CN112239488B

CN112239488B - Purification method of copper chelate of bleomycin compound

Info

Publication number: CN112239488B
Application number: CN201910649465.2A
Authority: CN
Inventors: 李继安; 林惠敏; 李亚军; 邓旭; 张建斌; 卢雪欢; 郭瑞玲; 孟宪纬
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2019-07-18
Filing date: 2019-07-18
Publication date: 2022-09-20
Anticipated expiration: 2039-07-18
Also published as: CN112239488A

Abstract

The invention discloses a purification method of a copper chelate of a bleomycin group compound. The purification method of the copper chelate of the bleomycin compound comprises the following steps of carrying out chromatography on fermentation enrichment concentrated solution of the copper chelate containing the bleomycin compound, wherein a chromatographic column filler is a monodisperse polystyrene/divinylbenzene chromatography medium, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 100-200 mu m, and eluent for the chromatography is water; collecting the eluent to obtain the aqueous solution of the copper chelate crude product containing the bleomycin compound. The copper chelate of the bleomycin compound with the purity of more than 50 percent and the yield of more than 80 percent can be obtained only by separating once.

Description

Purification method of copper chelate of bleomycin compound

Technical Field

The invention relates to a purification method of copper chelate of a bleomycin compound.

Background

The bleomycin family compounds including Pingyangmycin (Pingyangmycin, bleomycin A5), Boanmycin (Boanmycin, bleomycin A6) and Boningmycin (Boningmycin) are independently developed by Chinese scholars, and are collectively called bleomycin family glycopeptide antitumor antibiotics because the structures and the activities are very similar. Wherein the pingyangmycin is an antitumor antibiotic separated from an actinomycete culture solution in soil in Pinyang county of Zhejiang in China. The boanmycin is produced by a new Pingyang variety of the streptomycin verticillata and belongs to a regeneration product of Pingyang mycin. Boningmycin is an acetylate of the free amino group of the terminal amino body of boanmycin. Pingyangmycin and boanmycin have been put into clinical use. Mainly inhibits the incorporation of thymidine into DNA and the binding of thymidine with DNA to destroy it. In addition, it can break single strands of DNA and release some of the free bases, which may thus damage the DNA template and prevent DNA replication. Because the copper chelate of the bleomycin compound has good stability and is convenient to store, the bleomycin compound is stored in a form of copper chelate mostly, and when the bleomycin compound needs to be used, the copper chelate bulk drug can be obtained by decoppering and refining, and then the copper chelate bulk drug is prepared into a pharmaceutical preparation for clinical use.

Because the bleomycin compound and the copper chelate thereof are water-soluble components with large polarity, separation and purification are quite difficult, so that the purity of the copper chelate becomes a bottleneck which restricts the quality of the final product of the bleomycin compound. At present, the traditional production method of bleomycin compounds is to directly obtain a copper chelate by microbial fermentation, or to add copper salt after fermentation to form the copper chelate, to enrich the copper chelate bleomycin compounds (such as pingyangmycin, boanmycin and boningmycin) in fermentation liquor by using macroporous weak acid resin, to elute by hydrochloric acid, to neutralize by anion resin, to preliminarily purify by macroporous resin, to desorb by acetone-hydrochloric acid solution, to concentrate and freeze-dry the desorption solution to obtain a crude product. Then further purified by alumina chromatography, followed by a plug of CM-sephadex-C-25 of dextran backbone. Eluting ammonium formate, chromatography, concentrating with macroporous resin, desalting, and freeze drying to obtain blue lyophilized powder (Zhangrui, xu hong chapter, Louzhixian, etc. research on bleomycin II. separation, purification, physicochemical properties and identification of bleomycin [ J ] Microbiol, 1980(1): 78-83.). The method has the advantages of complicated procedures, long period, large using amount of solvent and water, low yield and low purity of extracted products.

Zhongjiafeng et al (CN 104231057A: Zhongjiafeng, Panjun Fang, Wan Qi Shi, et al. purification method of copper chelate of Pingyangmycin and its congeneric compound) starting from crude copper chelate of Pingyangmycin, method (1) uses monodisperse polystyrene/divinylbenzene (PS-DVB, PSD for short) chromatography medium alone, or method (2) uses monodisperse polystyrene/divinylbenzene (PS-DVB, PSD for short) chromatography medium and monodisperse polymethacrylate mixed type cation exchange chromatography medium in combination, as filler for chromatography. Compared with the traditional process, the efficiency is improved, the period is shortened, and the purity is greatly improved. However, the method still has certain disadvantages:

(1) is not suitable for the purification of crude raw materials with lower purity, and the crude copper chelate (with the purity of 20-60%) of pingyangmycin or homologous compounds thereof with higher purity must be used (see the paragraph [0015] of the specification). The lower purity crude copper chelate product of the bleomycin group contains more pigments and unknown impurities such as heteroprotein generated by microbial metabolism, and the impurities cannot be effectively removed by the patent methods disclosed by the ever-known Beacon and the like, and the separation chromatography medium adopted by the sample is easily polluted and poisoned by the lower purity sample, so that the regeneration effect is poor, and the subsequent industrial batch production is influenced.

(2) In the method (1) or (2), when the purification is performed by using a monodisperse polystyrene/divinylbenzene (PS-DVB, PSD for short) chromatography medium, the principle of PSD matrix microsphere separation sample is similar to that of a common macroporous resin, and generally, the PSD matrix microsphere separation sample is adsorbed on a column, and then eluted and separated by using organic solvent-water mixed solutions with different concentrations. The copper chelate of the bleomycin compound is a water-soluble component with large polarity, and the separation and purification are quite difficult; generally, the water phase is loaded on a column, and the pure water phase cannot be eluted; isocratic elution or gradient elution with organic solvents, such as methanol, ethanol, aqueous acetone, etc., is used. The sample of the bleomycin group is pure water phase when being loaded, and the elution is carried out by adopting a mixed system of an organic solvent and water phase, so that the collapse of a PSD matrix microsphere column bed is easily caused, and the chromatographic effect during the elution is influenced.

(3) The single purification effect is low, and the purity of more than 98 percent can be achieved by repeating the purification for many times.

In the method (1), a monodisperse polystyrene/divinylbenzene (PS-DVB, PSD for short) chromatographic medium is used independently; the purity is generally improved to 70-85% by single chromatography, and the recovery rate is more than 75%; the purity can reach more than 97% after 2-3 times of chromatography (see paragraph 0015 of the specification). It can be further confirmed from examples 1-5 and 8-9 that the purity of the crude product with purity lower than 55% can be only improved to below 89% in a single chromatography, and the purity of the crude product with purity higher than 98% can be achieved only by using PS-DVB alone at least three times; if a crude product with a purity higher than 55% is used, PS-DVB alone still needs two to three times to achieve a purity of more than 98%.

In the method (2), a monodisperse polystyrene/divinylbenzene (PS-DVB, PSD for short) chromatographic medium and a monodisperse polymethacrylate mixed type cation exchange chromatographic medium are combined for use; the monodisperse polymethacrylate mixed type cation exchange chromatography medium is used as a filler for chromatography, single chromatography is used for crude product purification, the purity can be improved by 20-55%, the recovery rate is more than 75%, and PS-DVB in the method (1) is used for desalination after elution. Therefore, it is usually necessary to perform four elutions to achieve a purity of 98% or more.

Disclosure of Invention

The invention aims to solve the technical problem of providing a purification method of a copper chelate of a bleomycin compound in order to overcome the defect of the existing purification method of the copper chelate of the bleomycin compound. The purification process of the invention directly starts from the enriched concentrated solution (the purity is lower than 15%) of the copper chelate crude product of the mithramycin compound prepared by the traditional process, and can obtain the product with the total yield of more than 55% and the purity of more than 98% by an HPLC area normalization method only by two times of chromatographic separation without recycling. The method can greatly reduce the waste water discharge amount of the prior process by more than 30 percent, effectively shorten the process period by about one half, has low separation medium cost and long service life compared with the prior process, is easy for industrial mass production, and has higher industrial application value.

The present invention solves the above-mentioned problems by the following technical means.

The invention provides a purification method of copper chelate of a bleomycin compound, which comprises the following steps of carrying out chromatography on an aqueous solution containing a crude copper chelate of the bleomycin compound, wherein a chromatography filler is a monodisperse polystyrene/divinylbenzene chromatography medium, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 10-60 mu m, and an eluent for chromatography is an aqueous solution of ammonium salt and water in sequence; collecting the eluent water to obtain an aqueous solution of a pure copper chelate product containing the bleomycin compound; the mass percentage concentration of the ammonium salt in the aqueous solution is 0.5-1.5%; in the aqueous solution containing the crude copper chelate of the bleomycin compound, the purity of the copper chelate of the bleomycin compound is more than 50%.

The copper chelate of the bleomycin group compound can be a chelate formed by copper ions and the bleomycin group compound (such as one or more of the bleomycin, the boanmycin and the boningmycin) which is obtained by fermenting streptomyces verticillata variant or a new variant thereof (such as streptomyces verticillatus var. pingyangensis CPCC200554, streptomyces verticillatus var. pingyangensis CPCC 200552) strains and is conventional in the field; in the invention, the copper chelate of the bleomycin compound can be one or more of a copper chelate of pingyangmycin, a copper chelate of boanmycin and a copper chelate of boningmycin; and for example, the copper chelate of pingyangmycin and/or the copper chelate of boanmycin.

