CN112480127A - Novel method for producing mitomycin - Google Patents
Novel method for producing mitomycin Download PDFInfo
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- CN112480127A CN112480127A CN202011448849.7A CN202011448849A CN112480127A CN 112480127 A CN112480127 A CN 112480127A CN 202011448849 A CN202011448849 A CN 202011448849A CN 112480127 A CN112480127 A CN 112480127A
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
- C07D487/14—Ortho-condensed systems
Abstract
The invention discloses a novel method for producing mitomycin, which comprises the following steps: s1, preprocessing mitomycin fermentation liquor; s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate; s3, filtering; s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain third eluent; s5, a refining step. The invention optimizes the extraction and purification production process of MMC on the basis of the prior art, adds partial separation and purification process steps, and ensures that the whole separation and purification process is more stable and controllable.
Description
Technical Field
The invention relates to the field of medicine extraction and purification, and particularly relates to a novel method for producing mitomycin.
Background
MMC is an antitumor drug obtained by fermentation culture and extraction and purification, and the chemical structural formula of the MMC is as follows:
the product is obtained by separating and purifying metabolites generated by fermentation culture of microorganisms (Streptomyces, caespitosus), belongs to quinone antibiotic substances, has three active groups acting on cells on the molecular structure, participates in the metabolism of tumor cells together, is an alkylating agent, and has toxic and side effects on cells. The product can cross-link with DNA, depolymerize DNA, prevent DNA replication process, and inhibit cancer cell division, so it has higher effect than general alkylating agent, and has toxicity similar to general alkylating agent, but far more effective than other alkylating agent.
Because the fermentation liquor is extracted, the feed liquid contains more impurities, the production period is long, the production process is greatly influenced by the fermentation condition, and the extraction and purification process needs to be optimized to stabilize the whole production process and produce the qualified MMC product.
Disclosure of Invention
It is an object of the present invention to provide a new method for producing mitomycin which solves one or more of the above mentioned problems of the prior art.
The invention provides a novel method for producing mitomycin, which comprises the following steps:
s1, mitomycin fermentation liquor pretreatment: transferring the mitomycin fermentation liquor to a pretreatment tank, stirring the fermentation liquor, adding perlite, and adjusting the pH of the feed liquid by using a sodium hydroxide solution with the mass concentration of 1%;
s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate;
s3, filtering: filtering the filtrate by using a bag filter so as to remove particle impurities in the filtrate;
s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain third eluent;
s5, refining: and concentrating, crystallizing, filtering, washing, drying and crushing the third eluent to obtain the mitomycin.
In certain embodiments, in step S1, the weight ratio of the mitomycin broth to perlite is from 100:2 to 100: 4.
In certain embodiments, the pH of the feed solution is adjusted in step S1 to range from 7 to 9.
In some embodiments, step S4 specifically includes the following steps:
carrying out primary adsorption on the filtrate obtained in the step S3 through macroporous resin, after the primary adsorption is finished, washing the resin with deionized water until the color of a washing liquid is lower than that of Y3#, blowing the washing liquid to dry the washing liquid under air pressure, introducing ethyl acetate to carry out primary elution, adjusting the pH value to 7-8 by using a phosphoric acid buffer solution, collecting primary eluent until the color is lower than that of Y5#, and stopping elution;
extracting the first eluent by a sodium chloride buffer solution to obtain an extract;
performing secondary adsorption on the extract liquor through macroporous resin, and performing secondary elution by using a methanol aqueous solution with the mass concentration of 5-30% to obtain secondary eluent;
purifying the secondary eluent by an industrial chromatographic column, washing with water, washing with methanol with the mass concentration of 10% and purifying with methanol with the mass concentration of 20-25% to obtain a feed liquid;
and (3) carrying out third adsorption on the feed liquid through macroporous resin, drying after the column loading is finished, and then carrying out third elution by using pure methanol to obtain third eluent.
In some embodiments, the primary resolving liquid should be allowed to stand before extraction to remove the underlying black liquid, which helps to improve purity.
Wherein: the primary resolving liquid is a solution after one elution.
In some embodiments, a portion of the volume of the primary desorption solution can be concentrated according to the volume of the field device before extraction, so that the usage amount of the sodium chloride buffer solution can be reduced, and the subsequent production time can be saved.
