CN104231057B - The purification process of the copper chelate of bleomycin A5 and its congeners - Google Patents
The purification process of the copper chelate of bleomycin A5 and its congeners Download PDFInfo
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Abstract
The invention discloses a kind of bleomycin A5 and its purification process of the copper chelate of congeners, monodisperse polystyrene/divinylbenzene chromatography media is used alone or is used with single dispersing polymethacrylates mixed type cation-exchange chromatography combination of media, chromatographed as filler.Instant invention overcomes former process cycle efficiency long is low, the deficiency that product purity is low, the bleomycin A5 of high-purity and its copper chelate of congeners are obtained, purity is determined with HPLC methods, by area percentage, reaches more than 97%.By the copper chelate further through decopper(ing) after, that is, obtain the bleomycin A5 and its congeners of high-purity, improve the security of clinical application.
Description
Technical field
The invention belongs to pharmaceutical chemistry and separating and purifying technology field, and in particular to a kind of bleomycin A5 and its congeners
Copper chelate purification process.
Background technology
Bleomycin A5（Pingyangmycin）And its congeners B1eomycin（Boanmycin）, boningmycin
（Boningmycin）Be by the antitumor antibiotics of Chinese scholar independent research and development, wherein bleomycin A5 and B1eomycin
Input clinical practice, their Dou Shu bleomycins families.The bleomycin of import（bleomycin）Main component is bleomycin
A2 and B2, bleomycin A5 is identical with the structure of Pingyangmycin, and B1eomycin is identical with the structure of boanmycin hydrochloride, boningmycin
It is the acetylate of B1eomycin end ammonia body free amine group.Bleomycin A5 is existing to be recorded in Pharmacopoeia of People's Republic of China two
（2010 editions）, it is the clinical more common cancer therapy drug of China.
Preclinical study shows that bleomycin A5 and its congeners B1eomycin, boningmycin are to a series of transplanting
The human body carcinoma of property tumour and nude mice model has significant curative effect, while being significantly lower than to the acute toxicity and pulmonary toxicity of mouse
Bleomycin.
Clinical research shows that bleomycin A5 has preferable curative effect to incidence carcinoma, lymthoma etc., particularly with the department of stomatology
Carcinoma has significant curative effect.Bleomycin A5 seldom suppresses spinal cord hemopoietic system and immunologic function, can be with other chemotherapy drugs in combination, can
For the patient being damaged by previous tretament hematopoiesis function.Current bleomycin A5 clinical practice has been further expanded, especially
It is in the pteryium effect is significant for treating hemangioma, lymphangioma and condyloma acuminatum and ophthalmology.Monoclonal in recent years
The conjugate of antibody and bleomycin A5 show in zoopery and acted on height curative effect, points out it in development antibody target medicine
In potential value【Zhen Yongsu etc., the research of antitumor antibiotics bleomycin A5 and clinical practice 40 years, antibiotic impurity, 2009,
Vol34 (10), 577~580】.B1eomycin has significant curative effect to head and neck neoplasm, to spinal cord hemopoietic system and immunologic function
Influence is small, and pulmonary toxicity is low, and also has stronger inhibitory action to HCC.Boningmycin is right in addition to antitumor action
Condyloma acuminatum, flat wart that psoriasis, seborrheic dermatitis and papillomavirus cause etc. play the role of good.
The current production technology of bleomycin A5 is first to produce bleomycin A5 etc. more than ten to plant component through microbial fermentation to constitute
Compound, be subsequently adding mantoquita formed bleomycin A5 copper chelate, then isolated and purified through ion-exchange chromatography repeatedly obtain compared with
The bleomycin A5 copper chelate of high-purity, then processed through decopper(ing), obtain bleomycin A5【Xu Hongzhang etc., the separation of bleomycin A5 and
Differentiate, Acta Pharmaceutica Sinica, 1980, vol15 (10), 609~614】.The production technology and bleomycin A5 of B1eomycin and boningmycin
It is similar, all it is, by microbial fermentation, then to form copper chelate, by decopper(ing) obtains B1eomycin and boningmycin again after purification
【Xu Hongzhang etc., bleomycin A6 and it in the status of bleomycin compound, Acta Pharmaceutica Sinica, 1988, vol23 (9), 667~
671】.Boningmycin can also obtain boningmycin cupric by B1eomycin cupric product by the method acetylation of chemical synthesis
Product, then decopper(ing) obtains boningmycin.
Bleomycin A5 copper chelate has bright-coloured blueness, and more former stability of compounds is bleomycin A5 and its chemical combination of the same clan
Important intermediate in thing production technology.Because bleomycin A5 and its congeners and copper chelate are very big water-soluble polarity
Property composition, isolate and purify suitable difficulty, therefore the purity of copper chelate turns into restriction bleomycin A5 and its congeners
The bottleneck of end product quality.Isolating and purifying for current their copper chelates uses ion-exchange gel filler always, predominantly
The fillers of the CM-sephadex of glucan skeleton-C 1.The filler swelling ratio is high, and Volume Changes are very in different salting liquids
Greatly, it is non-rigid filler, it is impossible to pressurize, to reach separating degree higher, post footpath is than one more than 30 so that each circulation
Cycle is very long, it is impossible to realize quick separating, and whole efficiency is low.Simultaneously because CM-sephadex-C 1 separation of itself
Degree is not very high, causes some impurity to remove so that the purity of final products is not high, and quality is relatively low.Chinese people's republicanism
Based on dry product, hydrochloric bleomycin A5 must not be less than the lower regulation this product of state's pharmacopeia two (2010 editions) bleomycin A5 hydrochloride
85.0%, hence it is evident that less than the requirement of one chemical drug.At present in the bleomycin A5 hydrochloride freeze drying powder injection of market circulation, with pharmacopeia
Liquid-phase condition detects that, according to area percentage, purity is in 92% or so (such as table 1 below and Fig. 1).B1eomycin situation is similar,
This is closely related with the technological level that copper chelate is isolated and purified with current bleomycin A5 and its congeners.
Table 1
PDACh1254nm4nm
| Peak # | Retention time | Area | Highly | Area % | Separating degree | Theoretical tray # |
| l | 12.367 | 3613 | 278 | 0.019 | 0.000 | 18408.173 |
| 2 | 23.288 | 48799 | 1840 | 0.254 | 20.330 | 17223.339 |
| 3 | 24.795 | 933l | 405 | 0.049 | 2.208 | 22878.556 |
| 4 | 25.893 | 12413 | 516 | 0.065 | 1.674 | 24910.097 |
| 5 | 27.823 | 98461 | 3180 | 0.513 | 2.537 | 16554.103 |
| 6 | 28.496 | 118256 | 5004 | 0.616 | 0.803 | 19745.637 |
| 7 | 28.970 | 174831 | 5511 | 0.9ll | 0.496 | 11122.725 |
| 8 | 29.879 | 17589010 | 502277 | 91.648 | 0.890 | 16023.010 |
| 9 | 31.262 | 415126 | 16171 | 2.163 | 1.581 | 24144.494 |
| l0 | 32.195 | 108182 | 3116 | 0.564 | 1.188 | 28196.639 |
| ll | 33.646 | 440848 | 21699 | 2.297 | 2.241 | 64950.931 |
| 12 | 34.478 | 32342 | 1472 | 0.169 | 1.488 | 54685.000 |
| 13 | 36.152 | 8020 | 467 | 0.042 | 3.15l | 93690.848 |
| 14 | 36.691 | 7602 | 360 | 0.040 | 1.039 | 67170.140 |
| 15 | 38.360 | 48577 | 2322 | 0.253 | 2.963 | 75078.577 |
| 16 | 39.417 | 76571 | 4269 | 0.399 | 2.03l | 107173.227 |
| Amount to | 19191981 | 568889 | 100.000 |
Adverse reaction monitoring annual report shows within 2012, and 56.7% adverse drug reaction comes from injection.Note
The impurities affect of agent bulk drug is penetrated to product quality, the security of clinical application is directly connected to.Therefore, innovation bleomycin A5 and
The separation purifying technique of its congeners and copper chelate, obtains the bleomycin A5 and its congeners and copper of high-purity
Chelate, so as to improve the quality of final products, reduces the adverse reaction of clinical application, as currently very exigence.
The content of the invention
The technical problems to be solved by the invention are the copper chelate for providing a kind of bleomycin A5 and its congeners
Purification process, by the bleomycin A5 and its congeners that prepare high-purity（Including B1eomycin, boningmycin）Copper
Chelate, so as to lift the product quality of bleomycin A5 and its congeners, improves the security of clinical application.
The present invention with bleomycin A5 and its congeners original production process through microbial fermentation, and add mantoquita chelate
The corresponding copper chelate crude product for arriving is raw material, and the bleomycin A5 and its of the same clanization of high-purity are obtained through specific separation purifying technique
The copper chelate of compound.Further post-processed through decopper(ing), drying etc., you can obtain the bleomycin A5 and its chemical combination of the same clan of high-purity
Produce product.