The ammonium salt can be any ammonium salt conventional in the art, such as one or more of ammonium chloride, ammonium acetate, ammonium formate, ammonium phosphate, ammonium dihydrogen phosphate, and ammonium hydrogen phosphate (e.g., ammonium chloride and/or ammonium acetate).

The mass percentage concentration of the aqueous solution of the ammonium salt is preferably 1.0%.

The pH of the aqueous solution of the crude copper chelate containing the compound of the bleomycin group can be a pH value conventional in the art, for example, 2.5 to 5.5, and in the present invention, the pH value is preferably 4.0 to 4.5 (for example, 4.1, 4.2, 4.25, 4.33 or 4.4). The pH can be adjusted using acids or bases as is conventional in the art. It will be appreciated by those skilled in the art that the anion of the acid is preferably the same as the anion of the ammonium salt of the eluent; the acid may be one or more of hydrochloric acid, acetic acid, formic acid and phosphoric acid (e.g. hydrochloric acid, again for example 6M hydrochloric acid) and the base may be one or more of an alkali metal hydroxide (e.g. sodium hydroxide and/or potassium hydroxide), an alkali metal carbonate (e.g. sodium carbonate and/or potassium carbonate) and an alkali metal bicarbonate (e.g. sodium bicarbonate and/or potassium bicarbonate) (again for example sodium hydroxide).

The monodisperse polystyrene/divinylbenzene chromatography medium preferably has a particle size of 30 μm.

The pore size of the monodisperse polystyrene/divinylbenzene chromatography medium can be any pore size conventional in the art, for example

(also e.g.

)。

The monodisperse polystyrene/divinylbenzene chromatography medium is preferably Poly ^RP -10、Poly ^RP -30、Poly ^RP -60、Poly ^RP -40、LXMS-15、LXMS-35、LXMS-60、UniPS ^TM -20、UniPS ^TM -30、UniPS ^TM -40 or UniPS ^TM -50, preferably UniPS ^TM -30、Poly ^RP -30 or LXMS-35. (Poly ^RP -10、Poly ^RP -30、Poly ^RP -40、Poly ^RP -60 is provided by Suzhou Susai Su Cuken technology, LXMS-15, LXMS-35, LXMS-60 is provided by Xian blue Xiao science and technology New Material, Inc., UniPS ^TM -20、UniPS ^TM -30、UniPS ^TM -40、UniPS ^TM -50 from Sozhou, Nami micro-technology, Inc.).

The column may be pretreated by methods conventional in the art, such as by pre-equilibration with an aqueous eluent ammonium salt solution, preferably pre-equilibration with a 2BV solution of the ammonium salt in the present invention.

The aspect ratio of the chromatographic column can be the aspect ratio conventional in the art, for example, 8:1 to 20:1, and in the present invention, preferably 10:1 to 15:1 (for example, 10:1, 12:1, 14: 1).

The flow rate of the eluent can be a flow rate conventional in the art, such as 0.05BV/h to 0.2BV/h (again, for example, 0.3BV/h to 0.5 BV/h).

When the eluent is an aqueous solution of an ammonium salt, the amount of the aqueous solution of the ammonium salt can be 4BV to 6BV (for example, 5 BV).

In the aqueous solution containing the crude copper chelate complex of the compound of the bleomycin family, the concentration of the copper chelate complex of the compound of the bleomycin family in the aqueous solution is preferably 20g/L to 65g/L (for example, 23g/L, 23.4g/L, 24g/L, 24.53g/L, 26g/L, 28.57g/L, 29g/L, 30.81g/L, 31g/L, 33.78g/L, 34.21g/L, 43.5g/L, 47.5g/L, 48.4g/L, 54.5g/L, 56.14g/L and 62.35 g/L).

The loading of the column may be that which is conventional in the art for this type of packing, i.e. the amount of sample per volume of packing that can be separated in a single elution; in the present invention, it is preferably 1 mg/mL-5 mg/mL (2.03g/L, 2.13g/L, 2.31g/L, 2.54g/L, 2.74g/L, 2.76g/L, 2.78g/L, 2.81g/L, 3.08g/L, 3.12g/L, 3.15g/L, 3.24g/L, 3.27g/L, 3.32g/L, 3.39g/L, 3.48g/L, 3.59 g/L).

The operation of collecting the eluate may be a routine operation in the art, such as HPLC trace monitoring, collecting the eluate containing the copper chelate of the compound of the bleomycin family; in the invention, the eluent containing the copper chelate of the bleomycin compound with HPLC purity higher than 95% is preferably collected; more preferably, the collection is stopped when the concentration of the copper chelate of the compound of the bleomycin group contained in the eluate is less than 50. mu.g/mL.

The purification method can also comprise the following post-treatment step, the obtained water solution containing the pure copper chelate of the bleomycin group compound is concentrated and freeze-dried to obtain the copper chelate of the bleomycin group compound; the concentration can be that the mass volume content of the copper chelate of the bleomycin compound in the aqueous solution is 30 g/L-50 g/L.

The purification method can also comprise the following steps of carrying out chromatography on the fermentation enrichment concentrated solution of the copper chelate containing the compound of the bleomycin family, wherein the chromatographic column filler is a monodisperse polystyrene/divinylbenzene chromatographic medium, the particle size of the monodisperse polystyrene/divinylbenzene chromatographic medium is 100-200 mu m, and the eluent for the chromatography is water; collecting the eluent to obtain the aqueous solution of the copper chelate crude product containing the bleomycin group compound; in the fermentation enrichment concentrated solution of the crude copper chelate containing the bleomycin group compound, the purity of the copper chelate of the bleomycin group compound is more than 5%.

The pH value of the fermentation enrichment concentrated solution containing the copper chelate of the bleomycin group compound can be the conventional pH value in the field, such as 2.5-5.5; the preferred range of the present invention is 4.1 to 4.5 (e.g., 4.2, 4.3, 4.4 or 4.5). The pH may be adjusted using acids or bases conventional in the art, such as one or more of hydrochloric acid, acetic acid, formic acid, and phosphoric acid (e.g., hydrochloric acid), and bases such as one or more of alkali metal hydroxides (e.g., sodium hydroxide and/or potassium hydroxide), alkali metal carbonates (e.g., sodium carbonate and/or potassium carbonate), and alkali metal bicarbonates (e.g., sodium bicarbonate and/or potassium bicarbonate) (e.g., again, sodium hydroxide).

In the chromatography of the fermentation enrichment concentrated solution, the eluent can be deionized water which is conventional in the field.

In the chromatography of the fermentation enriched concentrate, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is preferably 130 μm to 180 μm.

In the chromatography of the fermentation enriched concentrate, the monodisperse polystyrene/divinylbenzene chromatography medium can be chromatography No. 3, NM100, XR3SP or HZ20SS, such as XR3 SP. (chromatograph No. 3 and HZ20SS are available from Shanghai Huazhen science and technology, Inc., XR3SP is available from Shanghai Su Er chemical science and technology, Inc., and NM100 is available from Suzhou Na micro science and technology, Inc.).

In the chromatography of the fermentation-enriched concentrate, the column may be pretreated by methods conventional in the art, such as pre-equilibration with eluent, preferably pre-equilibration with 5BV of deionized water.

In the chromatography of the fermentation enriched concentrated solution, the height-to-diameter ratio of the chromatographic column can be the conventional height-to-diameter ratio in the field, such as 5: 1-15: 1; in the present invention, the ratio is preferably 5:1 to 10:1 (e.g., 6:1, 7:1, 8:1 or 9: 1).

In the chromatography of the fermentation enrichment concentrate, the flow rate of the eluent can be the conventional flow rate in the field, and the flow rate is preferably 0.05 BV/h-0.15 BV/h (for example 0.1BV/h) in the invention.

The mass volume content of the copper chelate of the bleomycin compound in the fermentation enriched concentrated solution can be the mass volume content conventional in the art, such as 25 g/L-35 g/L (such as 28g/L, 30g/L, 30.12g/L, 31.8g/L, 31.9g/L, 32.4g/L and 34.6 g/L).

In the chromatography of the fermentation enriched concentrated solution, the loading capacity of the chromatographic column can be the conventional loading capacity of the filler in the field (namely the amount of a sample which can be separated by the filler per unit volume in single elution; and can be 1 g/L-7 g/L (such as 1.96g/L, 2.08g/L, 2.15g/L, 2.66g/L, 3.64g/L, 3.98g/L, 4.79g/L, 4.87g/L, 4.89g/L, 4.98g/L and 5.13g/L) in the invention).

In the chromatography of the fermentation enriched concentrated solution, the operation of collecting the eluent can be the operation conventional in the field, for example, HPLC tracking monitoring, and the eluent containing the copper chelate of the compound of the bleomycin group is collected; in the invention, the eluent containing the copper chelate of the bleomycin compound with HPLC purity higher than 50% is preferably collected; more preferably, when the copper chelate of the bleomycin compound is a plurality of compounds, the copper chelates are collected respectively (for example, when the copper chelate of the bleomycin compound is a mixture of the copper chelate of pingyangmycin and the copper chelate of boanmycin, the copper chelate of pingyangmycin and the copper chelate of boanmycin are collected respectively).

The purification method of the copper chelate containing the bleomycin group compound can also further comprise the following fermentation liquor enrichment step,

step (1), carrying out acid pH value adjustment and alkali pH value adjustment on fermentation liquor of the copper chelate containing the bleomycin group compound, and filtering to obtain filtrate of the fermentation liquor of the copper chelate containing the bleomycin group compound;

and (2) subjecting the filtrate of the fermentation liquor containing the copper chelate of the bleomycin group compound in the step (1) to macroporous weak acid resin column enrichment chromatography, wherein an eluant is dilute hydrochloric acid, collecting the obtained eluate, adjusting the pH value with alkali, concentrating and filtering to obtain the fermentation enriched concentrated solution containing the copper chelate of the bleomycin group compound.

In step (1), the fermentation broth containing the copper chelate of the compound of the bleomycin group can be obtained by directly fermenting a streptomyces verticillata planus variant or a new variant thereof (such as streptomyces verticillata var. pingyangensis CPCC200554, streptomyces verticillata var. pingyangensis CPCC 200552) by a conventional fermentation method in the field, or can be formed by adding a copper salt into the fermentation broth containing the compound of the bleomycin group obtained after fermentation.

Alternatively, in the present invention, the fermentation liquid is preferably prepared by the following fermentation process, and it can be understood by those skilled in the art that the specific fermentation scale can be adjusted as required:

1 culture Medium

Slant medium (g/L): soluble starch 20.0, KNO ₃ 10.0，K ₂ HPO ₄ 5.0，MgSO ₄ ·7H ₂ O 5.0，NaCl 5.0，FeSO ₄ ·7H ₂ 0.1 of O, 5.0 of corn steep liquor, 20.0 of agar and 7.4 to 7.6 of pHs.

Seed fermentation medium (g/L): 30.0 parts of corn steep liquor, 20.0 parts of soybean cake powder, 2.0 parts of pomegranate flower yeast powder, 10.0 parts of glucose, 10.0 parts of maltodextrin and KH ₂ PO ₄ 1.0，ZnSO ₄ .7H ₂ O 0.5，CuSO ₄ .5H ₂ O 0.1，pH 6.5。

Basal fermentation medium (g/L): 7.5 parts of corn steep liquor, 35.0 parts of cold-pressed soybean cake powder, 2.0 parts of durian yeast, 5.0 parts of glucose, 60 parts of starch (alpha-amylase liquefaction) and NaNO ₃ 2.0；KH ₂ PO ₄ 1.0,ZnSO ₄ .7H ₂ O0.5，CuSO ₄ .5H ₂ O 0.1，pH 6.5。

Fermenter feed (g/L): 70.0 parts of corn steep liquor, 49.0 parts of cold-pressed bean flour, 21.0 parts of pomegranate flower yeast powder and KH ₂ PO ₄ 1.0,ZnSO ₄ .7H ₂ O 0.5，CuSO ₄ .5H ₂ O 0.1，NaNO ₃ 2.0, natural pH, 1.6 percent of material supplement amount each time and 12h of material supplement time interval.

2 culture conditions of strains

Slant culture: inoculating spore in freeze drying tube or glycerine tube onto slant culture medium (eggplant bottle, 60mL/300mL), culturing in 28.5-29 deg.C constant temperature incubator for 6-7 days, and terminating culture when colony is convex, surface is velvet, and spore is white and abundant.

Seed culture: transferring a certain amount of mature slant to a triangular shake flask filled with 80mL/500mL seed culture medium by using an inoculation shovel, wrapping with 8 layers of gauze, placing on a shaking table, and culturing at 220rmp and 28.5-29 ℃ for 24 h.

Shake flask fermentation culture: the well grown seed liquid is transferred to a triangular shaking flask filled with 35mL/250mL fermentation medium for culture at 220rmp and 28.5-29 ℃ for 168h at the inoculation amount of 5%.

Fermentation conditions of a 25L seed fermentation tank: the liquid loading amount is 16L, 80ml of cultured seed shake flask culture medium is inoculated into sterilized seed culture medium, the temperature is 28.5 ℃, the aeration ratio is 1.0vvm, the rotating speed is 0-14h and 250r/min, 14-18h and 300r/min, 18-24h and 350r/min, and 375r/min is obtained after 24 h; the fermentation period is 24-28 h.

Fermentation conditions of a 25L fermentation tank: the liquid loading amount is 16L, the inoculation amount is 40 percent, the temperature is 28.5 ℃, the aeration ratio is 1.0vvm, the rotating speed is 250r/min for 0-4h, 300r/min for 4-6h, 350r/min for 6-8h, 375r/min after 8h, and the fermentation period is 168-192 h.

In step (1), the acid may be an acid conventional in the enrichment step of such fermentation broth in the art, such as oxalic acid.

In step (1), the pH value adjusted by acid may be a pH value conventional in the enrichment step of such fermentation broth in the art, for example, 2.0 to 3.0 (e.g., 2.1, 2.3, 2.5, 2.6, 2.7, 2.8 or 2.9).

In step (1), the base may be a base conventional in the enrichment step of such fermentation broth in the art, such as sodium hydroxide, preferably 6M NaOH aqueous solution.

In step (1), the pH adjusted with a base may be a pH value customary in such fermentation broth enrichment steps of the art, for example 4.5-5.5 (again, for example, 4.6, 4.7, 4.8, 4.9 or 5.0).

In step (1), the filtration may be a conventional operation in the enrichment step of the fermentation broth in this type of the art, for example, filtration with an inorganic ceramic membrane of 10nm to 200 nm.

In step (2), the macroporous weak acid resin may be a macroporous weak acid resin which is conventional in the type of fermentation liquor enrichment step in the field, such as: d157, D152, D155, XR140, XR157, ZGD113 or ZGD115, preferably XR157, D157, ZGD115 or D155. (D157, D152, D155 are provided by Shanghai Huazhen science and technology, Inc., XR140, XR157 are provided by Shanghai Sungli chemical science and technology, Inc., ZGD113, ZGD115 are provided by Hangzhou resin, Inc.)

In step (2), the molar concentration of the eluent diluted hydrochloric acid can be the molar volume concentration conventional in the enrichment step of the fermentation liquid of the type in the field, for example, 0.1 mol/L-0.5 mol/L, preferably 0.3 mol/L.

In step (2), the pH value can be adjusted by using a base which is conventional in the enrichment step of the fermentation liquid of the type in the art, such as sodium hydroxide (e.g., 6M NaOH aqueous solution).

In step (2), the column is pre-equilibrated with deionized water, e.g., 5BV of deionized water.

The invention provides a purification method of a copper chelate of a bleomycin compound, which comprises the following steps of carrying out chromatography on fermentation enriched concentrated solution of the copper chelate containing the bleomycin compound, wherein a chromatographic column filler is a monodisperse polystyrene/divinylbenzene chromatography medium, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 100-200 mu m, and eluent for the chromatography is water; collecting the eluent to obtain the aqueous solution of the copper chelate crude product containing the bleomycin group compound; in the fermentation enrichment concentrated solution of the crude copper chelate containing the bleomycin group compound, the purity of the copper chelate of the bleomycin group compound is more than 5%.

In the chromatography of the fermentation enrichment concentrate, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is preferably 130 to 180 μm.

In the chromatography of the fermentation-enriched concentrate, the column may be pretreated by a method conventional in the art, such as pre-equilibration with an eluent, preferably pre-equilibration with 5BV of deionized water in the present invention.

In the chromatography of the fermentation enrichment concentrated solution, the height-diameter ratio of the chromatographic column can be the conventional height-diameter ratio in the field, such as 5: 1-15: 1; in the present invention, the ratio is preferably 5:1 to 10:1 (e.g., 6:1, 7:1, 8:1 or 9: 1).

In the chromatography of the fermentation enriched concentrated solution, the flow rate of the eluent can be the conventional flow rate in the field, and the flow rate is preferably 0.05 BV/h-0.15 BV/h (for example, 0.1BV/h) in the invention.

In the chromatography of the fermentation enriched concentrated solution, the operation of collecting the eluent can be the operation conventional in the field, for example, HPLC tracking monitoring, and the eluent containing the copper chelate of the compound of the bleomycin group is collected; in the invention, the eluent containing the copper chelate of the bleomycin compound with HPLC purity higher than 50% is preferably collected; more preferably, when the copper chelate of the bleomycin group compound is a plurality of compounds, the copper chelates are collected respectively (for example, when the copper chelate of the bleomycin group compound is a mixture of the copper chelate of pingyangmycin and the copper chelate of boanmycin, the copper chelate of pingyangmycin and the copper chelate of boanmycin are collected respectively).

1 culture Medium

Slant medium (g/L): soluble starch 20.0, KNO ₃ 10.0，K ₂ HPO ₄ 5.0，MgSO ₄ ·7H ₂ O 5.0，NaCl 5.0，FeSO ₄ ·7H ₂ 0.1 percent of O, 5.0 percent of corn steep liquor, 20.0 percent of agar and 7.4 to 7.6 of pHs.

2 culture conditions of strains

Shake flask fermentation culture: transferring the grown seed solution into a triangular shake flask filled with 35mL/250mL fermentation medium at 5% inoculation amount for culture at 220rmp and 28.5-29 ℃ for 168 h.

In step (1), the pH value adjusted by acid may be a pH value conventional in the enrichment step of such fermentation broth in the art, for example, 2.0-3.0 (e.g., 2.1, 2.3, 2.5, 2.6, 2.7, 2.8 or 2.9).

The invention also provides a bulk drug of the copper chelate of the bleomycin compound, which is prepared by the purification method of the copper chelate of the bleomycin compound.

In the present invention, BV is the volume of packing loaded in the column and is referred to as bed volume (bed volume).

In the present invention, BV/h means the volume of the solution passing through per hour is the volume of the packed bed, i.e., the flow rate. For example, the flow rate of the solution passing through the chromatographic column is 2 BV/h-4 BV/h, namely the volume of the solution passing through the chromatographic column per hour is 2-4 times of the volume of the packed bed.

