Method for purifying mitomycin C
By adding surfactants and guanidine hydrochloride to the fermentation broth, and combining a multi-step purification method with macroporous adsorption resin and C18 adsorption column, the problem of reduced mitomycin C content caused by streptomycin enzyme degradation was solved, and high-purity and high-yield mitomycin C preparation was achieved, which is suitable for industrial production.
Patent Information
- Application Number
- CN202410785307.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-18
- Publication Date
- 2025-12-19
AI Technical Summary
In the current technology for purifying mitomycin C, the enzymes secreted by Streptomyces cause a decrease in the content of mitomycin C in the fermentation broth, which affects the purification efficiency and yield.
Surfactants such as sodium dodecyl sulfate and guanidine hydrochloride were added to the fermentation broth to adjust the pH. Solid-liquid separation was then performed, followed by chromatographic separation using macroporous adsorption resin and a C18 adsorption column. Multi-step purification was carried out using different proportions of solvent and eluent, and finally, high-purity mitomycin C was obtained by crystallization.
High-purity and high-yield mitomycin C preparation has been achieved, making it suitable for large-scale industrial production.
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Figure BDA0004898449980000031
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of fermentation broth purification, in particular to a method for purifying mitomycin C. BACKGROUND
[0002] Mitomycin C is a broad-spectrum anti-tumor antibiotic extracted from the culture solution of Streptomyces verticillus, which has anti-cancer effect on various cancers. Its principle of action is to make the DNA of cells depolymerize and hinder the replication of DNA, thereby inhibiting the division of tumor cells.
[0003] In industry, mitomycin C is generally obtained by fermentation of Streptomyces. In order to improve the recovery rate of mitomycin C in the fermentation broth as much as possible, there are various methods in the prior art. 1) For example, activated carbon adsorption method is used by adding activated carbon to the culture filtrate obtained by separating the bacterial cells and adsorbing, and then eluting with an organic solvent. Or, the concentrated solution of mitomycin C obtained by dissolving the culture filtrate in an organic solvent is subjected to purification by alumina chromatography or countercurrent distribution method, and then the eluted mitomycin C solution is concentrated until dryness, a small amount of acetone is added, and thus the crystal is obtained. 2) The culture solution is adsorbed on a reversed-phase adsorption resin, and then eluted with acetone, methanol or ethanol, etc. The eluate is concentrated and the solvent is removed, saturated with salt, and then dissolved in chloroform. The chloroform extract is subjected to separation and elution by alumina column chromatography. The eluate is concentrated and made into a thick methanol solution, and then ether, petroleum ether, gasoline or crude oil is added to obtain pure crystals of mitomycin C. The mother liquor is further concentrated and the same operation is performed, and the obtained crude crystals are washed with a 10% methanol and ether mixture, and thus pure crystals of mitomycin C are obtained. 3) The culture solution is adsorbed on a reversed-phase adsorption resin, and then eluted with ethyl acetate. The eluate is distilled and the solvent is removed, and then the residue is adsorbed on a reversed-phase adsorption agent with small particle size, and then eluted with aqueous methanol. The eluate is adsorbed on a reversed-phase adsorption resin, and then eluted with methanol to wash mitomycin C from the resin. The eluate is concentrated, and mitomycin C is crystallized.
[0004] However, in the purification process, the enzyme that degrades mitomycin C is secreted from Streptomyces itself, so the content of mitomycin C in the fermentation broth is always reduced before the enzyme is inactivated or removed. SUMMARY
[0005] In order to overcome the above problems, the present application provides a method for purifying mitomycin C, comprising the following steps:
[0006] Step 1: adding a surfactant or / and guanidine hydrochloride to the fermentation broth, and then solid-liquid separation;
[0007] Step 2: separating by a chromatographic column to obtain a solution containing mitomycin C;
[0008] Step 3: obtaining mitomycin C solid from the mitomycin C solution.
[0009] Preferably, step 2 further comprises step 2-1 and step 2-2.
[0010] Step 2-1: solid-liquid separation to obtain supernatant, adsorption using macroporous adsorption resin, elution using eluent 1 to obtain elution product 1, removal of solvent from the elution product 1, preparation of column feed liquid, adsorption using a silica gel column, and elution using eluent 2 to obtain elution product 2.
[0011] Step 2-2: dissolution of the elution product 2 after removal of the solvent, adsorption using a C18 column, and elution using eluent 3 to obtain a solution containing mitomycin C.
[0012] Preferably, the eluent 1 is selected from acetone, ethanol, and methanol.
[0013] Preferably, the macroporous adsorption resin is T81 macroporous adsorption resin.
[0014] Preferably, the eluent 2 is a mixture of methanol and ethyl acetate.
