CN100418978C - Process for preparing sisomicin using film separation technology - Google Patents
Process for preparing sisomicin using film separation technology Download PDFInfo
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- CN100418978C CN100418978C CNB2006101352056A CN200610135205A CN100418978C CN 100418978 C CN100418978 C CN 100418978C CN B2006101352056 A CNB2006101352056 A CN B2006101352056A CN 200610135205 A CN200610135205 A CN 200610135205A CN 100418978 C CN100418978 C CN 100418978C
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- sisomicin
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- membrane separation
- separation technique
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Abstract
The present invention provides membrane separating process for preparing sisomicin. The membrane separating process includes ultrafiltering membrane depurating to eliminate protein, polysaccharide and other macromolecular impurities from material containing sisomicin, nanometer membrane separating to desalt, decompression concentrating to recover solvent, vacuum concentrating and drying to obtain crude sisomicin product. The process is simple, and has high product quality, low power consumption and low production cost.
Description
Technical field
The present invention relates to a kind of antibiotic green chemical industry production method, more specifically relate to a kind of method that adopts membrane separation technique to prepare the aminoglycoside antibiotics sisomicin.
Background technology
Aminoglycoside antibiotics is a class microbiotic of finding after penicillin early.Sisomicin (sisomicin is called for short SISO) is the biosynthetic a kind of aminoglycoside antibiotics of micromonospora, not only can be directly used in and produce the sisomicin injection, and be the raw material of semisynthetic antibiotics netilmicin (Netilmicin).Sisomicin and netilmicin are all listed China national essential drugs register in, most of gram-positive microorganisms and negative bacterium there are very strong anti-microbial effect [Chen Jianfeng etc., the optimization of Fermentation Conditions on Sisomicin Biosynthesis, process engineering journal, 2006:6 (3): 445-449].
Sisomicin has advantages such as has a broad antifungal spectrum, good effect, stable in properties and production technique are simple, the characteristics that many pathogenic bacterium had post antibiotic effect, be widely used in around clinical treatment septicemia, bacterial endocarditis, serious respiratory tract infection, kidney and urinary tract infections, skin histology and soft tissue infection, burn and the operation property infectation of bacteria etc., occupy suitable share on market, the sisomicin bulk drug that China produces accounts for 80% of world wide production.Therefore, the preparation method of research sisomicin has certain directive significance and vast market prospect.
Sisomicin is a kind of water-soluble, polynary weakly alkaline aminoglycoside antibiotics that contains two keys, molecular weight 447.26, molecular formula C
19H
37N
5O
7H
2O, 185～190 ℃ of fusing points (monohydrate) or 198～201 ℃ (semihydrate), specific rotatory power [α]
D 26(C=0.3%, H
2O)+188.90.The pure product of sisomicin are white amorphous powder, be soluble in the hydrophilic solvents such as hot water and methyl alcohol, ethanol, be slightly soluble in chloroform, insoluble in ether, sherwood oil, its vitriol is soluble in water, and [fourth tin is good etc. to be insoluble to organic solvent, the development of sisomycin, external pharmacy-microbiotic fascicle, 1984,6:510～516].
Membrane separation technique is meant that with outside energy or chemical potential difference be impellent, and separation, purifying and spissated a kind of technology of material are carried out in the selective permeation effect of dependence film, and its essence is similar to screening process.More specifically, membrane separation technique is according to the size in filter membrane aperture or the difference of the molecular weight of material size held back, under certain working pressure condition, material permeance or tunicle is held back, thereby reached the purpose of separating substances.
As a kind of emerging high efficiency separation means, membrane separation technique is considered to be one of the most rising new and high technology of 21 century, be widely used in food, medicine, chemical industry, biotechnology field, membrane separation technique commonly used mainly comprises four kinds (tables 1) such as micro-filtration, ultrafiltration, nanofiltration and reverse osmosiss.
Separatory membrane is of a great variety, can be divided into natural membranes and synthetic film from the mould material source, and synthetic film is divided into inorganic material film and organic high molecular layer two classes again.Macromolecule member material comprises: cellulose acetate class, polysulfones, polyamide-based, polyester, polyolefins, silicon-containing polymer, fluoropolymer and chitin kind etc.Inorganic material film comprises metallic membrane, ceramic membrane, glassy membrane and solid electrolyte film etc.
Compare with traditional isolation technique, membrane separation technique commonly used has following characteristics: 1. sepn process undergoing phase transition not can loss-rate have the partition method of phase change low.2. sepn process is carried out usually at normal temperatures, is particularly useful for the processing of heat-sensitive substances such as enzyme, medicine, fruit juice.3. membrane separation technique has from virus, bacterium to the extensive separating ranges of particulate, be not only applicable to organism and inorganics, but also be applicable to the separation of many singular solution systems, as separating of the separating of macromole in the solution and inorganic salt, some azeotropes or nearly boiling point system etc.4. because just with the impellent of pressure as membrane sepn, so separating device is simple, and is easy to operate, easily automatic control and maintenance.
But the present invention adopts membrane separation technique high-level efficiency, less energy-consumption ground to remove in the sisomicin solution small molecular weight impurities such as macromole impurity component such as protein, polysaccharide and inorganic salt, can reach the purpose of obvious raising sisomicin purity and quality.
Summary of the invention
The object of the present invention is to provide a kind of method that adopts membrane separation technique to prepare sisomicin, this method not only helps containing being further purified and preserving of material of sisomicin, and technology is simple, production energy consumption is little, be convenient to implement.
The method that the present invention adopts membrane separation technique to prepare sisomicin is achieved in that the material that contains sisomicin, remove macromole impurity such as protein and polysaccharide through the ultra-filtration membrane isolation technique after, effluent liquid is again through the desalination of nanofiltration membrane separation technology, concentrated solution through decompression and solvent recovery, vacuum concentration, be dried to the sisomicin crude product.
The present invention is than existing extracting method, adopt membrane separation technique to prepare sisomicin, its advantage is: remove macromole impurity such as protein and polysaccharide by the ultra-filtration membrane isolation technique after, effluent liquid is again through the desalination of nanofiltration membrane separation technology, thereby make the sisomicin extract obtain further separation and purification, and help the preservation of these goods.
Embodiment
The present invention adopts membrane separation technique to prepare the method for sisomicin, the step that comprises ultrafiltration removal of impurities and nanofiltration desalination, its concrete steps are: with the material that contains sisomicin is raw material, thin up or concentrated, control sisomicin solution ratio weighs 1.03～1.20, under 0.15～0.50MPa working pressure condition, through the ultrafiltration removal of impurities, after removing macromole impurity such as protein and polysaccharide, effluent liquid is under 0.50～1.0MPa working pressure condition, through nanofiltration desalination 3～5 times, concentrated solution-0.06～-0.09MPa, under 50～70 ℃ of conditions, through decompression and solvent recovery, vacuum concentration, be dried to the sisomicin crude product.
Described ultrafiltration removal of impurities process selects for use 6000 molecular weight cut-offs～150000 to hold back the separatory membrane of molecule.
Wherein, the separation membrane material of ultrafiltration removal of impurities process can be that pottery, polysulfones, polyvinylidene difluoride (PVDF), polyacrylonitrile, polymeric amide, chitosan or fine acetic acid are tieed up one or more in the plain film, preferentially selects ceramic membrane, polysulfone membrane or polyvinylidene fluoride film for use.
Described nanofiltration demineralising process is selected the separatory membrane of molecular weight cut-off 300～800 for use.
The epidermal area composition material of the composite nanometer filtering film of nanofiltration demineralising process can be one or more in aromatic polyamides class, SPSF class, sulfonated polyether sulfone class, poly-piperazine acidamide, polyvinylidene difluoride (PVDF) class, polypropylene nitrile, chitosan class or the cellulose family mould material, preferentially selects aromatic polyamides class, SPSF class or sulfonated polyether sulfone class mould material for use.
The above-mentioned material that contains sisomicin can be to derive from biosynthetic sisomicin fermented liquid, sisomicin crystalline mother solution, sisomicin extracting solution that other means obtain or in the elutriant one or more.
Physical and chemical parameter measuring method of the present invention is as follows:
(1) sisomicin Determination on content: adopting high performance liquid chromatography, is derivatization reagent with o-phthalaldehyde(OPA) and Thiovanic acid, and sisomicin is carried out column front derivation.Condition determination: Agilent 1100 type high performance liquid chromatographs (DAD diode-array detector), Waters Nova-Pak C
18Chromatographic column (Ф 4.6 * 150mm, 5 μ m), moving phase is methyl alcohol: water: acetate (include sodium heptanesulfonate 12mmol/L, pH 6.3)=70: 25: 5 (V/V/V), flow velocity 1.0ml/min, 30 ℃ of column temperatures, sample size 20 μ L detect wavelength 250nm.With sisomicin (purity 99%) (available from Sigma company) is contrast.
(2) determination of polysaccharide adopts the phenolsulfuric acid method, is contrast with glucose or D-semi-lactosi.Protein content determination adopts FoLin-phenol method, is contrast with the bSA.Inorganic ion content is measured and is adopted kit measurement, wherein SO
4 2-Mensuration adopt the bariumchloride precipitator method, Cl
-Measure and adopt the Silver Nitrate precipitator method, Ca
2+And Mg
2+Mensuration adopt methyl thymol blue complexometry.
Drying means of the present invention is as follows:
(1) the spraying drying condition is: feeding liquid concentration 10～20 degree Beaume (60 ℃), 160～250 ℃ of PG-5 type spray-drier inlet temperatures, 60～110 ℃ of temperature outs, centrifugal head operating pressure 1.6～3.0kgf/cm
2
(2) lyophilize condition is: drying temperature-10～-60 ℃, 35～70 ℃ of sublimation temperatures, pressure 0.05～0.18mbar, time of drying 20～40h.
(3) vacuum-drying condition is: 45～75 ℃ of drying temperatures, pressure-0.06～-0.095MPa, time of drying 15～50h.
Preparation method's of the present invention embodiment is presented below:
Embodiment 1
With the fermented liquid that the pH value is 6.76, proportion 1.03, sisomicin concentration are 0.7g/L is raw material, adopting molecular weight cut-off is 6000 polysulfone membrane, under the 0.50MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 1.0MPa working pressure through the polyamide nanofiltration membrane desalination of molecular weight cut-off 300 3 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, through decompression and solvent recovery, vacuum concentration, be spray dried to the sisomicin crude product.After measured, the rate of recovery 95.3% of sisomicin, the purity of sisomicin are 83.6%.
Embodiment 2
With pH value 9.89, proportion 1.20, sisomicin concentration is that the crystalline mother solution of 185.6g/L is a raw material, after 10 times of pure water dilutions, adopting membrane pore size is 0.22 μ m polyvinylidene fluoride film, under the 0.15MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 1.0MPa working pressure through the sulfonated polyether sulfone filtering film desalination of molecular weight cut-off 300 3 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, through decompression and solvent recovery, vacuum concentration, be spray dried to the sisomicin crude product.After measured, the rate of recovery 93.5% of sisomicin, the purity of sisomicin are 81.9%.
Embodiment 3
With pH value 11.32, proportion 1.03, sisomicin concentration is that the ammoniacal liquor elutriant of 132.9g/L is a raw material, after 10 times of pure water dilutions, adopting molecular weight cut-off is 30000 ceramic membranes, under the 0.35MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 1.0MPa working pressure through the SPSF nanofiltration membrane desalination of molecular weight cut-off 300 3 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, through decompression and solvent recovery, vacuum concentration, be spray dried to the sisomicin crude product.After measured, the rate of recovery 94.9% of sisomicin, the purity of sisomicin are 80.6%.
Embodiment 4
With pH value 7.23, proportion 1.06, sisomicin concentration is that the fermented liquid of 1.1g/L is a raw material, adopting molecular weight cut-off is 80000 polysulfone membrane, under the 0.25MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 0.85MPa working pressure through the polyamide nanofiltration membrane desalination of molecular weight cut-off 400 4 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, become the sisomicin crude product through decompression and solvent recovery, vacuum concentration, lyophilize.After measured, the rate of recovery 94.8% of sisomicin, the purity of sisomicin are 83.7%.
Embodiment 5
With pH value 9.36, proportion 1.18, sisomicin concentration is that the crystalline mother solution of 213.7g/L is a raw material, after 10 times of pure water dilutions, adopting molecular weight cut-off is 100000 polyvinylidene fluoride films, under the 0.20MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 0.60MPa working pressure through the sulfonated polyether sulfone filtering film desalination of molecular weight cut-off 500 4 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, become the sisomicin crude product through decompression and solvent recovery, vacuum concentration, lyophilize.After measured, the rate of recovery 93.9% of sisomicin, the purity of sisomicin are 84.2%.
Embodiment 6
With pH value 11.26, proportion 1.06, sisomicin concentration is that the ammoniacal liquor elutriant of 169.7g/L is a raw material, after 10 times of pure water dilutions, adopting molecular weight cut-off is 6000 ceramic membranes, under the 0.50MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 0.75MPa working pressure through the SPSF nanofiltration membrane desalination of molecular weight cut-off 600 4 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, become the sisomicin crude product through decompression and solvent recovery, vacuum concentration, lyophilize.After measured, the rate of recovery 95.6% of sisomicin, the purity of sisomicin are 82.2%.
Embodiment 7
With pH value 7.45, proportion 1.05, sisomicin concentration is that the fermented liquid of 1.5g/L is a raw material, adopting membrane pore size is 0.22 μ m polysulfone membrane, under the 0.15MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 0.50MPa working pressure through the polyamide nanofiltration membrane desalination of molecular weight cut-off 800 5 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, become the sisomicin crude product through decompression and solvent recovery, vacuum concentration, vacuum-drying.After measured, the rate of recovery 94.7% of sisomicin, the purity of sisomicin are 82.6%.
Embodiment 8
With pH value 10.38, proportion 1.20, sisomicin concentration is that the crystalline mother solution of 196.8g/L is a raw material, after 10 times of pure water dilutions, adopting molecular weight cut-off is 6000 polyvinylidene fluoride films, under the 0.50MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 0.50MPa working pressure through the sulfonated polyether sulfone filtering film desalination of molecular weight cut-off 800 5 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, become the sisomicin crude product through decompression and solvent recovery, vacuum concentration, vacuum-drying.After measured, the rate of recovery 92.9% of sisomicin, the purity of sisomicin are 81.7%.
Embodiment 9
With pH value 9.05, proportion 1.20, sisomicin concentration is that the ammoniacal liquor elutriant of 103.8g/L is a raw material, after 10 times of pure water dilutions, adopting membrane pore size is 0.22 μ m ceramic membrane, under the 0.15MPa working pressure, carry out the ultrafiltration removal of impurities, remove protein and polysaccharide, effluent liquid under the 0.50MPa working pressure through the SPSF nanofiltration membrane desalination of molecular weight cut-off 800 5 times, concentrated solution-0.06～-0.09MPa, 50～70 ℃ of conditions under, become the sisomicin crude product through decompression and solvent recovery, vacuum concentration, vacuum-drying.After measured, the rate of recovery 92.6% of sisomicin, the purity of sisomicin are 81.9%.
Above embodiment is intended to further describe for example the present invention, rather than limits the present invention by any way.
The present invention is novel, and technology is simple, the extraction efficiency height, and production cost is low, has bigger generalization.
Claims (6)
1. method that adopts membrane separation technique to prepare sisomicin, it is characterized in that: the step that comprises ultrafiltration removal of impurities and nanofiltration desalination: the material that contains sisomicin, remove macromole impurity such as protein and polysaccharide through the ultra-filtration membrane isolation technique after, effluent liquid is again through the desalination of nanofiltration membrane separation technology, concentrated solution through decompression and solvent recovery, vacuum concentration, be dried to the sisomicin crude product.
2. employing membrane separation technique according to claim 1 prepares the method for sisomicin, it is characterized in that: the step of described method is: with the material that contains sisomicin is raw material, thin up or concentrated, control sisomicin solution ratio weighs 1.03～1.20, under 0.15～0.50MPa working pressure condition, through the ultrafiltration removal of impurities, after removing macromole impurity such as protein and polysaccharide, effluent liquid is under 0.50～1.0MPa working pressure condition, through nanofiltration desalination 3～5 times, concentrated solution-0.06～-0.09MPa, under 50～70 ℃ of conditions, through decompression and solvent recovery, vacuum concentration, be dried to the sisomicin crude product.
3. employing membrane separation technique according to claim 1 and 2 prepares the method for sisomicin, it is characterized in that: described ultrafiltration removal of impurities process is selected the separatory membrane of 6000 molecular weight cut-offs～0.22 μ m membrane pore size for use.
4. employing membrane separation technique according to claim 3 prepares the method for sisomicin, it is characterized in that: the separation membrane material of described ultrafiltration removal of impurities process is one or more in mineral-type, polysulfones, fluoro-containing macromolecule material class, polyolefins, polyamide-based, chitin kind or the cellulose family mould material.
5. employing membrane separation technique according to claim 1 and 2 prepares the method for sisomicin, it is characterized in that: described nanofiltration demineralising process is selected the separatory membrane of molecular weight cut-off 300～800 for use.
6. employing membrane separation technique according to claim 5 prepares the method for sisomicin, it is characterized in that: the epidermal area composition material of the composite nanometer filtering film of described nanofiltration demineralising process is one or more in aromatic polyamides class, SPSF class, sulfonated polyether sulfone class, poly-piperazine acidamide, polyvinylidene difluoride (PVDF) class, polypropylene nitrile, chitosan class or the cellulose family mould material.
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| CN102796150A (en) * | 2012-08-31 | 2012-11-28 | 无锡福祈制药有限公司 | Method for separating and purifying high-purity sisomicin |
| CN107235972A (en) * | 2017-08-09 | 2017-10-10 | 安徽德信佳生物医药有限公司 | A kind of method that use membrane separation technique extracts hyoscine |
| CN109929008A (en) * | 2017-12-15 | 2019-06-25 | 江苏久吾高科技股份有限公司 | A kind of the UF membrane production method and device of pesticide |
| CN112409426B (en) * | 2020-11-25 | 2023-10-13 | 黑龙江格林赫思生物科技有限公司 | Preparation method of sisomicin sulfate |
| CN113209824A (en) * | 2021-04-29 | 2021-08-06 | 南京威尔生物科技有限公司 | Method for removing macromolecular substances in polysorbate80 |
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| CN1480226A (en) * | 2002-06-21 | 2004-03-10 | Antibiotic coating of porous body, its prepn. and application |
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