In some embodiments, the macroporous resin adsorption on the extract liquid requires that the sample loading proportion is determined according to the composition condition of the actual feed liquid, and the proper proportion is helpful for improving the yield and the purity.
In some embodiments, step S5 specifically includes the following steps:
concentrating the eluate for the third time under reduced pressure, cooling to below 25 deg.C for cooling crystallization after concentration, vacuum filtering the crystallized liquid until no liquid flows out, washing the filter cake twice by adding anhydrous ether, and stopping washing when the color of the washing liquid is lower than Y1 #;
after the completion of the washing, the resultant was dried in a vacuum to obtain a bluish-violet crystalline powder.
In some embodiments, the first adsorption is performed by controlling the flow rate of the upper column to be 1 to 4 times of the resin amount L/H, the deionized water is used for washing the resin by controlling the washing flow rate to be 1 to 4 times of the resin amount L/H, and the first elution is performed by controlling the elution flow rate to be 0.5 to 2 times of the resin amount L/H; the flow rate of the industrial chromatographic flow is controlled to be 0.5 to 5 times of the resin amount L/H; the flow rate of the upper column is controlled to be 1 to 4 times of the resin amount L/H during the third adsorption, and the elution flow rate is controlled to be 0.5 to 2 times of the resin amount L/H during the third elution.
In certain embodiments, the mass ratio of the amount of MMC to ODS resin on a single column of an industrial chromatograph is 4: 1000 to 12: 1000.
in certain embodiments, the phosphate buffer is formed from KH2PO4、Na2HPO4And deionized water, and KH2PO4、Na2HPO4And the weight ratio of the deionized water is 1: 15: 5000。
In certain embodiments, the sodium chloride buffer is KH2PO4、Na2HPO4NaCl and deionized water, and KH2PO4、Na2HPO4And the weight ratio of NaCl to deionized water is 1: 15: 250:5000.
In some embodiments, the temperature of the water bath during the concentration under reduced pressure is controlled to be 40 to 50 ℃, and the concentration of the crystal liquid is controlled to be 10 to 20 g/L.
In certain embodiments, the drying temperature is controlled between 50 and 60 ℃ and the drying time is controlled between 8 and 10 hours.
Has the advantages that: the invention adopts a new method for producing mitomycin, and has more ideal impurity removal effect on primary analysis solution. Most of fat-soluble protein and organic pigment impurities in the feed liquid are removed through extraction, and then the residual water-soluble protein impurities and other fermentation metabolites with longer retention time are removed through the macroporous resin, so that the purity of the feed liquid subjected to industrial chromatographic separation originally is improved to more than 60% from 20-30%, the pollution of chromatographic fillers is reduced, the single sample loading amount of the industrial chromatogram is increased, the separation process of the industrial chromatogram is more stable and controllable, and the quality of the separated qualified section feed liquid is also improved.
Detailed Description
The present invention will be described in further detail below with reference to embodiments.
Example 1
A novel process for producing mitomycin comprising the steps of:
s1, mitomycin fermentation liquor pretreatment: transferring the mitomycin fermentation liquor to a pretreatment tank, stirring the fermentation liquor while adding perlite, adjusting the pH of the feed liquid to 7 by using a sodium hydroxide solution with the mass concentration of 1%,
wherein: the weight ratio of the mitomycin fermentation liquor to the perlite is 100: 2;
s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate;
s3, filtering: filtering the filtrate by using a bag filter so as to remove particle impurities in the filtrate;
s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain a third eluent, which specifically comprises the following steps:
carrying out primary adsorption on the filtrate obtained in the step S3 through macroporous resin, after the primary adsorption is finished, washing the resin with deionized water until the color of a washing liquid is lower than that of Y3#, carrying out air pressure blow-drying after the washing is finished, then introducing ethyl acetate for primary elution, adjusting the pH value to 7 by using a phosphoric acid buffer solution, collecting the primary eluent until the color is lower than that of Y5#, and stopping elution;
extracting the first eluent by a sodium chloride buffer solution to obtain an extract;
performing secondary adsorption on the extract liquor through macroporous resin, and performing secondary elution by using a methanol aqueous solution with the mass concentration of 5% to obtain secondary eluent;
purifying the secondary eluent by an industrial chromatographic column, washing with water, washing with methanol with the mass concentration of 10% and purifying with methanol with the mass concentration of 20% to obtain a feed liquid;
carrying out third adsorption on the feed liquid by macroporous resin, drying after the column loading is finished, and then carrying out third elution by using pure methanol to obtain third eluent;
wherein: controlling the flow rate of the upper column to be 1 time of the resin amount L/H during the first adsorption, controlling the washing flow rate to be 1 time of the resin amount L/H during the deionized water resin washing, and controlling the elution flow rate to be 0.5 time of the resin amount L/H during the first elution; the flow of the industrial chromatographic flow is controlled to be 0.5 time of the resin amount L/H;
the mass ratio of the MMC resin to the ODS resin amount of the single column of the industrial chromatogram is 4: 1000, parts by weight;
the phosphate buffer solution is prepared from KH2PO4、Na2HPO4And deionized water, and KH2PO4、Na2HPO4And the weight ratio of the deionized water is 1: 15: 5000;
the sodium chloride buffer solution is prepared from KH2PO4、Na2HPO4NaCl and deionized water, and KH2PO4、Na2HPO4And the weight ratio of NaCl to deionized water is 1: 15: 250: 5000;
s5, refining: concentrating, crystallizing, filtering, washing, drying and crushing the third eluent to obtain the mitomycin, which specifically comprises the following steps:
concentrating the eluate for the third time under reduced pressure, cooling to below 25 deg.C for cooling crystallization after concentration, vacuum filtering the crystallized liquid until no liquid flows out, washing the filter cake twice by adding anhydrous ether, and stopping washing when the color of the washing liquid is lower than Y1 #;
drying in vacuum after washing to obtain blue-violet crystalline powder;
wherein: controlling the flow rate of the upper column to be 1 time of the resin amount L/H during the third adsorption, and controlling the elution flow rate to be 0.5 time of the resin amount L/H during the third elution;
the temperature of the water bath is controlled at 40 ℃ during the decompression concentration, and the concentration of the crystallization liquid is controlled at 10 g/L;
the drying temperature is controlled at 50 ℃, and the drying time is controlled at 8 h.
Example 2
A novel process for producing mitomycin comprising the steps of:
s1, mitomycin fermentation liquor pretreatment: transferring the mitomycin fermentation liquor to a pretreatment tank, stirring the fermentation liquor while adding perlite, adjusting the pH of the feed liquid to 8 by using a sodium hydroxide solution with the mass concentration of 1%,
wherein: the weight ratio of the mitomycin fermentation liquor to the perlite is 100: 3;
s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate;
s3, filtering: filtering the filtrate by using a bag filter so as to remove particle impurities in the filtrate;
s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain a third eluent, which specifically comprises the following steps:
carrying out primary adsorption on the filtrate obtained in the step S3 through macroporous resin, after the primary adsorption is finished, washing the resin with deionized water until the color of a washing liquid is lower than that of Y3#, carrying out air pressure blow-drying after the washing is finished, then introducing ethyl acetate for primary elution, adjusting the pH value to 8 by using a phosphoric acid buffer solution, collecting the primary eluent until the color is lower than that of Y5#, and stopping elution;
extracting the first eluent by a sodium chloride buffer solution to obtain an extract;
performing secondary adsorption on the extract liquor through macroporous resin, and performing secondary elution by using a methanol aqueous solution with the mass concentration of 10% to obtain secondary eluent;
purifying the secondary eluent by an industrial chromatographic column, washing with water, washing with methanol with the mass concentration of 10% and purifying with methanol with the mass concentration of 22% to obtain a feed liquid;
carrying out third adsorption on the feed liquid by macroporous resin, drying after the column loading is finished, and then carrying out third elution by using pure methanol to obtain third eluent;
wherein: controlling the flow rate of the upper column to be 2 times of the resin amount L/H during first adsorption, controlling the water washing flow rate to be 2 times of the resin amount L/H during deionized water resin washing, and controlling the elution flow rate to be 1 time of the resin amount L/H during first elution; the flow of the industrial chromatographic flow is controlled to be 1 time of the resin amount L/H;
the mass ratio of the MMC resin to the ODS resin amount of the single column of the industrial chromatogram is 6: 1000, parts by weight;
the phosphate buffer solution is prepared from KH2PO4、Na2HPO4And deionized water, and KH2PO4、Na2HPO4And the weight ratio of the deionized water is 1: 15: 5000;
the sodium chloride buffer solution is prepared from KH2PO4、Na2HPO4NaCl and deionized water, and KH2PO4、Na2HPO4And the weight ratio of NaCl to deionized water is 1: 15: 250: 5000;
s5, refining: concentrating, crystallizing, filtering, washing, drying and crushing the third eluent to obtain the mitomycin, which specifically comprises the following steps:
concentrating the eluate for the third time under reduced pressure, cooling to below 25 deg.C for cooling crystallization after concentration, vacuum filtering the crystallized liquid until no liquid flows out, washing the filter cake twice by adding anhydrous ether, and stopping washing when the color of the washing liquid is lower than Y1 #;
drying in vacuum after washing to obtain blue-violet crystalline powder;
wherein: controlling the flow rate of the upper column to be 2 times of the resin amount L/H during the third adsorption, and controlling the elution flow rate to be 1 time of the resin amount L/H during the third elution;
the temperature of the water bath is controlled at 42 ℃ during the decompression concentration, and the concentration of the crystallization liquid is controlled at 12 g/L;
the drying temperature is controlled at 52 ℃, and the drying time is controlled at 9 h.
Example 3
A novel process for producing mitomycin comprising the steps of:
s1, mitomycin fermentation liquor pretreatment: transferring the mitomycin fermentation liquor to a pretreatment tank, stirring the fermentation liquor while adding perlite, adjusting the pH of the feed liquid to 8 by using a sodium hydroxide solution with the mass concentration of 1%,
wherein: the weight ratio of the mitomycin fermentation liquor to the perlite is 100: 3;
s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate;
s3, filtering: filtering the filtrate by using a bag filter so as to remove particle impurities in the filtrate;
s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain a third eluent, which specifically comprises the following steps:
carrying out primary adsorption on the filtrate obtained in the step S3 through macroporous resin, after the primary adsorption is finished, washing the resin with deionized water until the color of a washing liquid is lower than that of Y3#, carrying out air pressure blow-drying after the washing is finished, then introducing ethyl acetate for primary elution, adjusting the pH value to 7 by using a phosphoric acid buffer solution, collecting the primary eluent until the color is lower than that of Y5#, and stopping elution;
extracting the first eluent by a sodium chloride buffer solution to obtain an extract;
performing secondary adsorption on the extract liquor through macroporous resin, and performing secondary elution by using a methanol aqueous solution with the mass concentration of 20% to obtain secondary eluent;
purifying the secondary eluent by an industrial chromatographic column, washing with water, washing with methanol with the mass concentration of 10% and purifying with methanol with the mass concentration of 24% to obtain a feed liquid;
carrying out third adsorption on the feed liquid by macroporous resin, drying after the column loading is finished, and then carrying out third elution by using pure methanol to obtain third eluent;
wherein: controlling the flow rate of the upper column to be 3 times of the resin amount L/H during first adsorption, controlling the washing flow rate to be 3 times of the resin amount L/H during deionized water resin washing, and controlling the elution flow rate to be 1.5 times of the resin amount L/H during first elution; the flow of the industrial chromatographic flow is controlled to be 4 times of the resin amount L/H;
the mass ratio of the MMC resin to the ODS resin amount of the single column of the industrial chromatogram is 10: 1000, parts by weight;
the phosphate buffer solution is prepared from KH2PO4、Na2HPO4And deionized water, and KH2PO4、Na2HPO4And the weight ratio of the deionized water is 1: 15: 5000;
the sodium chloride buffer solution is prepared from KH2PO4、Na2HPO4NaCl and deionized water, and KH2PO4、Na2HPO4And the weight ratio of NaCl to deionized water is 1: 15: 250: 5000;
s5, refining: concentrating, crystallizing, filtering, washing, drying and crushing the third eluent to obtain the mitomycin, which specifically comprises the following steps:
concentrating the eluate for the third time under reduced pressure, cooling to below 25 deg.C for cooling crystallization after concentration, vacuum filtering the crystallized liquid until no liquid flows out, washing the filter cake twice by adding anhydrous ether, and stopping washing when the color of the washing liquid is lower than Y1 #;
drying in vacuum after washing to obtain blue-violet crystalline powder;
wherein: controlling the flow rate of the upper column to be 3 times of the resin amount L/H during the third adsorption, and controlling the elution flow rate to be 1.5 times of the resin amount L/H during the third elution;
the temperature of the water bath is controlled at 48 ℃ during the decompression concentration, and the concentration of the crystallization liquid is controlled at 18 g/L;
the drying temperature is controlled at 58 ℃ and the drying time is controlled at 9 h.