The above preferred conditions may be combined arbitrarily to obtain preferred embodiments of the present invention without departing from the general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the purification process (1) of the invention directly starts from the enriched concentrated solution (the purity can be lower than 15%) of the copper chelate crude product of the bleomycin compound prepared by the traditional process, and the copper chelate of the bleomycin compound with the purity of more than 50% and the yield of more than 80% can be obtained only by primary separation through the chromatography of 100-200 mu m monodisperse polystyrene/divinylbenzene (PS-DVB) chromatography medium.

(2) Starting from the copper chelate of the bleomycin group compound with the purity of more than 50 percent, the copper chelate of the bleomycin group compound with the purity of more than 98 percent and the yield of more than 80 percent can be obtained by separating once through pure water phase chromatography separation of a monodisperse PS-DVB microsphere chromatography column with the purity of 10-60 mu m.

(3) By combining two steps, only one separation is needed in each step, and the effects of more than 50% of total yield and more than 98% of purity by an HPLC area normalization method can be achieved without recycling. The purification process of the invention does not use organic solvent, is easy for industrial scale-up production and has higher industrial application value.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

In the examples, the copper chelate of pingyangmycin and the copper chelate of boanmycin are abbreviated as pingyangmycin and boanmycin.

The instruments and reagents are shown in the following table:

TABLE 1 Instrument

TABLE 2 reagents

Reagent	Specification of	Manufacturer of the product
			NaOH	Analytical purity	Chinese medicine reagent
NH4Cl	Analytical purity	Chinese medicine reagent
			HCl	Analytical purity	Chinese medicine reagent
Deionized water		Self-made
			Methanol	Analytical purity	Chinese medicine reagent
Oxalic acid	Analytical purity	Chinese medicine reagent
			Ethanol	Analytical purity	Chinese medicine reagent

The detection method in the following examples is as follows:

chromatography column Agilent eclipse plus-18(5 μm,4.6 × 250 mm); using hexane sodium sulfonate solution (taking 7.53g of hexane sodium sulfonate and 3.72g of ethylene diamine tetraacetic acid, adding 0.08mol/L acetic acid solution to dissolve and dilute to 1000ml), and adjusting pH to 4.3 with ammonia solution) as mobile phase A; methanol-acetonitrile (7:3) is used as a mobile phase B, and the detection wavelength is 254 nm. Linear gradient elution was performed as per table one. The sample is diluted by 50% methanol water solution, and the supernatant is taken for injection after high-speed centrifugation at 12000 r/min.

TABLE 3 HPLC DETECTION TIME-GRADIENT METER

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	70	30
15	68	32
			35	55	45
36	70	30
			40	70	30

The evaluation method adopts a peak area normalization methodHPLC purity; the content of the bleomycin single-component compound in the solution is obtained by adopting an external standard method. The chromatographic peaks of the analytes are determined by comparison with the retention time and uv spectrum of a reference compound. PYM and BAM were quantified using a standard curve based peak area method. A standard curve was established with 5 experimental data points. The regression equations of pingyangmycin and boanmycin are Y-8.284X-21.3 (R) ² 0.9991) and Y10.17X-3.4 (R) ² 0.9993). Y and X are the peak area (mAU) and concentration (. mu.g/mL) of the analyte, respectively.

Pingyangmycin standard: pingyangmycin hydrochloride, standard of China institute, specification: 8mg, product batch number: 130424-201104.

Boanmycin control: for injection, the hydrochloric acid boanmycin (preparation), Jilin Aodong pharmaceutical group Yangji GmbH, Specification: 8.0mg, batch number: 20180301.

in the following examples, the fermentation broth is prepared by the following fermentation process (as will be understood by those skilled in the art, the specific scale of fermentation can be adjusted as required, and the specific species can be found in the examples):

1 culture Medium

Fermenter feed (g/L): corn steep liquor 70.0, cold-pressed bean flour 49.0, and durian yeastPowder 21.0, KH ₂ PO ₄ 1.0,ZnSO ₄ .7H ₂ O 0.5，CuSO ₄ .5H ₂ O 0.1，NaNO ₃ 2.0, natural pH, 1.6 percent of material supplement amount each time and 12h of material supplement time interval.

2 culture conditions of strains

And (3) shake flask fermentation culture: the well grown seed liquid is transferred to a triangular shaking flask filled with 35mL/250mL fermentation medium for culture at 220rmp and 28.5-29 ℃ for 168h at the inoculation amount of 5%.

Example 1

The main product of the pingyangensis single-component fermentation liquor is obtained by an aerobic fermentation method, the volume of a tank is 60L, the strain is streptomyces verticillius var. pingyangensis CPCC200554, the unit is 15 mu g/mL (the total content is about 900mg, the pH value is adjusted by oxalic acid to be 2.3, after stirring for 30min, the pH value is adjusted by 6M NaOH to be 4.9, then ceramic membrane filtration is carried out to obtain filtrate 68L (purified water is added in the middle for dilution), the total content of the filtrate is 853mg, 1L ZGD115 resin is added on the filtrate, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely added, the flow rate of 0.2BV/h of 0.3M hydrochloric acid solution is used for elution, the pH value of 6M NaOH is used for adjusting the pH value of 4.4 of the eluate, then rotary evaporation and concentration is carried out to be 28g/L, the filtrate is filtered to obtain 23mL, the content mg, the yield is 71.7%, and the HPLC purity is 8.95%.

Then chromatographic separation is carried out on a No. 3 chromatographic packed bed with the height-diameter ratio of 8:1 and the flow rate of 0.1BV/h, elution chromatographic separation is carried out by deionized water, HPLC tracking monitoring is carried out, and eluent with the HPLC purity higher than 50% is collected. Then concentrating and collecting the eluent until the concentration of 31g/L is 18mL, and the content is 556 mg. The yield of a single step is 86.2%, and the HPLC purity is 63.14%.

Then, 0.18g of ammonium chloride was added thereto, the pH was adjusted to 4.25 with 6M HCl, and 200mL of Poly (H/E) having a height-to-diameter ratio of 10:1 was added ^RP -30 chromatography columns at a flow rate of 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of the pingyangmycin HPLC area normalization method is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to a concentration of 31g/L and lyophilized to obtain high purity pingyangmycin copper chelate dry powder 511mg, single step yield 91.9%, total yield 56.7%, HPLC purity 99.16%.

Example 2

The strain is streptomyces verticillius var. pingyangensis CPCC200554, the monocomponent of the pingyangsu mycin is mainly produced, the strain is obtained by an aerobic fermentation method, the volume of a fermentation liquid placed in a tank is 58L, the unit is 17 mu g/mL, the total content is about 986mg, the pH value is adjusted to be 2.7 by oxalic acid, after stirring for 30min, the pH value is adjusted to be 4.7 by 6M NaOH, then, the ceramic membrane filtration is carried out to obtain 72L of filtrate (the middle is diluted by adding purified water), and the total content of the filtrate is 937 mg. The filtrate is put on 1L XR157 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely put on, 0.4M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h, the pH value of the eluent is adjusted to 4.4 by 6M NaOH, then the eluent is evaporated and concentrated to 30g/L in a rotary manner, and the filtrate is filtered to obtain 26mL of filtrate with the content of 798 mg. Single step yield 80.9% and HPLC purity 7.69%.

Then, the mixture is subjected to XR3SP 300mL packed bed chromatography separation with the height-diameter ratio of 10:1 and the flow rate of 0.1BV/h, deionized water elution chromatography separation is carried out, HPLC tracking monitoring is carried out, and eluent with the HPLC purity higher than 50% is collected. Then concentrating and collecting the eluent until the concentration of 31g/L is 22mL, and the content is 678 mg. Single step yield 85% with HPLC purity 64.82%.

Then 0.22g ammonium acetate was added, the pH was adjusted to 4.33 with 6M HCl, and 200mL LXMS-35 column with a height to diameter ratio of 12:1 was applied at a flow rate of 0.1 BV/h. Then, 0.5BV/h of 1% ammonium acetate aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of the pingyangmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to 42g/L concentration and lyophilized to obtain 613mg of high-purity pingyangmycin copper chelate powder with single-step yield of 90.4%, total yield of 62.1% and HPLC purity of 98.63%.

Example 3

The strain is streptomyces verticillius var. pingyangensis CPCC200554, and is mainly used for producing a pingyangmycin single component, 63L of pingyangmycin fermentation liquor is obtained by an aerobic fermentation method, the volume of the pingyangmycin fermentation liquor in a tank is 13 mu g/mL, the total content is about 819mg, the pH value is adjusted to 2.1 by oxalic acid, after stirring for 30min, the pH value is adjusted to 4.8 by 6M NaOH, and then the filtrate is filtered by a ceramic membrane to obtain 79L of filtrate (purified water is added in the middle for dilution), and the total content of the filtrate is 769 mg. The filtrate is coated with 1L D155 resin, the height-diameter ratio is 6:1, the flow rate is 2BV/h, after the materials are coated, 0.2M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h, the pH value of the eluent is adjusted to 4.3 by 6M NaOH, then the eluent is evaporated and concentrated to 23g/L, and the filtrate is filtered to obtain 25mL of filtrate with the content of 589 mg. The yield was 71.9%, and the HPLC purity was 9.16%.

Then, the mixture is subjected to XR3SP 300mL packed bed chromatography separation, the height-diameter ratio is 9:1, the flow rate is 0.1BV/h, deionized water elution chromatography separation and HPLC tracking monitoring are carried out, and eluent with the HPLC purity higher than 50% is collected. Then concentrating and collecting eluent until the concentration of 26g/L is 19.5mL, and the content is 507 mg. Yield of one step 86% and HPLC purity 59.14%.

0.20g of ammonium formate was then added, the pH was adjusted to 4.4 with 6M HCl and 200mL of an LXMS-60 column with an aspect ratio of 14:1 were applied at a flow rate of 0.1 BV/h. Then, 0.5BV/h of 1% ammonium formate aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of the pingyangmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluent is concentrated to the concentration of 34g/L and is lyophilized to obtain 439mg of high-purity pingyangmycin copper chelate dry powder, the single-step yield is 86.6%, the total yield is 53.6%, and the HPLC purity is 99.21%.

Example 4

The strain is streptomyces verticillius var. pingyangensis CPCC200554, the monocase of the pingyanensis is mainly produced, the aerobic fermentation method is adopted to obtain 62L of pingyangmycin fermentation liquor, the volume of the pingyangmycin fermentation liquor in a tank is 14 mu g/mL, the total content is about 868mg, the pH value is adjusted to 2.1 by oxalic acid, after stirring for 30min, the pH value is adjusted to 4.6 by 6M NaOH, and then ceramic membrane filtration is carried out to obtain 83L of filtrate (purified water is added in the middle for dilution), and the total content of the filtrate is 813 mg. The filtrate is coated with 1L D157 resin, the height-diameter ratio is 5:1, the flow rate is 2BV/h, after the materials are coated, 0.2M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h, the pH value of the eluent is adjusted to 4.3 by 6M NaOH, then the eluent is evaporated and concentrated to 26g/L, and the filtrate is filtered to obtain 23mL, the content of which is 624 mg. The yield was 71.9% and the HPLC purity was 8.67%.

Then, carrying out chromatographic separation on the eluate by using an HZ20SS 300mL packed bed, wherein the height-diameter ratio is 5:1, the flow rate is 0.1BV/h, eluting the eluate by using deionized water, carrying out chromatographic separation, carrying out HPLC tracking monitoring, and collecting the eluate with the HPLC purity higher than 50%. Then the eluate was concentrated and collected to 18mL of 23g/L concentrate, the content of which was 461 mg. Single step yield 73.9% with HPLC purity 68.97%.

Then, 0.18g of ammonium dihydrogen phosphate was added thereto, the pH was adjusted to 4.2 with 6M HCl, and 200mL of Poly (12: 1) was added thereto ^RP 40 chromatographic column with flow rate of 0.1 BV/h. Then, 0.5BV/h of 1% ammonium dihydrogen phosphate aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of the pingyangmycin HPLC area normalization method is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluent is concentrated to 43g/L concentration and is lyophilized to obtain 428mg of high-purity pingyangmycin copper chelate dry powder, the single step yield is 93%, the total yield is 59.3%, and the HPLC purity is 98.62%.

Example 5

The strain is streptomyces verticillius var. pingyangensis CPCC200554, the monocomponent of the pingyangmycin is mainly produced, 59L of the pingyangmycin fermentation liquor is obtained by an aerobic fermentation method, the volume of the pingyangmycin fermentation liquor in a tank is 15 mu g/mL, the total content is about 885mg, the pH value is adjusted to 2.1 by oxalic acid, after stirring for 30min, the pH value is adjusted to 4.6 by 6M NaOH, and then the filtrate is filtered by ceramic membrane to obtain 79L (the middle is diluted by adding purified water), and the total content of the filtrate is 824 mg. And (3) putting the filtrate on 1L XR140 resin, wherein the height-diameter ratio is 5:1, the flow rate is 2BV/h, eluting with 0.2M hydrochloric acid solution at the flow rate of 0.2BV/h after the materials are completely put on the filtrate, adjusting the pH value of the eluent to 4.3 by using 6M NaOH, performing rotary evaporation and concentration to 26g/L, and filtering to obtain 28mL of filtrate with the content of 728 mg. Yield 82.25% and HPLC purity 9.14%.

Following the second treatment of example 4, the packing was replaced with NM100 to yield 21mL of 24g/L concentrate with a content of 624 mg. Single step yield 85.7%, HPLC purity 55.24%.

The third treatment according to example 4 was carried out, the filler being replaced by Poly ^RP 60, obtaining 543mg of high-purity pingyangmycin copper chelate dry powder, wherein the single-step yield is 86.9 percent, the total yield is 61.35 percent, and the purity is 98.47 percent.

Example 6

Putting the fermentation liquor into a tank with the volume of 15.2L, putting streptomyces verticillius var. pingyangensis CPCC200552 strains, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangmycin and the unit of boanmycin are 158.61 mu g/mL and 42.17 mu g/mL respectively, the total content of pingyangmycin and 632.55mg boanmycin is 2379.15mg, adjusting the pH value to be 2.3 by oxalic acid, stirring for 30min, then filtering by a ceramic membrane with the thickness of 200nm to obtain 20L of filtrate (adding purified water for dilution), adjusting the pH value to be 4.9 by 6M NaOH, and the total content of pingyangmycin 2275.39mg and boanmycin 600.93mg in the filtrate. Putting the filtrate on 300mL of ZGD115 resin, eluting with 0.3M hydrochloric acid solution at the flow rate of 0.2BV/h after the materials are completely put on the resin and the height-diameter ratio is 4:1 and the flow rate is 2BV/h, adjusting the pH value of the eluent to 4.4 by 6M NaOH, then carrying out rotary evaporation and concentration to 28g/L, and filtering to obtain 87mL of filtrate with the content of 1934.08mg of pingyangmycin and 510.79mg of boanmycin. HPLC purity 9.15% and 8.16%, respectively, yield 81.29% and 80.75%.

And then, performing chromatographic separation on the mixture by using a No. 3 chromatographic packed bed with the height-diameter ratio of 8:1 and the flow rate of 0.1BV/h, eluting and chromatographic separation by using deionized water, performing HPLC tracking monitoring, respectively collecting eluent with the HPLC purity higher than 50% of pingyangmycin and boanmycin to respectively obtain eluent containing 1740.15mg of pingyangmycin and 462.18mg of boanmycin, and then respectively concentrating the eluent to 40mL and 15 mL. HPLC purity was 60.58% and 58.96%, respectively, and single step yield was 89.97% and 90.48%, respectively.

Then, 0.40g and 0.15g of ammonium chloride were added, the pH was adjusted to 4.25 with 6M HCl, and 500mL and 150mL of Poly (H/E) having a height to diameter ratio of 10:1 were added ^RP -30 chromatographic column, filler microspherical poreHas a diameter of

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to 20mL and 5mL and lyophilized to obtain high purity pingyangmycin and boanmycin copper chelate dry powders 1548.74mg and 405.72mg, respectively, with single step yields of 89.03% and 87.78%, and total yields of 65.09% and 64.14%, respectively. HPLC purity 98.14% and 99.08%, respectively.

Example 7

Placing fermentation liquor in a tank with the volume of 15.5L, carrying out aerobic fermentation on streptomyces verticillius var. pingyannis CPCC200552 strain to obtain the fermentation liquor, wherein the unit of pingyangmycin and boanmycin are 147.54 mu g/mL and 38.17 mu g/mL respectively, the total content of 2286.87mg pingyangmycin and 591.64mg boanmycin, adjusting the pH value to 2.8 by oxalic acid, stirring for 30min, then filtering by a ceramic membrane with the thickness of 200nm to obtain 20L of filtrate (diluting with purified water in the later filtering period), adjusting the pH value to 4.8 by 6M NaOH, and the total content of pingyangmycin 2218.26mg and boanmycin 567.96mg in the filtrate. The filtrate is put on 300mL XR157 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, 0.4M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h after the materials are completely put on, the pH value of the eluent is adjusted to 4.3 by 6M NaOH, then the eluent is evaporated and concentrated to 30.12g/L, and neutral filter paper is used for filtering to obtain 80mL filtrate with the content of 1913.15mg of pingyangmycin and 481.64mg of boanmycin. HPLC purity 8.78% and 7.55%, respectively, and yield 83.65% and 81.41%, respectively.

Then, the mixture is subjected to XR3SP 500mL packed bed chromatography separation, the height-diameter ratio is 8:1, the flow rate is 0.1BV/h, deionized water elution chromatography separation and HPLC tracking monitoring are carried out, eluent with the purity higher than 50% of Pingyangmycin and boanmycin HPLC are respectively collected, eluent containing 1635.74mg Pingyangmycin and 414.69mg boanmycin is respectively obtained, and then the eluent is respectively concentrated to 30mL and 18 mL. HPLC purity was 58.26% and 61.38%, respectively, single step yield was 85.49% and 86.10%, respectively.

Then, 0.30g and 0.18g of ammonium chloride were added thereto, respectively, and the pH was adjustedAdjusted to 4.4 with 6M HCl, and charged with 500mL and 150mL UniPS with a height to diameter ratio of 10:1 ^TM -50 chromatographic column with filler microsphere pore diameter of

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to about 18mL and 4mL and lyophilized to obtain high purity pingyangmycin and boanmycin copper chelate dry powders 1509.78mg and 374.47mg, respectively, with single step yields of 92.29% and 90.30%, and total yields of 66.02% and 63.29%, respectively. HPLC purity was 98.35% and 99.13%, respectively.

Example 8

Placing the fermentation liquor in a tank with the volume of 14.8L, placing streptomyces verteillius var. pingyangensis CPCC200552 strain, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangshensis and the unit of boanmycin are 161.27 mu g/mL and 46.88 mu g/mL respectively, the total content of the pingyangshycin and the boanmycin is 2386.79mg and 693.82mg, adjusting the pH value to be 2.6 by adopting oxalic acid, stirring for 30min, then filtering by a 50nm ceramic membrane to obtain 20L of filtrate (adding purified water for dilution at the later stage of filtration), adjusting the pH value to be 4.7 by adopting 6M NaOH, and the total content of the pingyangshacin 2243.58mg and the boanmycin 652.19mg in the filtrate. The filtrate is put on 300mL of D155 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely put on the filtrate, 0.5M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h, the pH value of the eluent is adjusted to 4.5 by 6M NaOH, then the eluent is evaporated and concentrated to 32.4g/L, and the medium-speed filter paper is used for filtering to obtain 77mL of filtrate with the content of 1929.48mg of pingyangmycin and 560.88mg of boanmycin. HPLC purity was 9.13% and 7.47%, respectively, and yield was 80.83% and 81.26%, respectively.

Then, chromatographic separation is carried out on the mixture by an HZ20SS 500mL packed bed, the height-diameter ratio is 8:1, the flow rate is 0.1BV/h, elution chromatographic separation is carried out by deionized water, HPLC tracking monitoring is carried out, eluents with purity higher than 50% of Pingyangmycin and boanmycin HPLC are respectively collected, eluents containing 1661.28mg Pingyangmycin and 472.91mg boanmycin are respectively obtained, and then the eluents are respectively concentrated to 35mL and 14 mL. HPLC purities of 61.85% and 59.27%, respectively, and single step yields of 86.09% and 84.31%, respectively.

Then respectively adding 0.35g and 0.14g of ammonium chloride, adjusting the pH to 4.1 by using 6M HCl, respectively loading the mixture on 500mL and 150mL LXMS-35 chromatographic columns with the height-diameter ratio of 10:1, wherein the pore diameter of the filler microspheres is

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluent is concentrated to about 22mL and 6mL, and the dry powder 1533.36mg and 435.73mg of high-purity pingyangmycin and boanmycin copper chelate are obtained by freeze-drying respectively, the single-step yield is 92.18 percent and 92.13 percent respectively, and the total yield is 64.24 percent and 62.80 percent respectively. HPLC purity 98.11% and 99.09%, respectively.

Example 9

Placing the fermentation liquor into a tank with the volume of 15.4L, placing streptomyces verteillius var. pingyangensis CPCC200552 strain, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangmycin and the unit of boanmycin are 137.63 mu g/mL and 31.54 mu g/mL respectively, the total content of pingyangmycin and 485.72mg of boanmycin, adjusting the pH value to be 2.9 by adopting oxalic acid, stirring for 30min, then filtering by a 50nm ceramic membrane to obtain 20L of filtrate (adding purified water for dilution at the later stage of filtration), adjusting the pH value to be 4.9 by adopting 6M NaOH, and the total content of pingyangmycin in the filtrate is 1899.07mg and boanmycin 435.21 mg. The filtrate is put on 300mL of D157 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely put on, 0.5M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h, the pH value of the eluent is adjusted to 4.3 by 6M NaOH, then the eluent is evaporated and concentrated to 31.8g/L, and the filtrate is filtered by medium-speed filter paper to obtain about 63mL of filtrate with the content of 1622.31mg of pingyangmycin and 371.24mg of boanmycin. HPLC purity 7.44% and 6.19% respectively, yield 76.54% and 76.43% respectively.

Then, chromatographic separation is carried out on the mixture by an HZ20SS 500mL packed bed, the height-diameter ratio is 10:1, the flow rate is 0.1BV/h, elution chromatographic separation is carried out by deionized water, HPLC tracking monitoring is carried out, eluents with purity higher than 50% of Pingyangmycin and boanmycin HPLC are respectively collected, eluents containing 1403.30mg Pingyangmycin and 318.89mg boanmycin are obtained, and then the eluents are respectively concentrated to 29mL and 13 mL. HPLC purity 58.97% and 60.24%, respectively, single step yield 86.50% and 85.89%, respectively.

Then, 0.29g and 0.13g of ammonium chloride were added thereto, the pH was adjusted to 4.4 with 6M HCl, and 500mL and 150mL of Poly (R) having a height to diameter ratio of 10:1 were added ^RP -60 chromatographic column with filler microsphere pore diameter of

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to about 18mL and 5mL and lyophilized to obtain high purity pingyangmycin and boanmycin copper chelate dry powders 1281.38mg and 292.13mg, respectively, with single step yields of 91.31% and 91.62%, and total yields of 60.45% and 60.13%, respectively. HPLC purity 98.43% and 99.15%, respectively.

Example 10

Placing fermentation liquor in a tank with the volume of 15.1L, placing streptomyces verteillius var. pingyangensis CPCC200552 strain, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangmycin and the unit of boanmycin are 157.59 mu g/mL and 43.03 mu g/mL respectively, the total content of the pingyangmycin and the boanmycin is 2379.61mg, adjusting the pH value to 2.8 by oxalic acid, stirring for 30min, then filtering by a 200nm ceramic membrane to obtain 20L of filtrate (adding purified water for dilution at the later stage of filtration), adjusting the pH value to 4.7 by 6M NaOH, and the total content of the pingyangmycin in the filtrate is 2286.81mg and the boanmycin is 625.71 mg. The filtrate is put on 300mL of ZGD113 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, 0.2M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h after the materials are completely put on, the pH value of the eluent is adjusted to 4.4 by 6M NaOH, then the eluent is concentrated to 34.6g/L by rotary evaporation, and the filtrate is filtered by medium-speed filter paper to obtain about 74mL of filtrate with the content of 2014.69mg of pingyangmycin and 548.12mg of boanmycin. HPLC purity was 9.18% and 8.69%, respectively, and yield was 84.66% and 84.35%, respectively.

Then, the mixture is subjected to XR3SP 500mL packed bed chromatography separation, the height-diameter ratio is 10:1, the flow rate is 0.1BV/h, deionized water elution chromatography separation and HPLC tracking monitoring are carried out, eluent with purities higher than 50% of pingyangmycin and boanmycin are respectively collected, eluent containing 1796.51mg pingyangmycin and 485.63mg boanmycin is respectively obtained, and then the eluent is respectively concentrated to 32mL and 17 mL. HPLC purity 67.58% and 68.36%, respectively, single step yield 89.17% and 88.59%, respectively.

Then, 0.32g and 0.17g ammonium chloride were added, respectively, and the pH was adjusted to 4.4 with 6M HCl, 500mL and 150mL UniPS with a height to diameter ratio of 12:1 ^TM -30 chromatographic column with filler microsphere pore diameter of

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. Concentrating the eluate to about 21mL and 7mL, and lyophilizing to obtain high-purity pingyangmycin and boanmycin copper chelate dry powders 1652.78mg and 441.92mg, respectively, with single-step yields of 91.99% and 90.82%, and total yields of 69.44% and 68.01%, respectively. HPLC purity 98.20% and 99.38%, respectively.

Example 11

Placing the fermentation liquor in a tank with the volume of 14.9L, placing streptomyces verteillius var. pingyangensis CPCC200552 strain, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangshensis and the unit of boanmycin are 161.76 mu g/mL and 39.17 mu g/mL respectively, the total content of the pingyangshycin and the boanmycin is 2410.22mg and 589.63mg, adjusting the pH value to be 2.5 by adopting oxalic acid, stirring for 30min, then filtering by a 50nm ceramic membrane to obtain 20L of filtrate (adding purified water for dilution at the later stage of filtration), adjusting the pH value to be 4.9 by adopting 6M NaOH, and the total content of the pingyangshacin 2265.61mg and the boanmycin 557.79mg in the filtrate. The filtrate is put on 300mL of D157 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely put on, 0.2BV/h hydrochloric acid solution is used for eluting at the flow rate of 0.2BV/h, 6M NaOH is used for adjusting the pH value of the eluent to be 4.2, then the eluent is evaporated and concentrated to 31.9g/L, and medium-speed filter paper is used for filtering to obtain about 74mL of filtrate with the content of 1948.42mg of pingyangmycin and 485.28mg of boanmycin. HPLC purity 8.23% and 7.14% respectively, yield 80.83% and 82.30% respectively.

Then, the mixture is subjected to XR3SP 500mL packed bed chromatography separation, the height-diameter ratio is 6:1, the flow rate is 0.1BV/h, deionized water elution chromatography separation and HPLC tracking monitoring are carried out, eluent with purities higher than 50% of pingyangmycin and boanmycin are respectively collected, eluent containing 1621.08mg pingyangmycin and 410.54mg boanmycin is respectively obtained, and then the eluent is respectively concentrated to 26mL and 12 mL. HPLC purity was 61.88% and 59.76%, respectively, single step yield was 83.19% and 84.51%, respectively.

Then respectively adding 0.26g and 0.12g of ammonium chloride, adjusting the pH to 4.2 by using 6M HCl, respectively putting the mixture into 500mL and 150mL LXMS-60 chromatographic columns with the height-to-diameter ratio of 14:1, wherein the pore diameter of the filler microspheres is

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to about 18mL and 5mL and lyophilized to obtain high purity pingyangmycin and boanmycin copper chelate dry powders 1496.27mg and 376.88mg, respectively, with single step yields of 92.31% and 91.80%, and total yields of 62.08% and 63.92%, respectively. HPLC purity 98.49% and 99.23%, respectively.

Example 12

Crude pingyangmycin with the purity of 51.32 percent is obtained by adopting the methods of examples 1 to 11 in any combination, and Poly is examined ^RP -30 pore diameter

And (5) purifying effect of the filler. The volume of the pingyangmycin concentrated solution is 28ml, the concentration is 29g/L, the content is about 812mg, 0.30g of ammonium chloride is added, the pH value is adjusted to 4.4 by adopting 6M HCl, a 400ml chromatographic column with the height-diameter ratio of 12:1 is arranged, and the flow rate is adjustedIs 0.08 BV/h.

Then, the mixture is eluted by 0.3BV/h flow rate of 1 percent ammonium chloride solution, the dosage is 5BV, then the mixture is eluted by deionized water at the same flow rate, the collection is started when the purity of the pingyangmycin is higher than 95 percent, and the collection is stopped when the unit is lower than 50 mu g/mL. Concentrating the eluate to 20ml, and lyophilizing to obtain 704mg of Pingyangmycin copper chelate dry powder. The yield was 86.7% and the HPLC purity was 98.15%.

Comparative example 1

Placing fermentation liquor in a tank with the volume of 15.1L, placing streptomyces verteillius var. pingyangensis CPCC200552 strain, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangmycin and the unit of boanmycin are 154.56 mu g/mL and 37.31 mu g/mL respectively, the total content of the pingyangmycin and the 563.38mg of the boanmycin is 2333.86mg of the pingyangmycin and 563.38mg of the boanmycin, adjusting the pH value to be 2.6 by adopting oxalic acid, stirring for 30min, then filtering by a 50nm ceramic membrane to obtain 21L of filtrate (adding purified water for dilution at the later stage of filtration), adjusting the pH value to be 4.7 by adopting 6M NaOH, and the total content of the pingyangmycin to be 2155.31mg and the boanmycin to be 527.67mg in the filtrate. The filtrate is put on 300mL of D157 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely put on, 0.2BV/h hydrochloric acid solution is used for elution, the pH value of the eluent is adjusted to 4.7 by 6M NaOH, and then the filtrate is filtered by rotary evaporation, concentration and medium-speed filter paper to obtain about 81mL of filtrate with the content of 1848.12mg of pingyangmycin and 464.79mg of boanmycin. Yields 8.45% and 7.32%, HPLC purities 79.18% and 82.50%.

Then the mixture is put into 500mL UniPSM 60-300 (provided by Suzhou nano-technology) packed bed for chromatographic separation, the height-diameter ratio is 8:1, the flow rate is 0.1BV/h, deionized water elution chromatographic separation is carried out, HPLC tracking monitoring is carried out, eluent with the purity higher than 50 percent of Pingyangmycin and boanmycin is respectively collected, eluent containing 943.89mg Pingyangmycin and 230.14mg boanmycin is respectively obtained, and then the eluent is respectively concentrated to 18mL and 8 mL. HPLC purity was 56.32% and 53.25%, respectively, single step yield was 51.07% and 49.51%, respectively. Pingyangmycin and boanmycin can not be effectively separated, and the single-step yield is obviously lower.

Comparative example 2

The pingyangmycin and boanmycin concentrates from comparative example 1 were then added with 0.18g and 0.08g, respectively, of ammonium chloride, the pH adjusted to 4.5 using 6M HCl, and 250mL and 80mL, respectively, of UniPMM 40-500 (available from Suzhou Na Microscience) with an aspect ratio of 14:1The aperture of the filler microspheres of the chromatographic column is

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to about 10mL and 5mL and lyophilized to give 512.34mg and 96.09mg of high purity pingyangmycin and boanmycin copper chelate dry powders, respectively, in single step yields of 54.27% and 41.75%, and HPLC purities of 95.27% and 95.66%, respectively. Both the single step yield and the final purity were low.

Comparative example 3

The fermentation liquor is put in a tank with the volume of 14.4L, streptomyces verticillius var. pingyangensis CPCC200552 strains are subjected to aerobic fermentation to obtain the fermentation liquor, the unit of the pingyangmycin and the unit of the boanmycin are respectively 168.12 mu g/mL and 39.35 mu g/mL, the total content of the pingyangmycin and the boanmycin is 2420.93mg, the pH value is adjusted to be 2.4 by oxalic acid, after stirring is carried out for 30min, the filtrate is filtered by a 50nm ceramic membrane to obtain 22L (purified water is added for dilution at the later stage of filtration), the pH value is adjusted to be 4.9 by 6M NaOH, and the total content of the pingyangmycin in the filtrate is 2243.33mg and the boanmycin is 524.56 mg. The filtrate is put on 300mL of ZGD115 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, 0.2M hydrochloric acid solution is eluted at the flow rate of 0.2BV/h after the materials are completely put on, the pH value of the eluent is adjusted to 4.6 by 6M NaOH, and then the filtrate is filtered by rotary evaporation and concentration filter paper to obtain about 62mL of filtrate with the content of 2024.71mg of pingyangmycin and 469.80mg of boanmycin. HPLC purity 8.54% and 7.13% with yields of 83.63% and 82.74%, respectively.

And then, performing chromatographic separation on the mixture by using an XR3SP 500mL packed bed, wherein the height-diameter ratio is 12:1, the flow rate is 0.1BV/h, the mixture cannot be eluted by using a 1% ammonium chloride aqueous solution, then, the mixture is eluted by using a 10% methanol solution for chromatographic separation, HPLC tracking and monitoring are performed, eluent with the HPLC purity higher than 50% of that of pingyangmycin and boanmycin is respectively collected, eluent containing 1293.38mg of pingyangmycin and 316.57mg of boanmycin is respectively obtained, and then, the eluent is respectively concentrated to 15mL and 10 mL. HPLC purity 53.76% and 56.28%, respectively, single step yield 63.87% and 67.44%, respectively. Pingyangmycin and boanmycin can not be effectively separated, and the yield is low.

Comparative example 4

The pingyangmycin and boanmycin concentrates obtained in comparative example 3 were then added with 0.15g and 0.10g, respectively, ammonium chloride, the pH was adjusted to 4.3 using 6M HCl, and 200mL and 100mL UniPS with an aspect ratio of 14:1 were added ^TM -30 chromatographic column with filler microsphere pore diameter of

The flow rate was 0.1 BV/h. And (3) adopting 2% ammonium chloride aqueous solution to be eluted, then adopting 10% methanol solution to elute at the same flow rate, starting to collect when the purity of Pingyangmycin or boanmycin by HPLC area normalization method is more than 95%, and stopping collecting when the unit is lower than 50 mu g/mL. The eluate was concentrated to about 10mL and 5mL and lyophilized to give high purity pingyangmycin and boanmycin copper chelate dry powders 652.44mg and 161.73mg, respectively, with single step yields of 50.44% and 51.09%. HPLC purity 96.13% and 95.39%, respectively. Pingyangmycin and boanmycin can not be effectively separated, and the purity and the yield are low.

Comparative example 5

Placing the fermentation liquor into a tank with the volume of 15.6L, placing streptomyces verteillius var. pingyannis CPCC200552 strain, carrying out aerobic fermentation to obtain the fermentation liquor, wherein the unit of pingyangmycin and the unit of boanmycin are 158.49 mu g/mL and 41.59 mu g/mL respectively, the total content of the pingyangmycin and the 648.80mg of the boanmycin is 2472.45mg, adjusting the pH value to be 2.9 by adopting oxalic acid, stirring for 30min, then filtering by a 50nm ceramic membrane to obtain 23L of filtrate (adding purified water for dilution at the later stage of filtration), adjusting the pH value to be 4.4 by adopting 6M NaOH, and the total content of the pingyangmycin in the filtrate is 2267.51mg and the boanmycin is 628.65 mg. The filtrate is put on 300mL of D157 resin, the height-diameter ratio is 4:1, the flow rate is 2BV/h, after the materials are completely put on, 0.2BV/h hydrochloric acid solution is used for elution, the pH value of the eluent is adjusted to 4.6 by 6M NaOH, and then the filtrate is obtained by rotary evaporation, concentration and filtration, wherein the filtrate is about 63mL, and the content of the pingyangmycin is 2040.55mg and the content of the boanmycin is 529.87 mg. HPLC purity 8.46% and 7.52% with yield 82.53% and 81.67%, respectively.

The bleomycin group compound has unstable structure at a high pH or in an alkaline state, for example, pH 6.5, and is prone to generate impurities during chromatography at a temperature higher than room temperature.

Adjusting the pH of the concentrated solution to 3.0 by using 6M HCl, performing chromatographic separation on a No. 3 500mL packed bed, performing elution chromatographic separation on the concentrated solution with a height-diameter ratio of 10:1 and a flow rate of 0.1BV/h by using deionized water, performing HPLC tracking monitoring, respectively collecting eluent with the HPLC purities of more than 50% of pingyangmycin and boanmycin, respectively obtaining eluent containing 894.67mg of pingyangmycin and 265.19mg of boanmycin, and then respectively concentrating the eluent to 15mL and 10 mL. HPLC purity was 53.14% and 56.26%, respectively, single step yield was 43.84% and 50.04%, respectively. The yields of pingyangmycin and boanmycin are both low.

Comparative example 6

The pingyangmycin and boanmycin concentrates from comparative example 5 were then added with 0.32g and 0.17g ammonium chloride, respectively, and the pH adjusted to 2.4 using 6M HCl, respectively, to 300mL and 100mL UniPS with an aspect ratio of 12:1 ^TM -30 chromatographic column with filler microsphere pore diameter of

The flow rate was 0.1 BV/h. Then, 0.5BV/h of 1% ammonium chloride aqueous solution is used for elution, the dosage is 5BV, then deionized water is used for elution at the same flow rate, when the purity of Pingyangmycin or boanmycin by HPLC area normalization is more than 95%, collection is started, and when the unit is less than 50 mug/mL, collection is stopped. The eluate was concentrated to about 12mL and 6mL and lyophilized to obtain high purity pingyangmycin and boanmycin copper chelate dry powders 531.70mg and 155.16mg, respectively, with single step yields of 59.43% and 58.51%. HPLC purity 95.67% and 96.28%, respectively. The purity and the yield are both low.

Claims

1. A purification method of a copper chelate of a bleomycin compound is characterized by comprising the following steps of carrying out chromatography on an aqueous solution containing a crude copper chelate of the bleomycin compound, wherein a chromatography filler is a monodisperse polystyrene/divinylbenzene chromatography medium, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 10-60 mu m, and an eluent for the chromatography is an aqueous solution of an ammonium salt and water in sequence; collecting the eluent water to obtain an aqueous solution of a pure copper chelate product containing the bleomycin compound; the mass percentage concentration of the ammonium salt in the aqueous solution is 0.5-1.5%; in the aqueous solution containing the crude copper chelate product of the bleomycin compound, the purity of the copper chelate of the bleomycin compound is more than 50 percent;

the operation of collecting the eluent is to collect the eluent containing the copper chelate of the bleomycin compound with HPLC purity of more than 95 percent.

2. The method of claim 1, wherein the copper chelate of the compound of the bleomycin group is a chelate of the compound of the bleomycin group obtained by fermentation of the Pingyang variety of Streptomyces verticillatus with copper ions;

and/or the ammonium salt is one or more of ammonium chloride, ammonium acetate, ammonium formate, ammonium phosphate, ammonium dihydrogen phosphate and ammonium hydrogen phosphate;

and/or the mass percentage concentration of the ammonium salt water solution is 1.0%;

and/or the pH value of the aqueous solution of the copper chelate crude product containing the bleomycin compound is 2.5-5.5;

and/or the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 30 mu m;

and/or the pore diameter of the monodisperse polystyrene/divinylbenzene chromatography medium is 100A-1000A;

and/or, the chromatographic column is equilibrated with the said eluent ammonium salt water solution in advance;

and/or the height-diameter ratio of the chromatographic column is 8: 1-20: 1;

and/or the flow rate of the eluent is 0.05 BV/h-0.2 BV/h;

and/or when the eluent is an ammonium salt aqueous solution, the using amount of the ammonium salt aqueous solution is 4 BV-6 BV;

and/or in the aqueous solution containing the crude copper chelate of the bleomycin group compound, the concentration of the copper chelate of the bleomycin group compound in the aqueous solution is 20-65 g/L;

and/or the loading capacity of the chromatographic column is 1 g/L-5 g/L.

3. The method of claim 2, wherein the copper chelate of the compound of the bleomycin group is a variant of the species Streptomyces verticillatusstreptomyces verticillius var.pingyangensis CPCC 200554And/orstreptomyces verticillius var.pingyangensis CPCC 200552Chelate formed by the bleomycin group compound obtained by fermentation of the strain and copper ions;

and/or the bleomycin group compound is one or more of pingyangmycin, boanmycin and boningmycin;

and/or the pH value of the aqueous solution of the copper chelate crude product containing the bleomycin group compound is 4.0-4.5;

and/or the pH value of the aqueous solution of the copper chelate containing the bleomycin compound is obtained by adopting acid or alkali regulation; the acid is one or more of hydrochloric acid, acetic acid, formic acid and phosphoric acid; the alkali is one or more of alkali metal hydroxide, alkali metal carbonate and alkali metal bicarbonate;

and/or the monodisperse polystyrene/divinylbenzene chromatography medium is Poly ^RP -10、Poly ^RP -30、Poly ^RP -60、Poly ^RP -40、LXMS-15、LXMS-35、LXMS-60、UniPS ^TM -20, chromatography No. 3, UniPS ^TM -40 or UniPS ^TM -50；

And/or, the chromatographic column is balanced by 2BV of the aqueous solution of the eluent ammonium salt in advance;

and/or the height-diameter ratio of the chromatographic column is 10: 1-15: 1;

and/or the flow rate of the eluent is 0.3 BV/h-0.5 BV/h;

and/or the dosage of the aqueous solution of the eluent ammonium salt is 5 BV;

and/or, when the copper chelate of the bleomycin compound is multiple, the operation of collecting the eluent is to collect the eluent with the HPLC purity higher than 95 percent and containing the copper chelate of the bleomycin compound, and the collection is stopped when the concentration of the copper chelate of the bleomycin compound contained in the eluent is lower than 50 mu g/mL;

and/or, the purification method also comprises the following post-treatment step, namely concentrating and freeze-drying the obtained water solution containing the pure copper chelate of the bleomycin group compound to obtain the copper chelate of the bleomycin group compound.

4. The method for purifying the copper chelate of a bleomycin compound according to any one of claims 1 to 3, further comprising the steps of subjecting a fermentation enriched concentrate containing the copper chelate of a bleomycin compound to chromatography, wherein a chromatography column filler is a monodisperse polystyrene/divinylbenzene chromatography medium, and the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 100 to 200 μm; eluent for chromatography is water; collecting the eluent to obtain an aqueous solution of a copper chelate crude product containing the bleomycin group compound; in the fermentation enrichment concentrated solution of the copper chelate crude product containing the bleomycin group compound, the purity of the copper chelate of the bleomycin group compound is more than 5 percent;

in the chromatography of the fermentation enriched concentrated solution, the operation of collecting the eluent is to collect the eluent of the copper chelate containing the bleomycin compound, the HPLC purity of which is higher than 50 percent.

5. The method for purifying the copper chelate of the bleomycin compound according to claim 4, wherein the pH value of the fermentation enriched concentrated solution of the copper chelate containing the bleomycin compound is 2.5 to 5.5;

and/or, in the chromatography of the fermentation enriched concentrated solution, the eluent is deionized water;

and/or in the chromatography of the fermentation enriched concentrated solution, the particle size of the monodisperse polystyrene/divinylbenzene chromatography medium is 130-180 μm;

and/or, in the chromatography of the fermentation enrichment concentrated solution, the chromatographic column is balanced by eluent in advance;

and/or in the chromatography of the fermentation enriched concentrated solution, the height-diameter ratio of the chromatographic column is 5: 1-15: 1;

and/or in the chromatography of the fermentation enrichment concentrated solution, the flow rate of the eluent is 0.05 BV/h-0.15 BV/h;

and/or the mass volume content of the copper chelate of the bleomycin compound in the fermentation enrichment concentrated solution is 25 g/L-35 g/L;

and/or the loading capacity of the chromatographic column is 0.5-10 g/L.

6. The method for purifying the copper chelate of the bleomycin compound according to claim 5, wherein the pH value of the fermentation enriched concentrated solution of the copper chelate containing the bleomycin compound is 4.1 to 4.5;

and/or the pH value of the fermentation enrichment concentrated solution containing the copper chelate compound of the bleomycin group is obtained by adjusting with acid or alkali, wherein the acid is one or more of hydrochloric acid, acetic acid, formic acid and phosphoric acid, and the alkali is one or more of alkali metal hydroxide, alkali metal carbonate and alkali metal bicarbonate;

and/or, the monodisperse polystyrene/divinylbenzene chromatography medium is chromatography No. 3, NM100, XR3SP, or HZ20 SS;

and/or the height-diameter ratio of the chromatographic column is 5: 1-10: 1;

and/or the flow rate of the eluent is 0.1 BV/h;

and/or, when the copper chelate of the bleomycin compound is a plurality of copper chelates, the operation of collecting the eluent is to collect the eluent containing the copper chelate of the bleomycin compound with HPLC purity higher than 50 percent respectively.

7. The method for purifying a copper chelate of a compound of the bleomycin family as set forth in claim 4, further comprising the fermentation broth enrichment step,

and (2) carrying out enrichment chromatography on the filtrate of the fermentation liquor containing the copper chelate of the bleomycin group compound in the step (1) by using a macroporous weak acid resin column, wherein an eluant is dilute hydrochloric acid, collecting the obtained eluate, adjusting the pH value of the eluate, concentrating and filtering to obtain the fermentation enriched concentrated solution containing the copper chelate of the bleomycin group compound.

8. The method of claim 7, wherein the fermentation broth of the copper chelate containing the compound of the bleomycin group is obtained by direct fermentation of a new variant of Streptomyces verticillatus Pingyang, or is formed by adding a copper salt to the fermentation broth containing the compound of the bleomycin group obtained after fermentation;

and/or, in the step (1), the acid is oxalic acid;

and/or, in the step (1), the pH value is adjusted to 2.0-3.0 by acid;

and/or, in the step (1), the alkali is 6M NaOH aqueous solution;

and/or, in the step (1), the pH value is adjusted to 4.5-5.5 by alkali;

and/or in the step (1), the filtering is inorganic ceramic membrane filtering of 10 nm-200 nm;

and/or, in the step (2), the alkali is 6M NaOH aqueous solution;

and/or, in the step (2), the macroporous weak acid resin is D157, D152, D155, XR140, XR157, ZGD113 or ZGD 115;

and/or in the step (2), the molar concentration of the eluent diluted hydrochloric acid is 0.1-0.5 mol/L;

and/or, in the step (2), the chromatographic column is balanced by 5BV of deionized water in advance.