[0015] Preferably, the eluent 2 is added in two times, and is recorded as eluent 2-1 and eluent 2-2 according to the order of addition, the proportion of methanol in the eluent 2-1 is 5% to 15% of the total volume, the proportion of methanol in the eluent 2-2 is 10% to 25% of the total volume, and the proportion of methanol in the eluent 2-2 is higher than that in the eluent 2-1.
[0016] Preferably, the eluent 2 is added in two times, and is recorded as eluent 2-1 and eluent 2-2 according to the order of addition, the proportion of methanol in the eluent 2-1 is 5% to 15% of the total volume, the proportion of methanol in the eluent 2-2 is 10% to 22% of the total volume, and the proportion of methanol in the eluent 2-2 is higher than that in the eluent 2-1.
[0017] Preferably, the eluent 2 is added in two times, and is recorded as eluent 2-1 and eluent 2-2 according to the order of addition, and is added in one of the following ways: the proportion of methanol in the eluent 2-1 is 5% of the total volume, and the proportion of methanol in the eluent 2-2 is 10% of the total volume; or, the proportion of methanol in the eluent 2-1 is 10% of the total volume, and the proportion of methanol in the eluent 2-2 is 20% of the total volume; or, the proportion of methanol in the eluent 2-1 is 12% of the total volume, and the proportion of methanol in the eluent 2-2 is 22% of the total volume; or, the proportion of methanol in the eluent 2-1 is 10% of the total volume, and the proportion of methanol in the eluent 2-2 is 15% of the total volume.
[0018] Preferably, the amounts of the eluent 2-1 and the eluent 2-2 are the same.
[0019] Preferably, the eluent 2-1 and the eluent 2-2 are eluted at a speed of 2 BV / h.
[0020] Preferably, the solvent in the elution product 1 is removed and ethanol is added to the elution product 1 to form the column loading solution.
[0021] Preferably, the solvent in the elution product 1 is removed and ethanol is added to the elution product 1 to form the column loading solution, and the concentration of the column loading solution is at least 200000 μg / ml.
[0022] Preferably, step 2-2; the solvent in the elution product 2 is removed and the elution product 2 is dissolved in solvent, and the solution is adsorbed by C18, and then eluted by elution solution 3 to obtain a solution containing mitomycin C.
[0023] Preferably, the flow rate of the high-pressure elution is 20-100 ml / min.
[0024] Preferably, the flow rate of the high-pressure elution is 20 ml / min or 30 ml / min or 40 ml / min or 50 ml / min or 100 ml / min.
[0025] Preferably, the elution solution 3 is a methanol solution.
[0026] Preferably, the solvent in the elution product 1 is removed and ethanol is added to the elution product 1 to form the column loading solution.
[0027] Preferably, the elution solution 3 is a methanol solution, and the concentration of methanol is not higher than 30% or not higher than 25% or not higher than 20% by volume.
[0028] Preferably, the surfactant is sodium dodecyl sulfate.
[0029] Preferably, the surfactant is 0.1% (w / v) sodium dodecyl sulfate.
[0030] Preferably, after the surfactant and / or guanidine hydrochloride are added to the fermentation broth, the pH is adjusted to 7-9, and then solid-liquid separation is performed.
[0031] Preferably, after the surfactant and / or guanidine hydrochloride are added to the fermentation broth, the pH is adjusted to 7-8.5, and then solid-liquid separation is performed.
[0032] Preferably, in step 1, the temperature for treating the fermentation broth is not higher than room temperature.
[0033] Preferably, in step 1, the temperature for treating the fermentation broth is not higher than 10°C or not higher than 9°C or not higher than 8°C or not higher than 7°C or not higher than 6°C.
[0034] Preferably, in step 3, after the concentrated dry mitomycin C solution is obtained, methanol is added to dissolve the solution, and crystallization is performed to obtain mitomycin C solid.
[0035] Preferably, in step 3, after the concentrated dry mitomycin C solution is obtained, pure methanol is added to dissolve the solution, and crystallization is performed to obtain mitomycin C solid.
[0036] The method provided by the present application can obtain high-purity mitomycin C with high yield, and is suitable for large-scale industrial production. DETAILED DESCRIPTION
[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified.
[0038] The materials and reagents used in the following examples are ordinary commercially available products and can be purchased in the market unless otherwise specified.
[0039] The present application will be further described by the following examples, which are not intended to limit the scope of the present application. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present application still fall within the scope of the present application.
[0040] Example 1
[0041] The stabilizer was added to the fermentation broth of Streptomyces, and the mixture was stirred at 4-8°C in the dark for 0-8h. The sample was taken every 1h, and the titer was determined.
[0042] The results are shown in the following table.
[0043]
[0044] In the above method, the degradation of mitomycin C was effectively inhibited after the addition of sodium dodecyl sulfate and guanidine hydrochloride to the fermentation broth for 1h.
