CN107674101B - A kind of purification process of tobramycin - Google Patents
A kind of purification process of tobramycin Download PDFInfo
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- CN107674101B CN107674101B CN201610629125.XA CN201610629125A CN107674101B CN 107674101 B CN107674101 B CN 107674101B CN 201610629125 A CN201610629125 A CN 201610629125A CN 107674101 B CN107674101 B CN 107674101B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/22—Cyclohexane rings, substituted by nitrogen atoms
- C07H15/222—Cyclohexane rings substituted by at least two nitrogen atoms
- C07H15/226—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings
- C07H15/234—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings attached to non-adjacent ring carbon atoms of the cyclohexane rings, e.g. kanamycins, tobramycin, nebramycin, gentamicin A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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Abstract
The present invention relates to a kind of purification process of tobramycin, this method is handled using nanofiltration crystallization mode by hydrolysis, decoloration, the filtered tobramycin solution of film, then column chromatography for separation is carried out using preparation chromatographic column to the crystal that crystallization obtains, the HPLC purity of the tobramycin adopted this method is up to 99.4% or more, and use nanofiltration crystallization mode, multiple exchange operations before avoiding crystallization in the prior art, have easy to operate, the low advantage of production cost；Product yield and quality are not only increased by the way of preparing column chromatography for separation, the production time is also reduced, reduces waste liquid amount, reduce environmental pollution.
Description
Technical field
The present invention relates to biofermentation pharmaceutical field more particularly to a kind of purification process of tobramycin.
Background technique
Tobramycin (structure is as follows), is a kind of aminoglycoside antibiotics, and clinic is mainly used for sensitive bacterial and draws
The severe infections risen, the infection of burn as caused by gram-negative bacteria especially Pseudomonas aeruginosa, Escherichia coli and pneumobacillus etc. lose
Mass formed by blood stasis, infection in respiratory system, urinary system infection contamination, gall-bladder infection of biliary tract and soft tissue severe infections etc..The antibacterial of tobramycin
Activity is similar to gentamicin, has good action to most gram negative bacillis and Pseudomonas aeruginosa, to staphylococcus not producing enzyme
The MIC of strain.
Although there are patent (CN201310621366.6) and paper publishing in the country, new engineering bacteria is constructed, can be fermented
Tobramycin is directly generated, avoids and is hydrolyzed from carbamyl tobramycin, but most of the country producer still passes through dark
Streptomycete fermentation produces carbamyl tobramycin, then is obtained based on the technique of tobramycin by hydrolysis.It is produced when as follows
The classical process of tobramycin:
The product assay that this fermentation method obtains is lower, and impurity level is relatively high, especially apramycin and aminoacyl
Two kinds of impurity (structure is as follows) of kalamycin B and product structure are very close, and separating difficulty is very big.
So current technique, wants 1-2 amberlite in above-mentioned classical technique " 711 resin decolorization " toward contact afterwards
Rouge carries out chromatographic isolation, can just obtain the tobramycin of 90% or more content.In order to guarantee separating effect, production was entirely chromatographed
The journey time is very long, to produce 2m diameter, for 4 meters of high chromatographic columns, substantially needs to complete for 20-30 days a batch
Production.Furthermore yield is not high, and the yield of 90% or more content of tobramycin is lower less than the yield of 60%, 95% or more content.
In addition, the crystallization of tobramycin is relatively difficult, it is easily created colloid, so the effect in order to guarantee crystallization, most of to tie
Crystalline substance is carried out using 90% or more high-purity tobramycin, this just determines that the tobramycin of fermenting and producing has to carry out
It could be crystallized after multiple cation exchange chromatography.
Summary of the invention
In light of the defects in the prior art, the purpose of the invention is to provide a kind of easy to operate, appropriate cloth purified
The tobramycin purification process of mycin purity is high.
