CN105481950B - A kind of Daptomycin extracting method - Google Patents
A kind of Daptomycin extracting method Download PDFInfo
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- CN105481950B CN105481950B CN201610058134.8A CN201610058134A CN105481950B CN 105481950 B CN105481950 B CN 105481950B CN 201610058134 A CN201610058134 A CN 201610058134A CN 105481950 B CN105481950 B CN 105481950B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Abstract
The present invention relates to antibiotics production technical fields, and in particular to a kind of Daptomycin extracting method.Include the following steps: that Daptomycin fermentation liquid is carried out ceramic membrane filter by step (1)；Filtered fluid is crossed macroporous absorbent resin and collects eluent by step (2)；Step (3) elutes NM-Q anion exchange resin on resulting eluent tune, collects eluent；Eluent obtained by step (3) is crossed reverse phase silica gel NMsil-C18 column by step (4), with ammonium acetate ethanol solution gradient elution pillar, collects eluent；The resulting Daptomycin eluent of step (4) is concentrated step (5), and calcium acetate and isopropanol is added, and places, crystallization, filters, dries to obtain Daptomycin crystal powder.The beneficial effects of the present invention are: Daptomycin extracting method of the invention, as eluent, is reduced to environment and producers' health pressure, and acid-base accommodation reagent reduces production cost using reducing without using the acetonitrile solution of hypertoxicity.
Description
Technical field
The present invention relates to antibiotics production technical fields, and in particular to a kind of Daptomycin extracting method.
Background technique
It is one of the severe challenge that the current whole world faces that pathogen, which generates drug resistance to antibiotic, and searching can effectively inhibit anti-
The antibiotic of medicine bacterium is the fundamental way for solving the problems, such as this.Vancomycin was once considered as the last of resisting gram-positive bacteria
One of defence line, however this more and more resistance to medicine bacterium are clinically had found in worldwide.
Daptomycin is a kind of first product for being known as cyclic ester peptides antibiotics family.It is from streptomyces parvus
(Streptomyces parvus) fermentation liquid extracts the cyclic lipopeptide of the structure novel of obtained 13 amino acid composition in the middle
Antibiotic, wherein ten Amino acid profile cyclic structures, capric acid and tryptophan are esterified outside ring.It not only has novel
Chemical structure, and its binding mode also from any to be approved antibiotic different.It can destroy bacterial cell membrane function in many aspects
Can, gram positive bacteria is thus killed rapidly.Daptomycin is more important in addition to it can act on most of clinically relevant gram positive bacterias
Be in vitro to being in that the drug resistances isolated strains such as methicillin (methicillin), vancomycin and Linezolid still have
Strong active.
Daptomycin (Daptomycin) is acknowledged as the best alternate antibiotic after vancomycin, is U.S.'s gift Lay
The novel lipopeptide antibiotic of discovery company's the 1980s, has apparent suppression to MRSA (resistance to first XiLin Staphylococcus aureus)
Production is used, and in addition to anti-MRSA, Daptomycin also has inhibiting effect to most of gram-positive bacteria.It is reported that up to support
Mycin deposits 2002~2005 years from 99% in 19615 plants of clinical gram-positive bacterias that each hospital of America & Canada collects
In inhibiting effect.Since the FDA of in September, 2003 ratifies Cubicin (Daptomycin injection) for treating complicated skin for the first time
Since skin structure infection, Daptomycin clinically apply it is more and more, it is more next to the demand of Daptomycin in the market
It is bigger.
But Daptomycin is tunning, the ferment filtrate obtained by fermented and cultured can generate in fermentation process big
The pigment of amount and the by-product similar with Daptomycin structure are such as dehydrated Daptomycin, therefore the extraction purification side of Daptomycin
Method is more complex.General Daptomycin producing strains, production capacity is unstable, and yield is lower, and fermentation byproduct is more, and impurity is more,
It is complex that work is extracted after causing, and substantially increases postorder purification difficulty, it is difficult to the final product for obtaining high-purity, from
And it can not industrialization production.
Cubist drugmaker in the U.S. grasps the exclusive exploitation in the whole world, production and the power of sale of Daptomycin at present, domestic
There are many mechanisms of family just under study for action, but is suitable for industrialized production, effective skill of the preparation high-purity daptomycin of production capacity
Art also compares shortage.
In the Chinese invention patent of Publication No. CN1404487A, extracted from fermentation liquid using n-butanol and water mould up to holding in the palm
Element, extraction are finished through HP-20 column, acetonitrile solution washing parsing, the parsing of eluent Poro D50 anion exchange resin.Its
Middle extraction step operation is more difficult, and fermentation liquid color is deeper, and layering can not differentiate, and the yield of extraction is lower；And the elution of HP-20 column
Liquid is the acetonitrile solution of severe toxicity, in addition Poros D50 anion exchange resin, and the country is without the type resin and without similar tree
Rouge, external price is very high, and for production cost angle, acetonitrile solution and Poros D50 anion exchange resin can not works
Industry.
The patent of invention etc. of Publication No. CN103724400A, CN102229651A, which has all been continued to use in CN1404487A, to be used
For acetonitrile solution as eluent, production process Poisoning is too big, and in the Chinese invention patent of Publication No. CN102924572A
Although having abandoned acetonitrile solution, the methanol for still using toxicity big is all unfavorable for Environmental security and production as eluent
Personnel health is not suitable for being applied to mass production.
The Chinese invention patent of Publication No. CN101830970A is disclosed with a kind of reverse phase silica gel material crude product essence
The Daptomycin of high-purity is made.This method is only the purification of Daptomycin crude product, without open how obtained from fermentation liquid
Finished product；This method uses buffer solution and Daptomycin crude product mixed liquor for sample solution, with compound YT-01 reverse phase silica gel filler
The Daptomycin that chromatographic purity is greater than 98% is obtained using highly polar aqueous solution as eluent for chromatography media.But silica gel base
Matter is unable to alkali resistance, and which has limited its application ranges.
The Chinese invention patent of Publication No. CN102276696A discloses a kind of purification process of Daptomycin, pure by half
The Daptomycin of change sephadex DEAE Sephadex A-25 or Ago-Gel DEAE Sepharose Fast
Flow purifying, the purity for obtaining Daptomycin is higher than 98%, but separating medium used in this method is polysaccharide skeleton, skeleton
It is softer, it can not be high pressure resistant；Moreover, sephadex DEAE Sephadex A-25 bed volume under different salinity can occur it is non-
Often apparent variation, and used acetonitrile solution as eluent in that patent, these have very big in industrial application
Limitation.
Documents and materials " research of Daptomycin fermentation liquid macroporous resin adsorption separation " (Chinese antibiotic magazine 2008 2
The 2nd phase of volume 33 moon) describe the method that Daptomycin fermentation liquid uses macroporous resin enrichment.This part of document is disclosed mould up to holding in the palm
There are operating procedure complexity, fermentation liquid pigment and the removal of impurity difficulty, final products color depth and purity for the method for extraction and purification of element
The problems such as not high, moreover, the different fermentations liquid that different strain fermentation generates is different surely with identical extracting method.
Therefore more effective discoloration method and easier method for extraction and purification are found, is possible to realize Daptomycin
Industrialized production.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of extraction side of Daptomycin environmental-friendly, at low cost
Method.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows: a kind of Daptomycin extracting method is provided,
Include the following steps:
Daptomycin fermentation liquid is carried out ceramic membrane filter by step (1), collects filtered fluid；
Filtered fluid obtained by step (1) is crossed the absorption of macroporous absorbent resin HZ801 column by step (2), and leakage is washed to after having adsorbed
Liquid obtains the eluent that Daptomycin unit is greater than 100mg/L with ethanol elution resin trap close to colourless out；
Step (3) is by the resulting eluent tune PH to 4.0-8.0 of step (2), upper NM-Q anion exchange resin, purified water
After top is washed, with sodium chloride solution gradient elution, the Daptomycin eluent that purity is greater than 90% is collected；
The resulting Daptomycin eluent of step (3) is crossed reverse phase silica gel NMsil-C18 column by step (4), adjusts its PH extremely
4.0-8.0, flow velocity 0.5-6BV/h upper prop, with 10-300mmol/L ammonium acetate ethanol solution gradient elution pillar, collection obtains pure
Degree is greater than 97% Daptomycin eluent；
It is 20000- that the resulting Daptomycin eluent of step (4) is concentrated to Daptomycin concentration by step (5)
80000mg/L obtains concentrate, and the calcium acetate of the 10%-90% of the Daptomycin quality of concentrate, volume of the concentrated liquid 50%- is added
90% isopropanol, places 36-48h, and crystallization filters, dries to obtain Daptomycin crystal powder.
Preferably, in above-mentioned Daptomycin extracting method, the step (1) specifically: by Daptomycin fermentation liquid into
Row ceramic membrane filter after being concentrated into volume half, while the side washing of water top is added to filter, keeps amount of water consistent with filtering traffic, continues
When measuring to 2-5 times that total filtrate volume is fermentating liquid volume, stop filtering, collects filtered fluid.
Preferably, in above-mentioned Daptomycin extracting method, the step (2) specifically: by filtered fluid obtained by step (1)
The absorption of macroporous absorbent resin HZ801 column is crossed, flow velocity 0.8BV/h is washed to omission timber close to colourless, first with volume hundred after adsorb
Score be 20% ethanol solution prewashing, then with percentage by volume be 60% ethanol solution carry out elution resin trap must reach support it is mould
Primitive unit cell is greater than the eluent of 100mg/L.
Preferably, in above-mentioned Daptomycin extracting method, " sodium chloride solution gradient elution is used " in the step (3) and is had
Body are as follows: successively use 25mM sodium chloride solution, 50mM sodium chloride solution, 60mM sodium chloride solution, 90mM sodium chloride solution carries out terraced
Degree elution.
Preferably, in above-mentioned Daptomycin extracting method, " sodium chloride solution gradient elution is used " in the step (3) and is had
Body are as follows: 10BV is first rinsed with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes impurity, then uses 50mmol/L sodium chloride solution
It rinses 6BV and simultaneously collects and be eluted lower Daptomycin, with 60mmol/L sodium chloride solution flushing 15BV, collect and be eluted
Daptomycin finally rinses 6BV with 90mmol/L sodium chloride solution.
Preferably, in above-mentioned Daptomycin extracting method, " 10-300mmol/L ammonium acetate second is used in the step (4)
Alcoholic solution gradient elution pillar " specifically: first pre- with the ethyl alcohol -20mmol/L ammonium acetate solution for being 20% containing volume fraction of ethanol
It washes 1BV, then with containing volume fraction of ethanol is that 38% ethyl alcohol -20mmol/L ammonium acetate solution elutes.
Preferably, in above-mentioned Daptomycin extracting method, " the resulting Daptomycin of step (4) is eluted in step (5)
It is 20000-80000mg/L " that liquid, which is concentrated to Daptomycin concentration, specifically: the resulting Daptomycin eluent of step (4) is dense
It is reduced to unit 50000mg/L, adjusts its PH to 4.8.
It preferably, further include step (6) in above-mentioned Daptomycin extracting method: by Daptomycin knot obtained by step (5)
Crystalline flour is dissolved in the water, and crosses the desalination of HZ016 resin, and eluent is added active carbon decoloring by 1/10 times of amount and removes endotoxin, filters, filter
Liquid be added 10 times amount isopropanols precipitated, by gained Daptomycin precipitated powder progress fluidized drying obtain Daptomycin at
Product.Wherein, a large amount of calcium acetates are added in crystallization in step 5, and obtained crystal powder ash content is exceeded, needs to re-dissolve desalination resin
Except deashing, then plus active carbon remove endotoxin, finally precipitating is dried to obtain finished product.
Preferably, above-mentioned Daptomycin extracting method specifically comprises the following steps:
Daptomycin fermentation liquid 30L, the content of the Daptomycin fermentation liquid Daptomycin are 2000mg/ by step (1)
L carries out ceramic membrane filter, after being concentrated into volume half, while the side washing of water top is added to filter, keeps amount of water consistent with filtering traffic,
Continue to total filtrate volume be fermentating liquid volume 5 times of amounts when, stop filter.Filtrate 160L is obtained, Daptomycin content is
340mg/L；
Step (2): the filtrate that step (1) obtains crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h has been adsorbed
After be washed to omission timber close to colourless, be first 20% ethyl alcohol prewashing with percentage by volume, then with percentage by volume be 60% ethyl alcohol
The eluent that elution resin trap Daptomycin unit is greater than 100mg/L is carried out, obtains eluent 9L, Daptomycin content is
5700mg/L；
Step (3): the eluent that step (2) obtains, after carrying out ultrafiltration with the ultrafiltration membrane of 20000 dalton, nanofiltration concentration,
Temperature control is concentrated into Daptomycin unit 15000-16000, tune pH value to 6.5, flow velocity in 15-20 degree when ultrafiltration and filtering
5BV/h upper NM-Q anion exchange resin after purified water top is washed, first uses 25mmol/L sodium chloride solution to rinse with 5BV/h flow velocity
10BV removes impurity, then rinses 6BV with 50mmol/L sodium chloride solution and collects the Daptomycin under being eluted, uses 60mmol/L
Sodium chloride solution rinses 15BV, collects the Daptomycin being eluted, and finally rinses 6BV with 90mmol/L sodium chloride solution,
The Daptomycin eluent that purity is greater than 90% is collected, eluent 25.2L is obtained, Daptomycin content is 1650mg/L；
Step (4): after the eluent concentrating daptomycin content to 30000mg/L that step (3) obtains, reverse phase silica gel is crossed
NMsil-C18, adjusts its PH6.5, flow velocity 4.2BV/h upper prop, first with 20% ethyl alcohol -20mmol/L ammonium acetate solution prewashing 1BV, then
Pillar is eluted with 38% ethyl alcohol -20mmol/L ammonium acetate solution, the Daptomycin eluent that purity is greater than 97% is collected, is washed
De- liquid 17.2L, Daptomycin content are 1790mg/L；
Step (5): the eluent concentrating daptomycin content that step (4) obtains to 50000mg/L obtains concentrate, is added dense
45% calcium acetate of the Daptomycin quality of contracting liquid, the isopropanol of the volume of the concentrated liquid 81% place crystallization for 24 hours, filter, and dry
It is dry, obtain Daptomycin crystal powder；
Step (6): the Daptomycin that step (5) obtains crystallizes wet-milling 80.03g, is dissolved in the water, and it is de- to cross HZ016 resin
Salt is added active carbon decoloring by 1/10 times of amount and removes endotoxin, filtering, and the isopropanol that 10 times of amounts are added in filtrate is precipitated, and filters,
Gained Daptomycin precipitated powder carries out fluidized drying and obtains Daptomycin finished product.
The beneficial effects of the present invention are: Daptomycin extracting method of the invention is made without using the acetonitrile solution of hypertoxicity
For eluent, reduce to environment and producers' health pressure；And during the extraction process, pH value maintains 7.0 or so, approaches
Neutrallty condition, to the corrosion of equipment, equipment depreciation is reduced, and acid-base accommodation reagent is reduced and is produced into using reducing
This, gained Daptomycin product purity is high, and for purity 98% or so, yield can reach 45% or so, mentions for the preparation of preparation
For more excellent raw material, the side effect in drug use process is reduced.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, it is explained below in conjunction with embodiment.
The most critical design of the present invention is: Daptomycin extracting method of the invention does not use the acetonitrile solution of hypertoxicity
As eluent, reduce to environment and producers' health pressure；And during the extraction process, pH value maintains 7.0 or so, connects
Near-neutral sulfite deinking, to the corrosion of equipment, equipment depreciation is reduced, and acid-base accommodation reagent reduces production using reducing
Cost.
Embodiment 1
Step (1): Daptomycin fermentation liquid 30L, the content of Daptomycin are 2000mg/L, carry out ceramic membrane filter, dense
After being reduced to volume half, while the side washing of drinking water top is added to filter, keeps amount of water consistent with filtering traffic, continue to total filtrate volume
For fermentating liquid volume 5 times of amounts when, stop filtering.Filtrate 160L is obtained, Daptomycin content is 340mg/L, yield 95.5%.
Step (2): the filtrate that step (1) obtains crosses (the macroporous absorbent resin HZ801 column purchase of macroporous absorbent resin HZ801 column
In Shanghai Huazhen Science and Technology Co., Ltd.) it adsorbs, flow velocity 0.8BV/h is washed to omission timber close to colourless, 20% ethyl alcohol after having adsorbed
Prewashing, 60% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L.Eluent 9L is obtained, it is mould up to holding in the palm
Cellulose content is 5700mg/L, yield 90.2%.
Step (3): the eluent that step (2) obtains, after carrying out ultrafiltration with the ultrafiltration membrane of 20000 dalton, nanofiltration concentration,
Temperature control is at 20 degree or less when ultrafiltration and filtering.It is concentrated into Daptomycin unit 15,000 or so, tune pH value to 6.5, flow velocity
5BV/h upper NM-Q anion exchange resin after purified water top is washed, first uses 25mmol/L sodium chloride solution to rinse with 5BV/h flow velocity
10BV removes impurity, then rinses 6BV with 50mmol/L sodium chloride solution and collects the Daptomycin under being eluted, uses 60mmol/L
Sodium chloride solution rinses 15BV, collects the Daptomycin being eluted, and finally rinses 6BV with 90mmol/L sodium chloride solution,
Collect the Daptomycin eluent that purity is greater than 90%.Eluent 25.2L is obtained, Daptomycin content is 1650mg/L, yield
81.0%.
Step (4): after the eluent concentrating daptomycin content to 30000mg/L that step (3) obtains, reverse phase silica gel is crossed
NMsil-C18 adjusts its PH6.5 flow velocity 4.2BV/h upper prop, first with 20% ethyl alcohol -20mmol/L ammonium acetate solution prewashing 1BV, then
Pillar is eluted with 38% ethyl alcohol -20mmol/L ammonium acetate solution, the Daptomycin eluent that purity is greater than 97% is collected, is washed
De- liquid 17.2L, Daptomycin content are 1790mg/L, yield 74.0%.It is dehydrated Daptomycin content 0.3%.
Step (5): the eluent concentrating daptomycin content that step (4) obtains to 50,000 units (i.e. 50000mg/L) obtains dense
Contracting liquid, is added 45% calcium acetate of the Daptomycin quality of concentrate, the isopropanol of the volume of the concentrated liquid 81%, and placement is analysed for 24 hours
Crystalline substance filters, drying.Obtain Daptomycin crystal powder, purity 98.6%, yield 91%.It is dehydrated Daptomycin content 0.02%.
Step (6): the Daptomycin that step (5) obtains crystallizes wet-milling 80.03g, is dissolved in the water, and it is de- to cross HZ016 resin
Salt is added active carbon decoloring by 1/10 times of amount and removes endotoxin, filtering, and the isopropanol that 10 times of amounts are added in filtrate is precipitated, and filters,
Daptomycin precipitated powder carries out fluidized drying.Obtain Daptomycin crystal powder, purity 98.3%, yield 95%.Dehydration reaches support
Mycin content 0.3%.
Embodiment 2
Fermentation liquid 30L, the content of Daptomycin are 2000mg/L, carry out ceramic membrane filter, after being concentrated into volume half, side
Add the side washing of drinking water top to filter, keep amount of water consistent with filtering traffic, continues to 2 times that total filtrate volume is fermentating liquid volume
When amount, stop filtering.
Filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber close to nothing after having adsorbed
Color, 10% ethyl alcohol prewashing, 80% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L.It is eluted
Liquid.
After the ultrafiltration membrane of 5,000 dalton of eluent carries out ultrafiltration, nanofiltration concentration, ultrafiltration exists with temperature control when filtering
20 degree or less.It is concentrated into Daptomycin unit 5,000 or so, adjusts pH value to 4.0, NM-Q anion exchange tree on flow velocity 2BV/h
Rouge after purified water top is washed, first rinses 10BV with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes impurity, then use 50mmol/
L sodium chloride solution rinses 6BV and collects the Daptomycin under being eluted, and rinses 15BV with 60mmol/L sodium chloride solution, collects
The Daptomycin being eluted finally rinses 6BV with 90mmol/L sodium chloride solution, and it is mould up to holding in the palm greater than 90% to collect purity
Plain eluent.
After eluent concentrating daptomycin content to 50000mg/L, reverse phase silica gel NMsil-C18 is crossed, its PH4.0 flow velocity is adjusted
0.5BV/h upper prop, first with 20% ethyl alcohol -10mmol/L ammonium acetate solution prewashing 1BV, then with 38% ethyl alcohol -30mmol/L acetic acid
Ammonium salt solution elutes pillar, collects the Daptomycin eluent that purity is greater than 97%.
Eluent concentrating daptomycin content to 20,000 units obtain concentrate, and Daptomycin total amount weight ratio 10% is added
Calcium acetate, the isopropanol of concentrate volume ratio 90% place crystallization for 24 hours, filter, drying.Obtain Daptomycin crystal powder.
Obtained Daptomycin crystallization wet-milling solution crosses the desalination of HZ016 resin in water, and it is de- that active carbon is added by 1/10 times of amount
Color removes endotoxin, filtering, and the isopropanol that 10 times of amounts are added in filtrate is precipitated, and is filtered, and it is dry that Daptomycin precipitated powder carries out boiling
It is dry, Daptomycin crystal powder is obtained, purity 97.2% is dehydrated Daptomycin 0.5%, to the total recovery 22.5% of fermentation liquid.
Embodiment 3
Fermentation liquid 30L, the content of Daptomycin are 2000mg/L, carry out ceramic membrane filter, after being concentrated into volume half, side
Add the side washing of drinking water top to filter, keep amount of water consistent with filtering traffic, continues to 2 times that total filtrate volume is fermentating liquid volume
When amount, stop filtering.
Filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber close to nothing after having adsorbed
Color, 30% ethyl alcohol prewashing, 50% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L.It is eluted
Liquid.
After the ultrafiltration membrane of 50,000 dalton of eluent carries out ultrafiltration, nanofiltration concentration, ultrafiltration is controlled with temperature when filtering
At 20 degree or less.It is concentrated into Daptomycin unit 20,000 or so, adjusts pH value to 8.0, the upper NM-Q anion friendship of flow velocity 0.5BV/h
Resin is changed, after purified water top is washed, 10BV is first rinsed with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes impurity, is then used
50mmol/L sodium chloride solution rinses 6BV and collects the Daptomycin under being eluted, and is rinsed with 60mmol/L sodium chloride solution
15BV collects the Daptomycin being eluted, and finally rinses 6BV with 90mmol/L sodium chloride solution, collects purity and is greater than
90% Daptomycin eluent.
After eluent concentrating daptomycin content to 5,000mg/L, reverse phase silica gel NMsil-C18 (the reverse phase silicon of C18 is crossed
Glue, model NMSil-C18), its PH8.0 flow velocity 2BV/h upper prop is adjusted, it is first pre- with 20% ethyl alcohol -10mmol/L ammonium acetate solution
1BV is washed, then elutes pillar with 38% ethyl alcohol -30mmol/L ammonium acetate solution, Daptomycin of the purity greater than 97% is collected and elutes
Liquid.
Daptomycin total amount weight ratio 60% is added to the concentrate of 80,000 units in eluent concentrating daptomycin content
Calcium acetate, the isopropanol of concentrate volume ratio 50% place crystallization for 24 hours, filter, drying.Obtain Daptomycin crystal powder.
Obtained Daptomycin crystallization wet-milling solution crosses the desalination of HZ016 resin in water, and it is de- that active carbon is added by 1/10 times of amount
Color removes endotoxin, filtering, and the isopropanol that 10 times of amounts are added in filtrate is precipitated, and is filtered, and it is dry that Daptomycin precipitated powder carries out boiling
It is dry, Daptomycin crystal powder is obtained, purity 96.3% is dehydrated Daptomycin 0.9%, and the total recovery to fermentation liquid is 26.4%.
Embodiment 4
Fermentation liquid 30L, the content of Daptomycin are 2000mg/L, carry out ceramic membrane filter, after being concentrated into volume half, side
Add the side washing of drinking water top to filter, keep amount of water consistent with filtering traffic, continues to 4 times that total filtrate volume is fermentating liquid volume
When amount, stop filtering.
Filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber close to nothing after having adsorbed
Color, 30% ethyl alcohol prewashing, 40% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L.It is eluted
Liquid.
After the ultrafiltration membrane of 30,000 dalton of eluent carries out ultrafiltration, nanofiltration concentration, ultrafiltration is controlled with temperature when filtering
At 20 degree or less.It is concentrated into Daptomycin unit 10,000 or so, adjusts pH value to 7.0, the upper NM-Q anion friendship of flow velocity 0.5BV/h
Resin is changed, after purified water top is washed, 10BV is first rinsed with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes impurity, is then used
50mmol/L sodium chloride solution rinses 6BV and collects the Daptomycin under being eluted, and is rinsed with 60mmol/L sodium chloride solution
15BV collects the Daptomycin being eluted, and finally rinses 6BV with 90mmol/L sodium chloride solution, collects purity and is greater than
90% Daptomycin eluent.
After eluent concentrating daptomycin content to 30,000mg/L, reverse phase silica gel NMsil-C18 (the reverse phase silicon of C18 is crossed
Glue, model NMSil-C18), its PH6.0 flow velocity 1.0BV/h upper prop is adjusted, first with 20% ethyl alcohol -10mmol/L ammonium acetate solution
Prewashing 1BV, then pillar is eluted with 38% ethyl alcohol -30mmol/L ammonium acetate solution, it collects Daptomycin of the purity greater than 97% and washes
De- liquid.
Eluent concentrating daptomycin content to 40,000 units obtain concentrate, and Daptomycin total amount weight ratio 35% is added
Calcium acetate, the isopropanol of concentrate volume ratio 70% place crystallization for 24 hours, filter, drying.Obtain Daptomycin crystal powder.
Obtained Daptomycin crystallization wet-milling solution crosses the desalination of HZ016 resin in water, and it is de- that active carbon is added by 1/10 times of amount
Color removes endotoxin, filtering, and the isopropanol that 10 times of amounts are added in filtrate is precipitated, and is filtered, and it is dry that Daptomycin precipitated powder carries out boiling
It is dry, Daptomycin crystal powder is obtained, purity 97.6% is dehydrated Daptomycin 0.4%, and the total recovery to fermentation liquid is 28.8%.
Embodiment 5
Fermentation liquid 30L, the content of Daptomycin are 2000mg/L, carry out ceramic membrane filter, after being concentrated into volume half, side
Add the side washing of drinking water top to filter, keep amount of water consistent with filtering traffic, continues to 5 times that total filtrate volume is fermentating liquid volume
When amount, stop filtering.
Obtained filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber after having adsorbed
Close to colourless, 20% ethyl alcohol prewashing, 60% ethanol elution resin trap Daptomycin unit is greater than the eluent of 100mg/L.
Obtained eluent, after carrying out ultrafiltration with the ultrafiltration membrane of 20,000 dalton, nanofiltration is concentrated, ultrafiltration and temperature when filtering
Degree control is at 20 degree or less.Be concentrated into Daptomycin unit 15,000 or so, adjust pH value to 6.5, on flow velocity 5BV/h NM-Q yin from
Sub-exchange resin after purified water top is washed, first rinses 10BV with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes impurity, then
6BV is rinsed with 50mmol/L sodium chloride solution and collects the Daptomycin under being eluted, and is rinsed with 60mmol/L sodium chloride solution
15BV collects the Daptomycin being eluted, and finally rinses 6BV with 90mmol/L sodium chloride solution, collects purity and is greater than
90% Daptomycin eluent.
After obtained eluent concentrating daptomycin content to 30,000mg/L, reverse phase silica gel NMsil-C18 excessively be (C18's
Reverse phase silica gel, model NMSil-C18), its PH6.5 flow velocity 4.2BV/h upper prop is adjusted, first with 20% ethyl alcohol -20mmol/L acetic acid
Ammonium salt solution prewashing 1BV, then pillar is eluted with 38% ethyl alcohol -20mmol/L ammonium acetate solution, collect the Da Tuo that purity is greater than 97%
Mycin eluent.
Obtained eluent concentrating daptomycin content to 60,000 units obtain concentrate, and Daptomycin total amount weight ratio is added
45% calcium acetate, the isopropanol of concentrate volume ratio 81% place crystallization for 24 hours, filter, drying.Obtain Daptomycin crystallization
Powder.
Obtained Daptomycin crystallization wet-milling 80.03g, is dissolved in the water, crosses the desalination of HZ016 resin, add by 1/10 times of amount
Enter active carbon decoloring except endotoxin, the isopropanol of filtering, filtrate 10 times of amounts of addition is precipitated, filtered, Daptomycin precipitated powder
Fluidized drying is carried out, Daptomycin crystal powder is obtained, purity 98.3% is dehydrated Daptomycin content 0.3%, to fermentation liquid
Total recovery is 44.6%.
Comparative example 1
It tests A (scheme in the Chinese invention patent of Publication No. CN 103724400A)
1L Daptomycin fermentation liquid is removed by the ceramic membrane filter in 0.01 μm of aperture to the water in Daptomycin fermentation liquid
The macromolecular substances such as dissolubility albumen, pigment obtain ceramic membrane filtrate, institute with the water of 3 times of volumes of fermentation liquid circulation washing ceramic membrane
Filtrate clear is obtained, and color is reddish yellow.
Filtrate is adjusted into pH to 6.0 with acetic acid diluted, crosses FPDA13 resin, washed resin and the 0.06N acetic acid for being 6.5 with pH
Sodium solution and the elution of 0.2N sodium chloride solution, collect the eluent that Daptomycin unit is greater than 100mg/L.
It tests B (correlation step of technical solution of the present invention: step (1) and (2))
1L Daptomycin fermentation liquid carries out ceramic membrane filter, after being concentrated into volume half, while the side washing of drinking water top is added to filter,
Keep amount of water it is consistent with filtering traffic, continue to total filtrate volume be fermentating liquid volume 3 times of amounts when, collect filter.
Filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber close to nothing after having adsorbed
Color, 20% ethyl alcohol prewashing, 60% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L.
It tests C (scheme in the Chinese invention patent of Publication No. CN 102924572A)
Taking fermentation unit is the Daptomycin fermentation liquid 100L of 1960 μ g/mL, and 3000g pearl is added in above-mentioned fermentation liquid
Rock stirs plate-frame filtering after 1h, obtains Daptomycin filtrate as filter aid.
Daptomycin filtrate is imported into macroreticular resin D312 column (loading amount 20L) with the flow velocity of 40L/h and carries out absorption enrichment,
Absorption, which finishes, is first washed with 35% ethanol water, then is desorbed with 72% ethanol water, desorbs flow control in 10L/
H starts to collect when Daptomycin concentration is higher than 200 μ g/mL in HPLC detection stripping liquid, when concentration is lower than 200 μ g/mL,
Stop collecting.
It tests D (scheme in the Chinese invention patent of Publication No. CN 102241736A)
Daptomycin Fermented Condensed liquid 1L is taken, adjusts pH3.6 with 1N hydrochloric acid.It is added with stirring 200g polyacrylic acid, adds 80g
Diatomite controls 10 DEG C of temperature and stirs 3 hours, filter cake is obtained by filtration, filter cake, which re-dissolves, is obtained by filtration filtrate.
Filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber close to nothing after having adsorbed
Color, 20% ethyl alcohol prewashing, 60% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L.
Experimental result: the Daptomycin yield for testing A is 78%, and eluent purity relatively low of last Daptomycin
Have 52%；The Daptomycin yield for testing B is 85%, obtained Daptomycin purity 70%；Experiment C Daptomycin yield be
65%, obtained Daptomycin purity 66%, Investigation on Plate Filtration is difficult to filter, time-consuming very long, loses larger；Experiment D's reaches
Tobramycin yield is 72%, obtained Daptomycin purity 70%.Experiment C and experiment D require that filter aid is added, and increase work
The complexity of skill；And test operating procedure of the A before and after FPDA13 resin and need to adjust pH value and 2 kinds of elutions, increase
The complexity of technique is added.
And test Daptomycin described in B slightly mention method do not need adjust pH value and be added filter aid, the reagent kind used
Class is few, easy to operate, and the rate of recovery is high, and purity is also high, and the Daptomycin better than experiment A, C and D slightly proposes method.
Comparative example 2 (correlation step of technical solution of the present invention: step (3))
After the experiment of the comparative example 1 resulting eluent 500ml of B carries out ultrafiltration with the ultrafiltration membrane of 20000 dalton, nanofiltration
Concentration, temperature control is at 20 degree or less when ultrafiltration and nanofiltration.It is concentrated into Daptomycin unit 10,000mg/L or so adjusts PH6.5
NM-Q anion exchange resin on flow velocity 5BV/h after purified water top is washed, elutes resin with sodium chloride solution, collects purity and be greater than
90% Daptomycin eluent.
Through overtesting, if be higher than 20 degree carries out ultrafiltration and nanofiltration at room temperature, Daptomycin is easy degradation, and dehydration is mould up to holding in the palm
Element and β-isomers increase.
Test E: sodium chloride solution elutes resin method particularly includes: first uses 25mM sodium chloride solution with 3mL/min flow velocity
It rinses 12.0BV and removes impurity, then rinse 7.0BV with 50mM sodium chloride solution and collect the Daptomycin under being eluted, finally use
500mM sodium chloride solution rinses 3.0BV (referring to Chinese patent CN102993273A).
Test F: sodium chloride solution elutes resin method particularly includes: first uses 25mmol/L sodium chloride molten with 5BV/h flow velocity
Liquid rinses 10BV and removes impurity, then rinses 6BV with 50mmol/L sodium chloride solution and collects the Daptomycin under being eluted, uses
60mmol/L sodium chloride solution rinses 15BV, collects the Daptomycin being eluted, and finally uses 90mmol/L sodium chloride solution
Rinse 6BV.
Experimental result: F is compared with testing E for experiment, and it is poor to be that the elution volume of eluent sodium chloride solution and concentration exist
Different, these differences cause the Daptomycin yield for testing E to be 55%；The Daptomycin yield for testing F is 80%, these differences are led
It causes the Daptomycin yield of experiment F to improve 45.5% than experiment E, improves nearly half, the poor yields dissenting views of Daptomycin
Method of the bright experiment F better than experiment E.
Comparative example 3 (correlation step of technical solution of the present invention: step (2) and (3))
Test G: step (2)+(3)
1L Daptomycin fermentation liquid carries out ceramic membrane filter, after being concentrated into volume half, while the side washing of drinking water top is added to filter,
Keep amount of water it is consistent with filtering traffic, continue to total filtrate volume be fermentating liquid volume 3 times of amounts when, collect filter.
Filtrate crosses the absorption of macroporous absorbent resin HZ801 column, and flow velocity 0.8BV/h is washed to omission timber close to nothing after having adsorbed
Color, 20% ethyl alcohol prewashing, 60% ethanol elution resin trap Daptomycin unit are greater than the eluent of 100mg/L；
After eluent carries out ultrafiltration with the ultrafiltration membrane of 20000 dalton, nanofiltration concentration, temperature control exists when ultrafiltration and nanofiltration
20 degree or less.It is concentrated into Daptomycin unit 10000 or so, adjusts NM-Q anion exchange resin on PH6.5 flow velocity 5BV/h, it is pure
After change water top is washed, resin is eluted with sodium chloride solution, collects the Daptomycin eluent that purity is greater than 90%.
Test H: step (3)+(2)
1L Daptomycin fermentation liquid carries out ceramic membrane filter, after being concentrated into volume half, while the side washing of drinking water top is added to filter,
Keep amount of water it is consistent with filtering traffic, continue to total filtrate volume be fermentating liquid volume 3 times of amounts when, collect filter.
After filtrate carries out ultrafiltration with 20000 dalton ultrafiltration membranes, adjust pH value to 6.5, the upper NM-Q anion friendship of flow velocity 5BV/h
Resin is changed, after purified water top is washed, resin is eluted with sodium chloride solution, collects the eluent that Daptomycin unit is greater than 100mg/L；
Eluent with 20000 all you ultrafiltration membranes carries out ultrafiltration after, nanofiltration concentration, temperature, which controls, when ultrafiltration and nanofiltration exists
20 degree or less.It is concentrated into Daptomycin unit 10000mg/L or so, crosses the absorption of macroporous absorbent resin HZ801 column, flow velocity 0.8BV/
H is washed to omission timber close to colourless, 20% ethyl alcohol prewashing after having adsorbed, 60% ethanol elution resin is collected and is greater than up to purity
90% Daptomycin eluent.
Experimental result: experiment G is only that the sequence of implementation steps is different with the difference for testing H, but implementation steps are not
It is same that the Daptomycin yield for testing G is caused to be 42%, and it is 68% that the Daptomycin yield for testing H, which is up to,；Test the mould up to holding in the palm of H
Plain yield improves 61.9% compared with testing G, and it is more to improve half.The yield of Daptomycin illustrates the implementation sequence for testing G
Implementation sequence better than experiment H.
Comparative example 4 (correlation step of technical solution of the present invention: step (4))
It tests I:(and refers to Chinese patent CN 102924572A)
Comparative example 3 tests the resulting eluent injection of polymer nanosphere UNIPS30RPC column (loading amount 1L) of G
Carry out chromatography, then successively use concentration be respectively 35%, 55% methanol aqueous solution as eluant, eluent to UNIPS30RPC
Column carries out gradient elution, and analyzes the HPLC content of eluent.According to HPLC testing result, collects HPLC purity and be more than or equal to
97% eluent.
Experiment J: embodiment 3 tests the resulting eluent of G and crosses reverse phase silica gel NMsil-C18 (reverse phase silica gel of C18, model
For NMSil-C18), adjust its pH value to 6.5, flow velocity 0.5~2BV/h upper prop, first with 20% ethyl alcohol -20mmol/L ammonium acetate solution
Prewashing 1BV, then eluted with 38% ethyl alcohol -20mmol/L ammonium acetate solution, collect the eluent that purity is greater than 97%.
Experimental result: the Daptomycin yield for testing I is 66%；The Daptomycin yield for testing J is 74%.It is excellent to test J
In experiment I.
Comparative example 5 (correlation step of technical solution of the present invention: step (4))
Embodiment 3 tests the resulting eluent of G and crosses reverse phase silica gel NMsil-C18, adjusts its pH value to 6.5, and flow velocity 0.5~
Resin is washed on 6BV/h upper prop, water top, with elution pillar, collects the eluent that purity is greater than 97%.
Table 1
| Eluent title | Yield | Purity | Color | It is dehydrated Daptomycin content |
| Eluent 1 | 74% | 97.8 | It is light yellow | 0.5 |
| Eluent 2 | 60% | 97.5 | Yellow | 0.6 |
| Eluent 3 | 48% | 97.1 | Yellow | 0.7 |
| Eluent 4 | 43% | 97.0 | Yellow | 0.8 |
| Eluent 5 | 59% | 97.5 | Yellow | 0.6 |
Each elution mode of table 1:
Eluent 1: first with 20% ethyl alcohol -20mmol/L ammonium acetate solution prewashing 1BV, then with 38% ethyl alcohol -20mmol/L
Ammonium acetate solution elution；
Eluent 2: it is eluted with 38% ethyl alcohol -20mmol/L ammonium acetate solution；
Eluent 3: ethanol elution is used；
Eluent 4: it is eluted with isopropanol；
Eluent 5: first with 20% isopropanol -20mmol/L ammonium acetate solution prewashing 1BV, then with 38% isopropanol -
The elution of 20mmol/L ammonium acetate solution；
It can be seen that the eluent collected purity when eluent 1-5 is eluted and be greater than 97%, ethanol acetate from the data of table 1
The content that Daptomycin is dehydrated in gradient and the yield and resulting Daptomycin of the elution of acetic acid isopropanol is all slightly better than other groups
, but in resulting Daptomycin color, the 1 resulting color of gradient gradient elution of eluent is most shallow, and decolorizing effect is optimal,
Therefore eluent of 1 gradient of eluent as reverse phase silica gel NMsil-C18 is selected.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright description are applied directly or indirectly in relevant technical field, are similarly included in this hair
In bright scope of patent protection.
Claims (7)
1. a kind of Daptomycin extracting method, which comprises the steps of:
Step (1): Daptomycin fermentation liquid is subjected to ceramic membrane filter, collects filtered fluid；
Step (2): filtered fluid obtained by step (1) is crossed into the absorption of macroporous absorbent resin HZ801 column, after having adsorbed, is washed to leakage
Liquid obtains the eluent that Daptomycin unit is greater than 100mg/L close to colourless, with ethanol elution resin trap；
Step (3): by the resulting eluent tune PH to 4.0-8.0 of step (2), upper NM-Q anion exchange resin, purified water top
After washing, 25mM sodium chloride solution is successively used, 50mM sodium chloride solution, 60mM sodium chloride solution, 90mM sodium chloride solution carries out terraced
Degree elution collects and obtains the Daptomycin eluent that purity is greater than 90%；
Step (4): the resulting Daptomycin eluent of step (3) is crossed into reverse phase silica gel NMsil-C18 column, adjusts its PH to 4.0-
8.0, flow velocity 0.5-6BV/h upper prop, first with 20% ethyl alcohol -20mmol/L ammonium acetate solution prewashing 1BV, then with 38% ethyl alcohol -
The elution of 20mmol/L ammonium acetate solution, collection obtain purity greater than 97% Daptomycin eluent；
Step (5): it is 20000-80000mg/L that the resulting Daptomycin eluent of step (4), which is concentrated to Daptomycin concentration,
Concentrate is obtained, the calcium acetate of the 10%-90% of the Daptomycin quality of concentrate is added, volume of the concentrated liquid 50%-90%'s is different
Propyl alcohol, places 36-48h, and crystallization filters, dries to obtain Daptomycin crystal powder.
2. Daptomycin extracting method as described in claim 1, which is characterized in that the step (1) specifically: by Daptomycin
Fermentation liquid carries out ceramic membrane filter, after being concentrated into volume half, while the side washing of water top is added to filter, keeps amount of water and filtering traffic one
It causes, when continuing to 2-5 times that total filtrate volume is fermentating liquid volume to measure, stops filtering, collect filtered fluid.
3. Daptomycin extracting method as described in claim 1, which is characterized in that the step (2) specifically: by step (1)
Gained filtered fluid crosses the absorption of macroporous absorbent resin HZ801 column, flow velocity 0.8BV/h, omission timber is washed to after having adsorbed close to colourless,
It is first 20% ethanol solution prewashing with percentage by volume, then carries out elution resin trap with percentage by volume for 60% ethanol solution
Obtain the eluent that Daptomycin unit is greater than 100mg/L.
4. Daptomycin extracting method as described in claim 1, which is characterized in that " with sodium chloride solution ladder in the step (3)
Degree elution " specifically: 10BV is first rinsed with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes impurity, then uses 50mmol/L
Sodium chloride solution rinses 6BV and collects the Daptomycin under being eluted, and rinses 15BV with 60mmol/L sodium chloride solution, collects quilt
The Daptomycin eluted finally rinses 6BV with 90mmol/L sodium chloride solution.
5. Daptomycin extracting method as described in claim 1, which is characterized in that " reached step (4) is resulting in step (5)
It is 20000-80000mg/L " that Tobramycin eluent, which is concentrated to Daptomycin concentration, specifically: step (4) is resulting mould up to holding in the palm
Plain eluent is concentrated to unit 50000mg/L, adjusts its PH to 4.8.
6. Daptomycin extracting method as described in claim 1, which is characterized in that further include step (6): will be obtained by step (5)
Daptomycin crystal powder is dissolved in the water, and crosses the desalination of HZ016 resin, and eluent is added active carbon decoloring by 1/10 times of amount and removes endogenous toxic material
Element, filtering, the isopropanol that 10 times of amounts are added in filtrate are precipitated, and gained Daptomycin precipitated powder progress fluidized drying must be reached
Tobramycin finished product.
7. Daptomycin extracting method as described in claim 1, which is characterized in that specifically comprise the following steps:
Step (1): by Daptomycin fermentation liquid 30L, the content of the Daptomycin fermentation liquid Daptomycin is 2000mg/L, into
Row ceramic membrane filter after being concentrated into volume half, while the side washing of water top is added to filter, keeps amount of water consistent with filtering traffic, continues
When to 5 times of amounts that total filtrate volume is fermentating liquid volume, stops filtering, obtain filtrate 160L, Daptomycin content is 340mg/
L；
Step (2): the filtrate that step (1) obtains crosses the absorption of macroporous absorbent resin HZ801 column, flow velocity 0.8BV/h, water after having adsorbed
Omission timber is washed till close to colourless, is first 20% ethyl alcohol prewashing with percentage by volume, then carried out with percentage by volume for 60% ethyl alcohol
The eluent that resin trap Daptomycin unit is greater than 100mg/L is eluted, obtains eluent 9L, Daptomycin content is
5700mg/L；
Step (3): the eluent that step (2) obtains, after carrying out ultrafiltration with the ultrafiltration membrane of 20000 dalton, nanofiltration concentration, ultrafiltration
With temperature control when filtering in 15-20 degree, it is concentrated into Daptomycin unit 15000-16000, tune pH value to 6.5, flow velocity 5BV/h
Upper NM-Q anion exchange resin after purified water top is washed, first rinses 10BV with 25mmol/L sodium chloride solution with 5BV/h flow velocity and removes
Then impurity rinses 6BV with 50mmol/L sodium chloride solution and collects the Daptomycin under being eluted, with 60mmol/L sodium chloride
Solution rinses 15BV, collects the Daptomycin being eluted, and finally rinses 6BV with 90mmol/L sodium chloride solution, collects pure
Degree is greater than 90% Daptomycin eluent, obtains eluent 25.2L, and Daptomycin content is 1650mg/L；
Step (4): after the eluent concentrating daptomycin content to 30000mg/L that step (3) obtains, reverse phase silica gel NMsil- is crossed
C18, adjusts its PH6.5, flow velocity 4.2BV/h upper prop, first with 20% ethyl alcohol -20mmol/L ammonium acetate solution prewashing 1BV, then with 38%
Ethyl alcohol -20mmol/L ammonium acetate solution elutes pillar, collects the Daptomycin eluent that purity is greater than 97%, obtains eluent
17.2L, Daptomycin content are 1790mg/L；
Step (5): the eluent concentrating daptomycin content that step (4) obtains to 50000mg/L obtains concentrate, and concentrate is added
Daptomycin quality 45% calcium acetate, the isopropanol of the volume of the concentrated liquid 81% places crystallization for 24 hours, filters, and drying obtains
To Daptomycin crystal powder；
Step (6): the Daptomycin that step (5) obtains crystallizes wet-milling 80.03g, is dissolved in the water, and crosses the desalination of HZ016 resin, presses
1/10 times of amount is added active carbon decoloring and removes endotoxin, filtering, and the isopropanol that 10 times of amounts are added in filtrate is precipitated, and filters, gained
Daptomycin precipitated powder carries out fluidized drying and obtains Daptomycin finished product.
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| CN107868120B (en) * | 2017-12-22 | 2021-06-01 | 苏州纳微科技股份有限公司 | Daptomycin purification method |
| CN109666065B (en) * | 2018-11-22 | 2022-02-25 | 北大方正集团有限公司 | Method for rapidly preparing high-purity daptomycin |
| CN110117310B (en) * | 2019-04-17 | 2023-06-27 | 华北制药华胜有限公司 | Purification method of daptomycin |
| CN112979756B (en) * | 2019-12-13 | 2023-01-03 | 浙江昌海制药有限公司 | Purification method of daptomycin |
| CN113004373A (en) * | 2019-12-19 | 2021-06-22 | 鲁南制药集团股份有限公司 | Daptomycin purification method |
| CN113563424B (en) * | 2021-09-15 | 2023-03-28 | 丽珠集团福州福兴医药有限公司 | Daptomycin purification method |
| CN113717253B (en) * | 2021-09-15 | 2023-03-21 | 丽珠集团福州福兴医药有限公司 | Purification method of daptomycin |
| CN114344447B (en) * | 2021-12-16 | 2023-08-25 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN115010792A (en) * | 2022-06-23 | 2022-09-06 | 丽珠集团新北江制药股份有限公司 | Purification method of vancomycin hydrochloride |
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