[0045] In addition, when the concentration of sodium dodecyl sulfate was 0.01%(m / v) and 0.5%(m / v), respectively, the titer of the supernatant was 37μg / ml and 41μg / ml, respectively, after centrifugation for 8h.
[0046] Example 2
[0047] In step (1), 0.1% sodium dodecyl sulfate was added to the 35L fermentation broth before discharge, and the pH was adjusted to about 7. The mixture was centrifuged at 10000rpm and 4°C to collect 30L supernatant with a titer of 48μg / ml.
[0048] In step (2), the supernatant was adsorbed by T81 macroporous adsorption resin, and then eluted with ethanol to collect the component-containing eluate.
[0049] In step (3), the eluate was concentrated and dried, then dissolved in ethanol to a concentration of at least 200000μg / ml, and then purified by silica gel column. The component-containing eluate was collected by elution with 5:95(methanol: ethyl acetate) 2BV, 10:90(methanol: ethyl acetate) 2BV, respectively, at a rate of 2BV / h under normal pressure. The eluate was concentrated and dried under vacuum at -0.08MPa and 30°C to obtain the concentrate.
[0050] Step (4), the concentrated solution was dissolved in ethanol to a concentration of no less than 200000 μg / ml, and the solution was filtered through a 0.45 μm filter to obtain a preparation column liquid. The preparation column liquid was subjected to C18 high pressure preparation. The high pressure preparation eluent was 15% methanol aqueous solution, the flow rate was 20 ml / min, and the intermediate product with a purity of more than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0051] Step (5), the 95% purity intermediate product was combined, and the solvent was concentrated under vacuum at -0.08 MPa and 30°C. Pure methanol was added to the concentrated solution at a volume to weight ratio of 2:1 (ml:g) of the material before crystallization, and the solution was completely dissolved and crystallized at 2°C overnight. The crystals were filtered and dried at -0.08 MPa and 30°C under vacuum to obtain the finished product 0.61 g with a purity of more than 99% and a yield of 42.36%.
[0052] Example 3:
[0053] Step (1), 0.1% sodium dodecyl sulfate was added before the fermentation broth was discharged from a 36L fermenter, and the pH was adjusted to about 8. The broth was subjected to tube centrifugation at 13000 rpm and 8°C, and the supernatant 32L was collected with a titer of 45 μg / ml.
[0054] Step (2), the centrifuged supernatant was adsorbed by T81 macroporous adsorption resin and then eluted with acetone to collect the component-containing eluent.
[0055] Step (3), the eluent was concentrated and dried, and then dissolved in ethanol to a concentration of at least 200000 μg / ml. The solution was subjected to purification on a silica gel column, eluted with 15:85 (methanol: ethyl acetate) 2BV and 22:78 (methanol: ethyl acetate) 2BV in sequence at a flow rate of 2BV / h under normal pressure, and the component-containing eluent was collected. The eluent was concentrated and dried under vacuum at -0.07 MPa and 35°C to obtain a concentrated solution.
[0056] Step (4), the concentrated solution was dissolved in ethanol to a concentration of no less than 200000 μg / ml, and the solution was filtered through a 0.45 μm filter to obtain a preparation column liquid. The preparation column liquid was subjected to C 18 high pressure preparation, the high pressure preparation eluent was 20% methanol aqueous solution, the flow rate was 40 ml / min, and the intermediate product with a purity of more than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0057] Step (5), the 95% purity intermediate product was combined, and the solvent was concentrated under vacuum at -0.09 MPa and 35°C. Pure methanol was added to the concentrated solution at a volume to weight ratio of 10:1 (ml:g) of the material before crystallization, and the solution was completely dissolved and crystallized at 4°C overnight. The crystals were filtered and dried at -0.07 MPa and 30°C under vacuum to obtain the finished product 0.64 g with a purity of more than 99% and a yield of 44.4%.
[0058] Example 4:
[0059] Step (1), before the 33L fermentation broth was discharged, 0.1% sodium dodecyl sulfate was added, and the pH was adjusted to about 7.5, and the supernatant was collected by tube centrifugation at 11000 rpm and 6°C, 29L, with a titer of 41 μg / ml.
[0060] Step (2), the supernatant was adsorbed by T81 macroporous adsorption resin, and then eluted with methanol, and the component-containing eluate was collected.
[0061] Step (3), after the eluate was concentrated and dried, ethanol was added to dissolve at least 200000 μg / ml, and the silica gel column was purified, and the component-containing eluate was collected by eluting with 10:90 (methanol: ethyl acetate) 2BV, 20:80 (methanol: ethyl acetate) 2BV, respectively, at a rate of 2BV / h under normal pressure, and the eluate was concentrated and dried under vacuum at -0.07 MPa and 20°C to obtain a concentrate.