To achieve the goals above, it specifically adopts the following technical scheme that
(1) tobramycin crude product is soluble in water, obtain crude product aqueous solution；Methanol or second are added into the crude product aqueous solution
Alcohol obtains mixed liquor；
(2) make the mixed liquor through horizontally disposed nanofiltration membrane, after observing that crystal is precipitated, stop nanofiltration, stand analysis
It is brilliant；
(3) crystal that step (2) are precipitated carries out preparation chromatographic isolation, with 0.5%~1.5% trifluoroacetic acid aqueous solution
To flow A phase, methanol is flowing B phase, and the mixed liquor of the A and B that are 10-100:0-90 according to volume ratio are that mobile phase carries out gradient
Elution, collect rich in tobramycin efflux to get.
Wherein, in step (1), in the crude product aqueous solution concentration of tobramycin be 300-350g/L, the methanol or
The additional amount of ethyl alcohol is 4-5 times of the crude product aqueous solution volume.Tobramycin crude product of the present invention can be solids crude
Product can also be the liquid crude product containing tobramycin.When the crude product is liquid, the common hand diluted or be concentrated can be used
The concentration of tobramycin in solution is adjusted to 300-350g/L by section, then adds methanol or ethyl alcohol prepares mixed liquor, such
The mixed liquor of concentration facilitates the slow precipitation of crystal in subsequent nanofiltration crystallization operation, and obtained crystal purity is good, yield
It is high.
Preferably, the crude product aqueous solution obtains by the following method: using cationic exchange resin adsorption method from production
Carbamyl amine tobramycin is extracted in the fermentation liquid of carbamyl amine tobramycin, and it is mould to hydrolyze the appropriate cloth of carbamyl amine
Element, decolourized to gained hydrolyzate, after film filtration treatment to get.
Wherein, the carbamyl amine tobramycin fermentation liquid is as known to those skilled in the art by streptomyces tenebrarius
Fermentation generates.The cation exch ange adsorption, hydrolyze, decoloration, membrane filtration operation with purify tobramycin in the prior art
Operation early period it is similar (referred in specific visible background technology classical operation), can be according to the condition of the prior art at
Reason.
Specifically, the cationic exchange resin adsorption method is using 732 cation exchange resins as stationary phase, with 1-3%
Ammonium hydroxide be strippant (preferably 2% ammonium hydroxide).
The condition of the hydrolysis are as follows: 80-90 DEG C, pH is 9.5~10, is hydrolyzed 2-3 hours.
The decoloration uses 711 resins.
The film filtering uses the nanofiltration membrane of aperture 80-150 dalton.
In a kind of specific embodiment, the crude product aqueous solution obtains by the following method: fermentation liquid puts tank,
The filtrate containing carbamyl tobramycin is obtained by filters pressing, gained filtrate is adsorbed using 732 cation exchange resins,
Then it is desorbed with the ammonium hydroxide of 1-3%, stripping liquid is in 80-90 DEG C, and hydrolysis obtains tobramycin under conditions of pH is 9.5~10
Solution；It is decolourized using 711 resins to the tobramycin solution, obtained destainer uses aperture for 80-150 dalton
Rolling nanofiltration membrane machine be concentrated, controlled concentration be 300-350g/L to get.
Wherein, step (2) carries out crystallization treatment to the mixed liquor that step (1) obtains using nano filtering process, keeps mixed liquor slow
Through horizontally disposed nanofiltration membrane, it is easier to penetrate the characteristic of nanofiltration membrane than organic solvent using water, slowly changes crystallizing system
The ratio of middle water and organic solvent, so that tobramycin smoothly crystallizes out.Such method improves the content of tobramycin, together
When avoid the trouble for needing the processing of multiple ion-exchange chromatography before crystallization in the prior art, enormously simplify operation, reduction
Production cost.
Inventor is the study found that there is larger impact in the aperture of nanofiltration membrane to the crystallization of step (2), when nanofiltration membrane aperture is larger
When, target compound (tobramycin) can penetrate nanofiltration membrane, and yield is caused to reduce；When membrane aperture is smaller, nanofiltration operation is slow
Slowly, inefficiency.Therefore, preferably, in step (2), the nanofiltration uses the nanofiltration of aperture 100-200 dalton to the present invention
Film can not only guarantee that the speed of permeate when nanofiltration operation was unlikely to too slow at this time, but also can guarantee the transmission that product will not be excessive
Film causes yield losses.
It is further preferred that the nanofiltration carries out under 1.2-1.5MPa pressure.
It preferably, further include continuing nanofiltration until the mixeding liquid volume subtracts after observing that crystal is precipitated in step (2)
As little as it is equivalent to 0.5-1 times of the mixeding liquid volume, then the step of stopping nanofiltration.Inventor it has been investigated that, at such
It is crystallized under part, the moisture in solution can be removed to the greatest extent, keep crystallization more abundant, and be unlikely to crystallization excessively, made
At the reduction of purity.
Invention also provides a kind of cup type collecting and filtering apparatus convenient for step (2) nanofiltration operation, the cup type collecting and filtering apparatus packets
Open staving is included, and the bung being covered in the opening, the staving and bung form a closed space；The bucket
It is provided with the nanofiltration membrane parallel with the cross section of the staving, the nanofiltration membrane, bung and bucket wall between the two in vivo
It is formed for holding the material fluid bath to nanofiltration liquid, the nanofiltration membrane, bucket bottom and bucket wall between the two form waste collection
Slot；The agitating paddle protruded into the material fluid bath, observation window, pressure gauge and valve are provided on the bung.
The cup type collecting and filtering apparatus is made of stainless steel material, is able to bear the pressure of 2Mpa or more.
In a preferred embodiment, the lower part of the nanofiltration membrane is additionally provided with a supporting course, in the supporting course
Between be equipped with micropore, top nanofiltration membrane permeate entered in waste collection slot by micropore.
In another preferred embodiment, be provided on the outside of the material fluid bath for the nanofiltration liquid heating or
The collet of cooling.
The method that nanofiltration operation is carried out using this cup type collecting and filtering apparatus are as follows:
(1) mixed liquor of step (1) is transferred in material fluid bath；
(2) it covers bung and forms a confined space；
(3) start the agitating device on feed liquid bung, stirring rate is 50-120 revs/min；
(4) by the valve on bung, compressed nitrogen is passed through into staving, air pressure maintains 1.2-1.5Mpa.
(5) it is observed by form, after having the precipitation of apparent crystal in feed liquid, continues the solution body in nanofiltration to material fluid bath
Product is 0.5-1 times of mixeding liquid volume, stops nanofiltration, pressure in bucket is slowly decreased to normal pressure, after being stirred to crystallization, material
Liquid is transferred to separation equipment separation, collects crystal, spare.
Nanofiltration operation of the invention is suitable for using horizontally disposed nanofiltration membrane, and in such cases, crystal can slowly be precipitated,
Ensure that is crystallized goes on smoothly, nanofiltration of the invention is carried out using commercially available tubular nanofiltration membrane or rolling nanofiltration membrane and is operated, effect
It is poor.
Wherein, the commercially available acquisition of C18 bonded silica gel described in step (3) is newly created production present invention preferably employs HuaPu
XAqua C18 filler, 10-12 μm of partial size.
Preferably, in step (3), the trifluoroacetic acid aqueous solution that A phase is 1% is flowed.
Preferably, in step (3), the preparation chromatography is using C18 bonded silica gel as stationary phase, according to 0-10min 100%
A, 10-20min A:B=(10-30): the gradient of (70-90) is eluted.
It is further preferred that condition of gradient elution are as follows: 0-10min 100%A, 10-20min A:B=20:80.
The present invention is isolated and purified using preparative chromatography is final to crystal progress, and preparative chromatography has splendid separation
Effect, and the separation used time is shorter, compared with conventional column chromatography, not only increases product yield and quality, also greatly reduces
Production time, wastewater flow rate also significantly reduce, and reduce environmental pollution.
Preferably, purification process of the present invention further includes first carrying out nanofiltration processing to efflux described in step (3),
Then obtained nanofiltration liquid is freeze-dried again, the step of to obtain tobramycin solid pure product.
Wherein, the aperture of the nanofiltration membrane is 80-120 dalton.
As the optimal technical solution of the present invention, the purification process of tobramycin includes the following steps:
(1) tobramycin crude product is soluble in water, obtain the crude product aqueous solution that concentration is 300-350g/L, Xiang Suoshu crude product
The methanol or ethyl alcohol of 4-5 times of volume are added in aqueous solution, obtains mixed liquor；
(2) under 1.2-1.5MPa pressure, make the mixed liquor through horizontally disposed membrane aperture 100-200 dalton
Nanofiltration membrane, observe crystal be precipitated after, continue nanofiltration until the mixed liquor volume reduce it is water-soluble to the crude product is equivalent to
Liquid product 0.5-1 times when, stop nanofiltration, pressure release, stand crystallization；
(3) crystal that step (3) are precipitated carries out preparation chromatographic isolation, and the preparation chromatography is solid with C18 bonded silica gel
Determine phase, 1% trifluoroacetic acid aqueous solution is flowing A phase, and methanol is flowing B phase, according to 0-10min 100%A, 10-20min
The gradient of A:B=20:80 is eluted, collect rich in tobramycin efflux to get；
(4) it uses aperture to carry out nanofiltration processing to the efflux for the nanofiltration membrane of 80-120 dalton, is freeze-dried institute
Nanofiltration liquid is obtained to get tobramycin solid pure product.
The present invention improves the purity of product using the method for nanofiltration crystallization, and preparation chromatographic column is recycled to divide product
From greatly reducing the time of separation, the product purity of acquisition is up to 99.4% or more, entire purification process operating process letter
Single, environmental pollution is smaller.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined with each other each preferably to get the present invention
Embodiment.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..It is related in following embodiment
Reagent or raw material be ordinary commercial products, commercially available acquisition；The operation being related to is that this field is normal unless otherwise specified
Rule operation.The cup type collecting and filtering apparatus being related to is to be mentioned in specification, certainly, it will be understood by those skilled in the art that using
It is different from the device of cup type collecting and filtering apparatus of the present invention, nanofiltration crystallization equally may be implemented, and can obtain essentially identical
As a result.
Embodiment 1
A kind of purification process of tobramycin, includes the following steps:
(1) fermentation liquid puts tank, obtains the filtrate containing carbamyl tobramycin by filters pressing, is exchanged using 732 cations
Resin adsorbs gained filtrate, is then desorbed with 1% ammonium hydroxide, and stripping liquid hydrolyzes to obtain tobramycin solution；Make
It is decolourized with 711 resins to the tobramycin solution, obtained destainer uses aperture for the rolling nanofiltration of 80 dalton
Film machine is concentrated, and obtaining concentration is 300g/L, and volume is the tobramycin aqueous solution of 1L, and high performance liquid chromatography detection purity is
72.3%.
(2) tobramycin aqueous solution is transferred in cup type collecting and filtering apparatus, is slowly added to the methanol solution of 4L thereto.Close cup
Formula nanofiltration machine cap, starting speed of agitator are 50 revs/min.High pressure nitrogen pipeline is connected, the pressure in cup type collecting and filtering apparatus is maintained
For 1.2Mpa.It is observed, is found after there is apparent crystal in crystal solution by eyepiece, continue to stop after nanofiltration goes out the waste water of 500ml
Only.Continue crystallization 10 hours, separates crystalline solid.It is 83.4% that efficient liquid phase, which detects chromatography detection purity,.
(3) dissolving crystallized body is taken water as a solvent, is separated with column is prepared, the filler for preparing column is that HuaPu newly creates
The XAquaC18 filler of production, 10 microns of partial size, flowing A phase is the trifluoroacetic acid aqueous solution of 1% (percent by volume), flows B phase
For methanol, detector is evaporative light scattering detector (ELSD), is eluted according to the condition of such as the following table 1, collects and is rich in appropriate cloth
The efflux of mycin is lyophilized with freeze dryer is entered after the rolled film concentration that aperture is 100 dalton, obtains white powder 195g,
High performance liquid chromatography detection purity is 99.6%.
Table 1: elution requirement
| Time min | A% | B% |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 10.1 | 20 | 80 |
| 20 | 20 | 80 |
Embodiment 2
A kind of purification process of tobramycin, includes the following steps:
(1) fermentation liquid puts tank, obtains the filtrate containing carbamyl tobramycin by filters pressing, is exchanged using 732 cations
Resin adsorbs gained filtrate, is then desorbed with 2% ammonium hydroxide, and stripping liquid hydrolyzes to obtain tobramycin solution；Make
It is decolourized with 711 resins to told tobramycin solution, obtained destainer uses aperture for the rolling nanofiltration of 150 dalton
Film machine is concentrated, and obtaining concentration is 350g/L, and volume is the tobramycin aqueous solution of 1.5L, high performance liquid chromatography detection purity
It is 73.1%.
(2) tobramycin aqueous solution is transferred in cup type collecting and filtering apparatus, is slowly added to the ethanol solution of 7.5L thereto.It closes
Cup type nanofiltration machine cap, starting speed of agitator are 120 revs/min.High pressure nitrogen pipeline is connected, the pressure in cup type collecting and filtering apparatus is maintained
Power is 1.5Mpa.It is observed, is found after there is apparent crystal in crystal solution by eyepiece, after continuation nanofiltration goes out the waste water of 500ml
Stop.Continue crystallization 12 hours, separates crystalline solid.It is 83.4% that efficient liquid phase, which detects chromatography detection purity,.
(3) dissolving crystallized body is taken water as a solvent, is separated with column is prepared, column and separation method is prepared with embodiment 1, obtains
Enter freeze dryer after to the rolled film concentration that the efflux aperture rich in tobramycin is 120 dalton to be lyophilized, obtains white
Powder 364g, high performance liquid chromatography detection purity are 99.5%.
Embodiment 3
A kind of purification process of tobramycin, includes the following steps:
(1) fermentation liquid puts tank, obtains the filtrate containing carbamyl tobramycin by filters pressing, is exchanged using 732 cations
Resin adsorbs gained filtrate, is then desorbed with 3% ammonium hydroxide, and stripping liquid hydrolyzes to obtain tobramycin solution；Make
It is decolourized with 711 resins to told tobramycin solution, obtained destainer uses aperture for the rolling nanofiltration of 100 dalton
Film machine is concentrated, and obtaining concentration is 320g/L, and volume is the tobramycin aqueous solution of 1.2L, high performance liquid chromatography detection purity
It is 72.7%.
(2) tobramycin aqueous solution is transferred in cup type collecting and filtering apparatus, is slowly added to the ethanol solution of 7.5L thereto.It closes
Cup type nanofiltration machine cap, starting speed of agitator are 100 revs/min.High pressure nitrogen pipeline is connected, the pressure in cup type collecting and filtering apparatus is maintained
Power is 1.3Mpa.It is observed, is found after there is apparent crystal in crystal solution by eyepiece, after continuation nanofiltration goes out the waste water of 500ml
Stop.Continue crystallization 11 hours, separates crystalline solid.It is 83.4% that efficient liquid phase, which detects chromatography detection purity,.
(3) dissolving crystallized body is taken water as a solvent, is separated with column is prepared, column and separation method is prepared with embodiment 1, obtains
Enter freeze dryer after to the rolled film concentration that the efflux aperture rich in tobramycin is 80 dalton to be lyophilized, obtains white powder
Last 256g, high performance liquid chromatography detection purity are 99.7%.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (9)
1. a kind of purification process of tobramycin, which comprises the steps of:
(1) methanol or ethyl alcohol are added into the tobramycin crude product aqueous solution of fermenting and producing, obtains mixed liquor；
Wherein, the tobramycin crude product aqueous solution obtains by the following method: using cationic exchange resin adsorption method from life
It produces and extracts carbamyl amine tobramycin in the fermentation liquid of carbamyl amine tobramycin, it is mould to hydrolyze the appropriate cloth of carbamyl amine
Element, decolourized to gained hydrolyzate, after film filtration treatment to get；
(2) make the mixed liquor through horizontally disposed nanofiltration membrane, after observing that crystal is precipitated, continue nanofiltration until the mixing
Liquid product is reduced to being equivalent to 0.5-1 times of the crude product aqueous solution volume, stops nanofiltration, standing crystallization；
(3) crystal that step (2) are precipitated carries out preparation chromatographic isolation, is stream with 0.5%~1.5% trifluoroacetic acid aqueous solution
Dynamic A phase, methanol are flowing B phase, and the mixed liquor of the A and B that are 10-100:0-90 according to volume ratio are that mobile phase progress gradient is washed
It is de-, collect the efflux rich in tobramycin to get.
2. purification process according to claim 1, it is characterised in that: the concentration of tobramycin is in the crude product aqueous solution
The additional amount of 300-350g/L, the methanol or ethyl alcohol is 4-5 times of the crude product aqueous solution volume.
3. purification process according to claim 1, it is characterised in that: the cationic exchange resin adsorption method is with 732 sun
Ion exchange resin is stationary phase, using the ammonium hydroxide of 1-3% as strippant；And/or the hydrolysis is at 80-90 DEG C, pH is 9.5~
It is carried out under conditions of 10；And/or the decoloration uses 711 resins；And/or the film filtering uses aperture 80-150 dalton
Nanofiltration membrane.
4. purification process according to claim 1, it is characterised in that: step (2) nanofiltration uses the road aperture 100-200
The nanofiltration membrane that you pause.
5. purification process according to claim 4, it is characterised in that: the nanofiltration carries out under 1.2-1.5MPa pressure.
6. described in any item purification process according to claim 1~5, it is characterised in that: in step (3), the preparation chromatography
Using C18 bonded silica gel as stationary phase, according to 0-10min 100%A, 10-20min A:B=(10-30): the gradient of (70-90)
It is eluted.
7. purification process according to claim 6, it is characterised in that: further include first to efflux described in step (3) into
Row nanofiltration processing, is then again freeze-dried obtained nanofiltration liquid, the step of to obtain tobramycin solid pure product.
8. purification process according to claim 7, it is characterised in that: the nanofiltration is received using aperture 80-120 dalton
Filter membrane.
9. purification process according to claim 1, characterized by the following steps:
(1) methanol or ethyl alcohol of 4-5 times of volume are added into the tobramycin crude product aqueous solution that concentration is 300-350g/L, obtains mixed
Close liquid；
(2) under 1.2-1.5MPa pressure, the mixed liquor is made to penetrate the nanofiltration of horizontally disposed membrane aperture 100-200 dalton
Film after observing that crystal is precipitated, continues nanofiltration until the volume of the mixed liquor is reduced to being equivalent to the crude product aqueous liquid
At long-pending 0.5-1 times, stop nanofiltration, pressure release stands crystallization；
(3) to step (2) be precipitated crystal carry out preparation chromatographic isolation, the preparation chromatography using C18 bonded silica gel as stationary phase,
1% trifluoroacetic acid aqueous solution is flowing A phase, and methanol is flowing B phase, according to 0-10min 100%A, 10-20min A:B=
The gradient of 20:80 is eluted, collect rich in tobramycin efflux to get；
(4) aperture is used to carry out nanofiltration processing to the efflux for the nanofiltration membrane of 80-120 dalton, freeze-drying gained is received
Filtrate is to get tobramycin solid pure product.
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Citations (3)
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|---|---|---|---|---|
| CN102477052A (en) * | 2010-11-26 | 2012-05-30 | 北大方正集团有限公司 | Method for extracting apramycin |
| CN105126650A (en) * | 2015-08-16 | 2015-12-09 | 哈尔滨工业大学宜兴环保研究院 | Antibiotic separating nanofiltration membrane preparation method |
| CN105732738A (en) * | 2016-03-09 | 2016-07-06 | 丽珠集团新北江制药股份有限公司 | Tobramycin purification method |
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2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477052A (en) * | 2010-11-26 | 2012-05-30 | 北大方正集团有限公司 | Method for extracting apramycin |
| CN105126650A (en) * | 2015-08-16 | 2015-12-09 | 哈尔滨工业大学宜兴环保研究院 | Antibiotic separating nanofiltration membrane preparation method |
| CN105732738A (en) * | 2016-03-09 | 2016-07-06 | 丽珠集团新北江制药股份有限公司 | Tobramycin purification method |
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