Described bleomycin A5 and its purification process of the copper chelate of congeners, may be used to lower two ways and enter
OK：
Method one：Chromatographed as filler from monodisperse polystyrene/divinylbenzene chromatography media, particle diameter 20~
60 μm, particle diameter fluctuation range<5 μm, apertureIt is preferred that the UniPS of Suzhou Nano-Micro Bio-technology Co., Ltd.TM-
20、UniPSTM-30、UniPSTM- 40 or UniPSTM- 50 serial filler products, more preferably UniPSTM- 40 series.Aperture is preferablyOrMore preferably aperture
Take bleomycin A5 or the copper chelate crude product of its congeners（Purity 20%~60%）It is added to ultrapure water dissolves
With on ultra-pure water or the chromatographic column that balances each other of flowing, pillar height is with post footpath than 15~40：1, chromatographic column 5~50mg/ml of carrying capacity, with 0
~20% methyl alcohol, ethanol, aqueous acetone solution isocratic elution or gradient elution（Such as 0~10%-20% linear gradients）, preferably 10%
Methyl alcohol isocratic elution, elution flow rate：0.5~2ml/ (mincm2).Single is chromatographed, and purity can bring up to 70~85%, the rate of recovery
>75%, single 2~6 hours time of chromatography.Again through 2~3 chromatographies, purity can reach more than 97%, not consider recycling chromatography, return
Yield>25%, it is considered to recycling chromatography, the accumulation rate of recovery can reach more than 80%.If using CM-sephadex-C-25 fillers, to reach
To similar purification effect, single at least 60~100 hours time of chromatography.Use monodisperse polystyrene/divinyl benzene layer
Analysis filler, compared to CM-sephadex-C-25 fillers, efficiency can improve 10~16 times.
Method two：By the medium used by method one（Monodisperse polystyrene/divinylbenzene chromatography media）With single dispersing
Polymethacrylates mixed type cation-exchange chromatography combination of media, is chromatographed as filler.
Described single dispersing polymethacrylates mixed type cation-exchange chromatography medium, 30~70 μm of particle diameter, particle diameter
Fluctuation range<5 μm, apertureIt is preferred that the UniMSP-30 or UniMSP- of Suzhou Nano-Micro Bio-technology Co., Ltd.
50 serial filler products, more preferably UniMSP-50 series.Aperture is preferablyOrMore preferably
Its application method is exemplified below：
Take the copper chelate crude product of bleomycin A5 or its congeners or with the sample after the medium preliminary purification of method one
Product, are added on the chromatographic column with ultrapure water balance with water dissolves, and pillar height is with post footpath than 15~40：1, with single dispersing polymethyl
Acid esters mixed type cation-exchange chromatography medium is chromatographed for filler, 5~50mg/ml of chromatographic column carrying capacity.With various concentrations
The aqueous solution gradient elution of ammonium chloride or ammonium formate（Such as use 0.05M~0.5M ammonium chlorides gradient elution, 0.1M~1.0M ammonium chlorides
Gradient elution, or 0.05~1M ammonium formate gradient elutions）.Elution flow rate：0.5~2ml/ (mincm2).Single is chromatographed, and is used for
Purifying crude, purity can improve 20~55%, the rate of recovery>75%, single 7~15 hours time of chromatography.With in method one after desalination
Medium chromatograph again.Than CM-sephadex-C-25 filler for single chromatography, efficiency at least improves 7~10 times.For
The Sample Purification on Single of purity 90% or so, purity can improve more than 5%, the rate of recovery>50%.Cupric product chelate crude product is reclaimed, through the above
Combination recycling chromatography 1~3 time, can obtain the bleomycin A5 of purity more than 97% or the copper chelate of its congeners.
Two kinds of filler combinations are used, it is possible to reduce the consumption of organic solvent, beneficial to environmental protection.And be applied in combination, can make
The content of single impurity is lower.Such as following is applied in combination mode, can neatly combine in actual applications.
By bleomycin A5 and its copper chelate crude product of congeners（Purity 20%~60%）First with the poly- methyl of single dispersing
After acrylic ester mixed mould assembly cation-exchange chromatography medium purification 1~3 time, then chromatographed with rnonodisperse polystyrene/divinylbenzene
Medium purification, until purity reaches more than 97%.
Or by bleomycin A5 and its copper chelate crude product of congeners（Purity 20%~60%）First with dispersion polyphenyl
Ethene/divinylbenzene chromatography media is purified 1~3 time, then with single dispersing polymethacrylates mixed type cation exchange layer
Analysis medium purification, until purity reaches more than 97%.
The bleomycin A5 and its copper chelate of congeners obtained using above-mentioned purification process of the invention, use HPLC methods
Detection, according to area percentage, its purity is 97%~100%.
The high-purity that the above method is obtained（Purity 97%~100%）The copper chelating of bleomycin A5 and its congeners
Thing is through decopper(ing), and the post processing such as dries, you can obtain the bleomycin A5 and its congeners product of purity more than 97%.
The purity of the purity and their corresponding copper chelates of bleomycin A5 and its congeners, refers to use in the present invention
HPLC methods are determined, by the purity that area normalization method is calculated.
It should be noted that purification process of the invention is also applied for, other tools of specific name are not referred in the present invention
There is the compound of the bleomycin family similar with bleomycin A5 structure.
Beneficial effect：
Using purification process of the invention, the bleomycin A5 of high-purity and its copper chelate of congeners can be obtained
（Purity 97%~100%）, then post-processed through decopper(ing), drying etc., you can obtain the bleomycin A5 and its congeners of high-purity
Product, can increase substantially finished product quality, so as to improve the security of clinical application.
Purification process efficiency high of the invention, not only filler have separating degree higher to principal component and impurity, can obtain
The product of higher purity；Simultaneously because the rigid of filler is good, the coefficient of expansion is low, pressure-resistant, the cycle of each circulation in separation
Greatly shorten, production efficiency improves 7~16 times.
Brief description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of commercially available bleomycin A5 hydrochloride sample in 2011.
Fig. 2 is the HPLC collection of illustrative plates of bleomycin A5 copper chelate obtained in embodiment 3.
Specific embodiment
Technical scheme is described in further detail below in conjunction with specific embodiment.But these embodiments are only examples
Property, any limitation is not constituted to the scope of the present invention.It will be understood by those within the art that, without departing from this hair
The details of technical scheme and form can be modified or equivalent under bright spirit and scope, but these are repaiied
Change or replace and each fall within the scope of the present invention.
Embodiment 1
Take 3.0 grams of bleomycin A5 copper chelate crude products（Content 55%）, ultrapure water dissolves are used, ultrapure level is up to used in advance
Weighing apparatus equipped with UniPSTM-30The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).0%-15% linear gradients are pressed with ethanol-water system elute 4 column volumes（The volume of filler is referred here to, under
Together）, then 1 column volume is washed with 15% ethanol, all of colour band on post is all got off with 30% ethanol finally.Single chromatographs the time
About 4.5 hours, the stream part of purity more than 85% is collected, be concentrated under reduced pressure into dry, measure purity and the rate of recovery.Purity 87.6%, returns
Yield 82%.By sample again up to ultrapure water balance equipped with UniPSTM-30 The chromatographic column of serial filler, together
Operate above, obtain sample, purity 94.5%, the rate of recovery 76%.Repeat once, the stream part of purity more than 97% is collected, after merging
It is concentrated under reduced pressure to be evaporated, obtain sample, purity 99.2%, the rate of recovery 60%.The sample of not up to purity requirement is merged, the same method is anti-
Multiple mistake is equipped with UniPSTM-30The chromatographic column of serial filler, obtains the bleomycin A5 copper chelate of purity 99.3%, adds up
The rate of recovery 84%.
Embodiment 2
Take 10 grams of bleomycin A5 copper chelate crude products（Content 48%）, ultrapure water dissolves are used, ultrapure water balance is up to used in advance
Equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).0%-15% linear gradients are pressed with methanol-water solution and elute 4 column volumes, then wash 1 cylinder with 15% methyl alcohol
Product, is finally all got off all of colour band on post with 50% methyl alcohol.The single chromatography time is about 4.5 hours, collects purity 85%
Stream part above, is concentrated under reduced pressure into dry, measure purity and the rate of recovery.Purity 86.1%, the rate of recovery 76%.Sample is up to used again
Ultrapure water balance equipped with UniPSTM-40The chromatographic column of serial filler, operates more than, obtains sample, purity
94.8%, the rate of recovery, 75%.Repeat once, collect the stream part of purity more than 97%, being concentrated under reduced pressure after merging is evaporated, and obtains sample
Product, purity 98.7%, the rate of recovery 61%.The sample of not up to purity requirement is merged, the same method is crossed and UniPS is housed repeatedlyTM-40The chromatographic column of serial filler, obtains the bleomycin A5 copper chelate of purity 99.0%, adds up the rate of recovery 85%.
Embodiment 3
Take 40 grams of bleomycin A5 copper chelate crude products（Content 56%）, ultrapure water dissolves are used, it is up to flat with 10% methyl alcohol in advance
Weighing apparatus equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 5.0cm, 150cm high.Chromatographic flow rates 1.8ml/
(min·cm2).With 10% methanol aqueous solution isocratic elution, 4 column volumes are washed altogether, finally wash post with 50% methyl alcohol.When single is chromatographed
Between be 6 hours, collect purity more than 85% stream part, be concentrated under reduced pressure into it is dry, determine purity and the rate of recovery.Purity 89.3%, reclaims
Rate 84%.By sample again up to after 10% equilibrium methanol equipped with UniPSTM-40 The chromatographic column of serial filler, together
Operate above, collect the stream part of purity more than 97%, being concentrated under reduced pressure after merging is evaporated, and obtains sample, purity 98.7%（Referring to figure
2）, the rate of recovery 56%.The sample of not up to purity requirement is merged, the same method is crossed and UniPS is housed repeatedlyTM-40Series
The chromatographic column of filler, obtains the bleomycin A5 copper chelate of purity 99.1%, adds up the rate of recovery 89%.
Embodiment 4
Take 3.0 grams of bleomycin A5 copper chelate crude products（Content 43%）, ultrapure water dissolves are used, ultrapure level is up to used in advance
Weighing apparatus equipped with UniPSTM-50The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).0%-15% linear gradients are pressed with acetone-water system and elute 3 column volumes, then wash 1 column volume with 15% acetone,
Finally post is washed with 30% acetone.The single chromatography time is 4 hours, collects the stream part of purity more than 85%, is concentrated under reduced pressure into dry, measure
Purity and the rate of recovery.Purity 86.5%, the rate of recovery 83%.By sample again up to ultrapure water balance equipped with UniPSTM-50The chromatographic column of serial filler, operates more than, obtains sample, purity 95.3%, the rate of recovery 78%.Repeat once, obtain
To sample, purity 98.8%, the rate of recovery 68%.The sample of not up to purity requirement is merged, the same method is crossed and UniPS is housed repeatedlyTM-
50The chromatographic column of serial filler, obtains the bleomycin A5 copper chelate of purity 98.4%, adds up the rate of recovery 87%.
Embodiment 5
Take 3.0 grams of bleomycin A5 copper chelate crude products（Content 45%）, ultrapure water dissolves are used, it is up to flat with 10% methyl alcohol in advance
Weighing apparatus equipped with UniPSTM-20The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.5ml/
(min·cm2).With 10% methanol aqueous solution isocratic elution, 4 column volumes are washed altogether, finally wash post with 50% methyl alcohol.When single is chromatographed
Between about 6 hours, collect purity more than 85% stream part, be concentrated under reduced pressure into it is dry, determine purity and the rate of recovery.Purity 87.6%, returns
Yield 86%.By sample again up to 10% equilibrium methanol equipped with UniPSTM-20 The chromatographic column of serial filler, together
Operate above, obtain sample, purity 94.9%, the rate of recovery 80%.Repeat once, obtain sample, purity 97.6%, the rate of recovery
56%.The sample of not up to purity requirement is merged, the same method is crossed and UniPS is housed repeatedlyTM-20The chromatography of serial filler
Post, obtains the bleomycin A5 copper chelate of purity 98.2%, adds up the rate of recovery 82%.
Embodiment 6
Take 3.0 grams of bleomycin A5 copper chelate crude products（Content 50%）, ultrapure water dissolves are used, ultrapure level is up to used in advance
Weighing apparatus equipped with UniMSP-50The chromatographic column of serial filler, filler diameter 2.4cm, 50cm high.Chromatographic flow rates 0.8ml/
(min·cm2).First use 0.05M NH4Cl is eluted, and washes 1 column volume, and 0.1M NH are then used successively4Cl elutes 2 column volumes,
0.2M NH4Cl elutes 4 column volumes, 0.3M NH4Cl elutes 2 column volumes, 0.4M NH4Cl elutes 2 column volumes, uses 0.5M
NH4Cl is washed till all of colour band on post and all gets off.The single chromatography time is 12 hours, collects the stream part of purity more than 85%,
Up to ultrapure water balance equipped with UniPSTM-40The chromatographic column of serial filler（Diameter 2.5cm 40cm high）, loading
Chromatographic column is rinsed with purified water after finishing, treats silver nitrate detection without Cl-Afterwards, eluted with 20% methyl alcohol, collect blue ribbon.Decompression is dense
It is reduced to dry, measure purity and the rate of recovery.Purity 86%, the rate of recovery 83%.By the ultrapure water dissolves of sample, ultrapure water balance is up to used
Equipped with UniMSP-50The ibid method operation of the chromatographic column of serial filler, it is dry with being concentrated under reduced pressure into after method desalination, obtain
The sample of purity 95.2%, the rate of recovery 84%.By the ultrapure water dissolves of sample, up to 10% equilibrium methanol equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/ (mincm2).Use 10% first
Alcohol solution isocratic elution, washes 4 column volumes altogether, finally washes post with 50% methyl alcohol.The stream part of purity more than 97% is collected, is depressurized dense
It is reduced to dry, measure purity and the rate of recovery.Purity 99.1%, the rate of recovery 65%.The sample of not up to purity requirement is merged, the same method
After equipped with UniMSP-50The chromatographic column of serial filler and equipped with UniPSTM-40The chromatography of serial filler
Post, obtains the bleomycin A5 copper chelate of purity 99.2%, adds up the rate of recovery 86%.
Embodiment 7
Take 3.0 grams of bleomycin A5 copper chelate crude products（Content 50%）, ultrapure water dissolves are used, it is up to flat with 10% methyl alcohol in advance
Weighing apparatus equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).With 10% methanol aqueous solution isocratic elution, 4 column volumes are washed altogether, finally wash post with 50% methyl alcohol.When single is chromatographed
Between be 3.5 hours, collect purity more than 85% stream part, be concentrated under reduced pressure into it is dry, determine purity and the rate of recovery.Purity 88%, reclaims
Rate 85%.By sample with after ultrapure water dissolves up to 10% equilibrium methanol equipped with UniPSTM-40Serial filler
Chromatographic column, ibid method operation, obtains the sample of purity 94.7%, the rate of recovery 78%.By the ultrapure water dissolves of sample, up to ultrapure
Water balance equipped with UniMSP-50The chromatographic column of serial filler, filler diameter 2.4cm, 50cm high.Chromatographic flow rates
0.77ml/(min·cm2).First use 0.05M NH4Cl is eluted, and washes 1 column volume, and 0.1M NH are then used successively4Cl elutes 2
Column volume, 0.2M NH4Cl elutes 4 column volumes, 0.3M NH4Cl elutes 2 column volumes, 0.4M NH4Cl elutes 2 cylinders
Product, with the NH of 0.5M4Cl is washed till all of colour band on post and all gets off.The stream part of purity more than 97% is collected, ultra-pure water is up to used
Balance equipped with UniPSTM-40 The chromatographic column of serial filler（2.5×40cm）, loading uses ultrapure water after finishing
Chromatographic column, treats silver nitrate detection without Cl-Afterwards, eluted with 20% methyl alcohol, collect blue ribbon.Be concentrated under reduced pressure into it is dry, determine purity and return
Yield.Purity 99.3%, the rate of recovery 61%.The sample of not up to purity requirement is merged, the same method is after equipped with UniPSTM-40The chromatographic column of serial filler and equipped with UniMSP-50The chromatographic column of serial filler, obtains purity 99.1%
Bleomycin A5 copper chelate, adds up the rate of recovery 87%.
Embodiment 8
Take 1.0 grams of B1eomycin copper chelate crude products（Content 42%）, ultrapure water dissolves are used, it is up to flat with 10% methyl alcohol in advance
Weighing apparatus equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).With 10% methyl alcohol isocratic elution, 4 column volumes are washed altogether, finally with 50% methyl alcohol by all of colour band whole on post
Get off.Single chromatography the time be about 3.5 hours, collect purity more than 85% stream part, be concentrated under reduced pressure into it is dry, determine purity and return
Yield.Purity 88.7%, the rate of recovery 88%.By sample again up to after ultrapure water balance equipped with UniPSTM-40System
The chromatographic column of row filler, operates more than, obtains sample, purity 96.4%, the rate of recovery, 82%.Repeat once, collect purity
More than 97% stream part, is concentrated under reduced pressure into dry after merging, obtain sample, purity 99.3%, the rate of recovery 65%.Not up to purity will want
The sample asked merges, and the same method is crossed and UniPS is housed repeatedlyTM-40The chromatographic column of serial filler, obtains purity 99.4%
B1eomycin copper chelate, adds up the rate of recovery 82%.
Embodiment 9
Take 3.0 grams of boningmycin copper chelate crude products（Content 30%）, ultrapure water dissolves are used, ultrapure level is up to used in advance
Weighing apparatus equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).0%-15% linear gradients are pressed with methanol-water solution and elute 4 column volumes, then elute 1 cylinder with 15% methyl alcohol
Product, finally washes post with 50% methyl alcohol.The single chromatography time is 4.5 hours, collects the stream part of purity more than 85%, is concentrated under reduced pressure into
It is dry, determine purity and the rate of recovery.Purity 85.5%, the rate of recovery 84%.By sample again up to being equipped with after ultrapure water balance
UniPSTM-40The chromatographic column of serial filler, operates more than, obtains sample, purity 95.4%, the rate of recovery 77%.Weigh again
It is multiple once to collect the stream part of purity more than 97%, it is concentrated under reduced pressure into dry after merging, obtain sample, purity 98.7%, the rate of recovery
63%.The sample of not up to purity requirement is merged, the same method is crossed and UniPS is housed repeatedlyTM-40The chromatography of serial filler
Post, obtains the boningmycin copper chelate of purity 99.2%, adds up the rate of recovery 85%.
Embodiment 10
Take 3.0 grams of B1eomycin copper chelate crude products（Content 51%）, ultrapure water dissolves are used, it is up to flat with 10% methyl alcohol in advance
Weighing apparatus equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter 2.5cm, 40cm high.Chromatographic flow rates 0.8ml/
(min·cm2).With 10% methanol aqueous solution isocratic elution, 4 column volumes are washed altogether, finally wash post with 50% methyl alcohol.When single is chromatographed
Between be 3.5 hours, collect purity more than 85% stream part, be concentrated under reduced pressure into it is dry, determine purity and the rate of recovery.Purity 87%, reclaims
Rate 84%.By sample with after ultrapure water dissolves up to 10% equilibrium methanol equipped with UniPSTM-40The layer of serial filler
Analysis post, ibid method operation, obtains the sample of purity 93.8%, the rate of recovery 75%.By the ultrapure water dissolves of sample, 0.05M is up to used
Ammonium formate balance equipped with UniMSP-50The chromatographic column of serial filler, filler diameter 2.4cm, 50cm high.Chromatography stream
Fast 0.8ml/ (mincm2).First eluted with 0.1M ammonium formates, wash 1 column volume, then elute 1 with 0.2M ammonium formates successively
Column volume, 0.4M ammonium formates elute 1 column volume, and 0.5M ammonium formates elute 3 column volumes, and 0.6M ammonium formates elute 1 cylinder
Product, 0.8M ammonium formates elute 2 column volumes, and 0.9M ammonium formates elute 2 column volumes, are washed till on post with the ammonium formate of 1.0M and owned
Colour band all get off.The stream part of purity more than 97% is collected, up to ultrapure water balance equipped with UniPSTM-40System
The chromatographic column of row filler（2.5×40cm）, loading uses ultrapure water chromatographic column after finishing, after desalination, eluted with 20% methyl alcohol,
Collect blue ribbon.It is concentrated under reduced pressure into dry, measure purity and the rate of recovery.Purity 98.7%, the rate of recovery 60%.By not up to purity requirement
Sample merge, the same method is after equipped with UniPSTM-40The chromatographic column of serial filler and equipped with UniMSP-50The chromatographic column of serial filler, obtains the B1eomycin copper chelate of purity 98.7%, adds up the rate of recovery 86%.
Embodiment 11
Take 3.0 grams of boningmycin copper chelate crude products（Content 30%）, ultrapure water dissolves are used, 0.1M is up to used in advance
NH4Cl balance equipped with UniMSP-50The chromatographic column of serial filler, filler diameter 2.4cm, 50cm high.Chromatographic flow rates
0.8ml/(min·cm2).First use 0.1M NH4Cl is eluted, and washes 1 column volume, and 0.2M NH are then used successively4Cl elutes 2 posts
Volume, 0.4M NH4Cl elutes 2 column volumes, 0.6M NH4Cl elutes 3 column volumes, 0.8M NH4Cl elutes 2 column volumes,
0.9M NH4Cl elutes 2 column volumes, with the NH of 1.0M4Cl is washed till all of colour band on post and all gets off.Single chromatography the time be
12 hours, the stream part of purity more than 80% is collected, up to ultrapure water balance equipped with UniPSTM-40Serial filler
Chromatographic column（2.5×40cm）, loading finish after with purified water rinse chromatographic column, treat silver nitrate detection without Cl-Afterwards, 20% methyl alcohol is used
Wash-out, collects blue ribbon.It is concentrated under reduced pressure into dry, measure purity and the rate of recovery.Purity 83%, the rate of recovery 82%.By sample with ultrapure
Water dissolves, up to 10% equilibrium methanol equipped with UniPSTM-40The chromatographic column of serial filler, filler diameter
2.5cm, 40cm high.Chromatographic flow rates 0.8ml/ (mincm2).With 10% methanol aqueous solution isocratic elution, 4 column volumes are washed altogether,
Finally post is washed with 50% methyl alcohol.The stream part of purity more than 90% is collected, dry, measure purity and the rate of recovery are concentrated under reduced pressure into after merging.
Purity 94.7%, the rate of recovery 79%.By the ultrapure water dissolves of sample, up to ultrapure water balance equipped with UniMSP-50
The same method operation of chromatographic column of serial filler, it is dry with being concentrated under reduced pressure into after method desalination, obtain the sample of purity 98.6%, the rate of recovery
62%.The sample of not up to purity requirement is merged, the same method is after equipped with UniMSP-50The chromatographic column of serial filler
And equipped with UniPSTM-40The chromatographic column of serial filler, obtains the boningmycin copper chelate of purity 99.1%, adds up back
Yield 83%.
Embodiment 12 prepares high-purity hydrochloric acid bleomycin A5 by high-purity copper chelate
The bleomycin A5 copper chelate 1g of the high-purity that will be prepared according to the inventive method（Purity 99.1%）, add
25ml methyl alcohol dissolves, and it is 2.5 methanolic acid to be added dropwise and adjusts pH value, is passed through H2S gases（About 0.5L/min）, 20min, standing 0.5h, mistake
Filter, methyl alcohol washing filter cake, merging filtrate, plus 3 times of acetone precipitations of volume, are placed 2 hours, and filtering is drained, and filter cake is molten with methyl alcohol
Solution, plus 2% decolorizing with activated carbon, filtering, filtrate add 3 times of acetone precipitations of volume, place, and filtering is drying to obtain.HPLC is determined, pure
Spend is 99.2%.
Embodiment 13 prepares high-purity hydrochloric acid B1eomycin by high-purity copper chelate
The B1eomycin copper chelate 1g of the high-purity that will be prepared according to the inventive method（Purity 98.7%）, add
25ml methyl alcohol dissolves, and it is 2.5 methanolic acid to be added dropwise and adjusts pH value, is passed through H2S gases（About 0.5L/min）, 20min, standing 0.5h, mistake
Filter, methyl alcohol washing filter cake, merging filtrate, plus 3 times of acetone precipitations of volume, are placed 1 hour, and filtering is drained, and filter cake is molten with methyl alcohol
Solution, plus 2% decolorizing with activated carbon, filtering, filtrate add 3 times of acetone precipitations of volume, place, and filtering is drying to obtain.HPLC is determined, pure
Spend is 98.5%.
Embodiment 14 prepares high-purity hydrochloric acid boningmycin by high-purity copper chelate
The boningmycin copper chelate 1g of the high-purity that will be prepared according to the inventive method（Purity 99.1%）, add
25ml methyl alcohol dissolves, and it is 2.5 methanolic acid to be added dropwise and adjusts pH value, is passed through H2S gases（About 0.5L/min）, 20min, standing 0.5h, mistake
Filter, methyl alcohol washing filter cake, merging filtrate, plus 3 times of acetone precipitations of volume, are placed 1.5 hours, and filtering is drained, filter cake methyl alcohol
Dissolving, plus 2% decolorizing with activated carbon, filtering, filtrate add 3 times of acetone precipitations of volume, place, and filtering is drying to obtain.HPLC is determined,
Purity is 98.9%.
Claims (8)
1. the purification process of the copper chelate of a kind of bleomycin A5 and its congeners, it is characterised in that
Chromatographed as filler from monodisperse polystyrene/divinylbenzene chromatography media, 20~60 μm of particle diameter, particle diameter ripple
Dynamic scope<5 μm, aperture
The copper chelate crude product for taking bleomycin A5 and its congeners is added to ultra-pure water or is flowed equal with ultrapure water dissolves
On the chromatographic column of weighing apparatus, pillar height is with post footpath than 15~40：1, chromatographic column 5~50mg/ml of carrying capacity, with 0~20% methyl alcohol, ethanol,
Aqueous acetone solution isocratic elution or gradient elution, 0.5~2ml/ of elution flow rate (mincm2), the single chromatography time 2~6 is small
When；After single chromatography, reclaim, ibid method obtains final product the bleomycin A5 and its congeners of high-purity again through 2~3 chromatographies
Copper chelate, purity is 97%~100%.
2. method according to claim 1, it is characterised in that aperture isOr
3. method according to claim 1, it is characterised in that with 10% methyl alcohol isocratic elution.
4. the purification process of the copper chelate of a kind of bleomycin A5 and its congeners, it is characterised in that
By monodisperse polystyrene/divinylbenzene chromatography media and single dispersing polymethacrylates mixed type cation exchange
Chromatography media is combined, and is chromatographed as filler；
Described monodisperse polystyrene/divinylbenzene chromatography media, 20~60 μm of particle diameter, particle diameter fluctuation range<5 μm, hole
FootpathDescribed single dispersing polymethacrylates mixed type cation-exchange chromatography medium, the μ of particle diameter 30~70
M, particle diameter fluctuation range<5 μm, aperture
5. method according to claim 4, it is characterised in that described monodisperse polystyrene/divinylbenzene chromatography
The aperture of medium isOrDescribed single dispersing polymethyl
The aperture of acid esters mixed type cation-exchange chromatography medium is preferablyOr
6. method according to claim 4, it is characterised in that the copper chelate for taking bleomycin A5 and its congeners is thick
Product or with the sample after monodisperse polystyrene/divinylbenzene chromatography media preliminary purification, are added to super with ultrapure water dissolves
On the chromatographic column of pure water equilibrium, pillar height is with post footpath than 15~40：1, handed over single dispersing polymethacrylates mixed type cation
Chromatography media is changed for filler is chromatographed, chromatographic column 5~50mg/ml of carrying capacity, with the ammonium chloride or the water of ammonium formate of various concentrations
Solution gradient is eluted, 0.5~2ml/ of elution flow rate (mincm2), single chromatography, after desalination, then with monodisperse polystyrene/
Divinylbenzene chromatography media is chromatographed again；Crude product containing copper chelate is reclaimed, is chromatographed 1~3 time with method recycling through combination of the above,
Obtain final product the bleomycin A5 of high-purity and its copper chelate of congeners.
7. method according to claim 4, it is characterised in that the copper chelate of bleomycin A5 and its congeners is thick
Product first with after single dispersing polymethacrylates mixed type cation-exchange chromatography medium purification 1~3 time, then with disperseing polyphenyl second
Alkene/divinylbenzene chromatography media purifying, until purity reaches more than 97%.
8. method according to claim 4, it is characterised in that the copper chelate of bleomycin A5 and its congeners is thick
Product are first purified 1~3 time with monodisperse polystyrene/divinylbenzene chromatography media, then mixed with single dispersing polymethacrylates
Mould assembly cation-exchange chromatography medium purification, until purity reaches more than 97%.
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| CN102134298A (en) * | 2011-01-11 | 2011-07-27 | 浙江大学 | Preparation method and using method for special absorbent for purifying catechines |
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