Example 4
A novel process for producing mitomycin comprising the steps of:
s1, mitomycin fermentation liquor pretreatment: transferring the mitomycin fermentation liquor to a pretreatment tank, stirring the fermentation liquor while adding perlite, adjusting the pH of the feed liquid to 9 by using a sodium hydroxide solution with the mass concentration of 1%,
wherein: the weight ratio of the mitomycin fermentation liquor to the perlite is 100: 4;
s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate;
s3, filtering: filtering the filtrate by using a bag filter so as to remove particle impurities in the filtrate;
s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain a third eluent, which specifically comprises the following steps:
carrying out primary adsorption on the filtrate obtained in the step S3 through macroporous resin, after the primary adsorption is finished, washing the resin with deionized water until the color of a washing liquid is lower than that of Y3#, carrying out air pressure blow-drying after the washing is finished, then introducing ethyl acetate for primary elution, adjusting the pH value to 8 by using a phosphoric acid buffer solution, collecting the primary eluent until the color is lower than that of Y5#, and stopping elution;
extracting the first eluent by a sodium chloride buffer solution to obtain an extract;
performing secondary adsorption on the extract liquor through macroporous resin, and performing secondary elution by using a methanol aqueous solution with the mass concentration of 30% to obtain secondary eluent;
purifying the secondary eluent by an industrial chromatographic column, washing with water, washing with methanol with the mass concentration of 10% and purifying with methanol with the mass concentration of 25% to obtain a feed liquid;
carrying out third adsorption on the feed liquid by macroporous resin, drying after the column loading is finished, and then carrying out third elution by using pure methanol to obtain third eluent;
wherein: controlling the flow rate of the upper column to be 4 times of the resin amount L/H during first adsorption, controlling the washing flow rate to be 4 times of the resin amount L/H during deionized water resin washing, and controlling the elution flow rate to be 2 times of the resin amount L/H during first elution; the flow of the industrial chromatographic flow is controlled to be 5 times of the resin amount L/H;
the mass ratio of the MMC resin to the ODS resin amount of the single column of the industrial chromatogram is 12: 1000, parts by weight;
the phosphate buffer solution is prepared from KH2PO4、Na2HPO4And deionized water, and KH2PO4、Na2HPO4And the weight ratio of the deionized water is 1: 15: 5000;
the sodium chloride buffer solution is prepared from KH2PO4、Na2HPO4NaCl and deionized water, and KH2PO4、Na2HPO4And the weight ratio of NaCl to deionized water is 1: 15: 250: 5000;
s5, refining: concentrating, crystallizing, filtering, washing, drying and crushing the third eluent to obtain the mitomycin, which specifically comprises the following steps:
concentrating the eluate for the third time under reduced pressure, cooling to below 25 deg.C for cooling crystallization after concentration, vacuum filtering the crystallized liquid until no liquid flows out, washing the filter cake twice by adding anhydrous ether, and stopping washing when the color of the washing liquid is lower than Y1 #;
drying in vacuum after washing to obtain blue-violet crystalline powder;
wherein: controlling the flow rate of the upper column to be 4 times of the resin amount L/H during the third adsorption, and controlling the elution flow rate to be 2 times of the resin amount L/H during the third elution;
the temperature of the water bath is controlled at 50 ℃ during the decompression concentration, and the concentration of the crystallization liquid is controlled at 20 g/L;
the drying temperature is controlled at 60 ℃, and the drying time is controlled at 10 h.
In summary, the following steps: the invention optimizes the extraction and purification production process of MMC on the basis of the prior art, adds partial separation and purification process steps, and ensures that the whole separation and purification process is more stable and controllable.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these should be considered as within the scope of the present invention.
Claims (10)
1. A novel method for producing mitomycin, comprising the steps of:
s1, mitomycin fermentation liquor pretreatment: transferring the mitomycin fermentation liquor to a pretreatment tank, stirring the fermentation liquor, adding perlite, and adjusting the pH of the feed liquid by using a sodium hydroxide solution with the mass concentration of 1%;
s2, plate and frame filtration: pressing the feed liquid into a polypropylene filter cloth in a plate frame for filtering, and collecting filtrate;
s3, filtering: filtering the filtrate by using a bag filter so as to remove particle impurities in the filtrate;
s4, extraction step: carrying out first adsorption and first elution on the filtrate obtained in the step S3 through macroporous resin to obtain a first eluent; extracting the first eluent to obtain extract liquor; carrying out second adsorption and second elution on the extract liquor through macroporous resin to obtain a second eluent; purifying the second eluent by industrial chromatography to obtain feed liquid; carrying out third adsorption and third elution on the feed liquid through macroporous resin to obtain third eluent;
s5, refining: and concentrating, crystallizing, filtering, washing, drying and crushing the third eluent to obtain the mitomycin.
2. The novel method for producing mitomycin according to claim 1, wherein in step S1, the weight ratio of the mitomycin broth to perlite is from 100:2 to 100: 4.
3. The novel process for producing mitomycin according to claim 1, wherein in step S1 the pH of the feed solution is adjusted to the range of 7 to 9.
4. The novel method for producing mitomycin according to claim 1, characterized in that step S4 comprises the following steps:
carrying out primary adsorption on the filtrate obtained in the step S3 through macroporous resin, after the primary adsorption is finished, washing the resin with deionized water until the color of a washing liquid is lower than that of Y3#, blowing the washing liquid to dry the washing liquid under air pressure, introducing ethyl acetate to carry out primary elution, adjusting the pH value to 7-8 by using a phosphoric acid buffer solution, collecting primary eluent until the color is lower than that of Y5#, and stopping elution;
extracting the first eluent by a sodium chloride buffer solution to obtain an extract;
performing secondary adsorption on the extract liquor through macroporous resin, and performing secondary elution by using a methanol aqueous solution with the mass concentration of 5-30% to obtain secondary eluent;
purifying the secondary eluent by an industrial chromatographic column, washing with water, washing with methanol with the mass concentration of 10% and purifying with methanol with the mass concentration of 20-25% to obtain a feed liquid;
and (3) carrying out third adsorption on the feed liquid through macroporous resin, drying after the column loading is finished, and then carrying out third elution by using pure methanol to obtain third eluent.
5. The novel method for producing mitomycin according to claim 1, characterized in that step S5 comprises the following steps:
concentrating the eluate for the third time under reduced pressure, cooling to below 25 deg.C for cooling crystallization after concentration, vacuum filtering the crystallized liquid until no liquid flows out, washing the filter cake twice by adding anhydrous ether, and stopping washing when the color of the washing liquid is lower than Y1 #;
after the completion of the washing, the resultant was dried in a vacuum to obtain a bluish-violet crystalline powder.
6. The novel process for producing mitomycin according to claim 4, wherein the first adsorption is carried out by controlling the flow rate through the column at 1 to 4 times the amount of resin L/H, the deionized water is used to wash the resin at 1 to 4 times the amount of resin L/H, and the first elution is carried out by controlling the flow rate at 0.5 to 2 times the amount of resin L/H; the flow rate of the industrial chromatographic flow is controlled to be 0.5 to 5 times of the resin amount L/H; the flow rate of the upper column is controlled to be 1 to 4 times of the resin amount L/H during the third adsorption, and the elution flow rate is controlled to be 0.5 to 2 times of the resin amount L/H during the third elution.
7. The process for producing mitomycin according to claim 4, wherein the phosphate buffer consists of KH2PO4、Na2HPO4And deionized water, and KH2PO4、Na2HPO4And the weight ratio of the deionized water is 1: 15: 5000.
8. the process for producing mitomycin according to claim 4, wherein the sodium chloride buffer is KH buffer2PO4、Na2HPO4NaCl and deionized water, and KH2PO4、Na2HPO4And the weight ratio of NaCl to deionized water is 1: 15: 250:5000.
9. The novel process for producing mitomycin according to claim 5, wherein the temperature of the water bath during concentration under reduced pressure is controlled to 40 to 50 ℃ and the concentration of the crystal is controlled to 10 to 20 g/L.
10. The novel process for producing mitomycin according to claim 5, wherein the drying temperature is controlled to 50 to 60 ℃ and the drying time is controlled to 8 to 10 hours.
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| CN115785106A (en) * | 2022-12-09 | 2023-03-14 | 无锡福祈制药有限公司 | Novel method for increasing MMC extraction yield |
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