[0062] Step (4), a suitable amount of ethanol was added to the concentrate to dissolve to not less than 200000 μg / ml, and after the solution was clear, it was filtered through a 0.45 μm filter membrane to obtain a preparation column liquid, which was loaded into a C 18 High pressure preparation, the high pressure preparation eluent was 20% methanol aqueous solution, the flow rate was 50 ml / min, and the intermediate product with a purity of more than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0063] Step (5), the 95% pure intermediate product was combined, and the solvent was concentrated and dried under vacuum at not less than -0.07 MPa and 20°C, and then pure methanol was added, with a volume: weight of the material before crystallization = 3:1 (ml:g), until completely dissolved, and then stirred overnight at 2°C, filtered, and the crystals were dried under vacuum at not less than -0.07 MPa and 30°C to obtain the finished product 0.50 g with a purity of more than 99% and a yield of 42.1%.
[0064] Example 5:
[0065] Step (1), before the 40L fermentation broth was discharged, 0.1% sodium dodecyl sulfate was added, and the pH was adjusted to about 8.5, and the supernatant was collected by tube centrifugation at 12000 rpm and 6°C, 35L, with a titer of 39 μg / ml.
[0066] Step (2), the supernatant was adsorbed by T81 macroporous adsorption resin, and then eluted with ethanol, and the component-containing eluate was collected.
[0067] Step (3), the concentrated dry eluent was dissolved in ethanol to at least 200000 μg / ml, and purified by silica gel column, eluted with 12:88 (methanol: ethyl acetate) 2BV, 22:78 (methanol: ethyl acetate) 2BV, respectively, at 2BV / h under normal pressure, and the eluent containing the components was collected. The eluent was concentrated dry solvent under vacuum at -0.09 MPa and 30°C to obtain a concentrate.
[0068] Step (4), the concentrate was dissolved in an appropriate amount of ethanol to not less than 200000 μg / ml, and filtered through a 0.45 μm filter after dissolving to obtain a preparation column liquid. The preparation column liquid was loaded onto a C 18 High pressure preparation, the high pressure preparation eluent was 20% methanol aqueous solution, the flow rate was 50 ml / min, and the intermediate product with a purity greater than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0069] Step (5), the 95% pure intermediate product was combined and concentrated dry solvent under vacuum at not less than -0.09 MPa and 30°C. Pure methanol was added to the concentrate at a volume: weight of the material before crystallization = 3:1 (ml:g) until complete dissolution. The mixture was stirred at 2°C overnight, filtered, and the crystals were dried under vacuum at not less than -0.09 MPa and 30°C to obtain the finished product 0.55 g with a purity greater than 99% and a yield of 40.29%.
[0070] Example 6:
[0071] Step (1), 0.1% sodium dodecyl sulfate was added before the 32L fermentation liquid was discharged, and the pH was adjusted to about 8. Tube centrifugation was performed at 13000 rpm and 6°C, and the supernatant 27L was collected with a titer of 47 μg / ml.
[0072] Step (2), the centrifuged supernatant was adsorbed by T81 macroporous adsorption resin and then eluted with pure acetone to collect the eluent containing the components.
[0073] Step (3), the concentrated dry eluent was dissolved in ethanol to at least 200000 μg / ml, and purified by silica gel column, eluted with 12:88 (methanol: ethyl acetate) 2BV, 22:78 (methanol: ethyl acetate) 2BV, respectively, at 2BV / h under normal pressure, and the eluent containing the components was collected. The eluent was concentrated dry solvent under vacuum at -0.09 MPa and 30°C to obtain a concentrate.
[0074] Step (4), the concentrate was dissolved in an appropriate amount of ethanol to not less than 200000 μg / ml, and filtered through a 0.45 μm filter after dissolving to obtain a preparation column liquid. The preparation column liquid was loaded onto a C 18 High pressure preparation, the high pressure preparation eluent was 20% methanol aqueous solution, the flow rate was 50 ml / min, and the intermediate product with a purity greater than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0075] Step (5), 95% purity intermediate product was combined, and the solvent was concentrated under vacuum at not less than -0.09 MPa and 30°C. Pure methanol was added to the concentrated solvent in a volume: weight of the material before crystallization = 3:1 (ml: g) until complete dissolution. The mixture was stirred overnight at 2°C, filtered, and the crystals were dried in a vacuum at not less than -0.09 MPa and 30°C to obtain the finished product 0.54 g with a purity greater than 99% and a yield of 42.55%.
[0076] Comparative Example 1:
[0077] Step (1), 0.1% sodium dodecyl sulfate was added before the 36 L fermentation broth was discharged, and the pH was adjusted to about 8. Tubular centrifugation was performed at 12000 rpm at room temperature, and the supernatant 31 L was collected with a titer of 49 μg / ml.
[0078] