage tank, obtains mixed liquor, and mixed liquor progress ultrafiltration impurity elimination is obtained ultrafiltration mixed liquor；Ultrafiltration is mixed again
It closes liquid and carries out nanofiltration removal of impurities, obtain nanofiltration mixed liquor；
(3) the nanofiltration mixed liquor salt acid for adjusting pH value to acidity that will be obtained in (2) stands 60-120 minutes, is filtered under diminished pressure simultaneously
Filter cake is collected, filter cake is dissolved in sodium carbonate-bicarbonate buffer solution, A40926 sample solution is made；Sodium carbonate-the carbonic acid
The pH value of hydrogen sodium buffer solution is 10-11 and the ethyl alcohol containing 5-15%；
(4) by the A40926 sample solution upper prop high pressure preparative liquid chromatography in (3), chromatographic column using HILIC hydrophilic chromatographic column into
Row isolates and purifies, and collects the preparation chromatography efflux that purity is greater than 98%；
(5) it collects the preparation chromatography efflux that (4) obtain and obtains high-purity A40926 B0 by being concentrated under reduced pressure and being lyophilized.
2. the preparation method of the high-purity A40926B0 of suitable industrialized production as described in claim 1, it is characterised in that: described
Low-carbon alcohols are one of methanol, ethyl alcohol, propyl alcohol or two or more mixtures, the preferably mixing of methanol, ethyl alcohol or both
Object.
3. the preparation method of the high-purity A40926B0 of suitable industrialized production as claimed in claim 1 or 2, it is characterised in that:
The filter operation selects filtering membrane material for metal film or ceramic membrane or natural macromolecule membrane or synthetic polymeric membrane,
The pore diameter range of the filtering membrane material is 50-200 nm；It is metal film or pottery that the ultrafiltration, which selects ultrafiltration membrane material,
Porcelain film or natural macromolecule membrane or synthetic polymeric membrane, the ultrafiltration carry out twice, ultrafiltration membrane material retention used
Molecular weight is respectively 25-30 KD and 5-10 KD；The nanofiltration operation selects nanofiltration membrane material as polystyrene or gathers inclined fluorine
Ethylene or polytetrafluoroethylene (PTFE), the nanofiltration membrane material molecular cut off are 300-1000.
4. the preparation method of the high-purity A40926B0 of suitable industrialized production as claimed in claim 1 or 2, it is characterised in that:
In step (3), nanofiltration mixed liquor after the merging salt acid for adjusting pH value of 5-10% to 3.0-5.0.
5. the preparation method of the high-purity A40926B0 of suitable industrialized production as claimed in claim 1 or 2, it is characterised in that:
Chromatographic column filler used in the high pressure preparative liquid chromatography is that perhaps the partial size of the carbon 18 of carbon 8 or carbon 8 is 5-10 μ to carbon 18
M, the chromatography column internal diameter is 20-30 millimeters, column length is 250-500 millimeters；Mobile phase used in the preparative liquid chromatography
For the aqueous solution of acetonitrile or the aqueous solution of methanol, the ratio of acetonitrile or methanol and water is 10:90-30:70, and Detection wavelength is
210-230 nm carries out chromatography peak detection using diode array detector.
6. the preparation method of the high-purity A40926B0 of suitable industrialized production as claimed in claim 1 or 2, it is characterised in that:
The temperature of the reduced pressure is controlled at 50-60 DEG C.
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Application publication date: 20190823 |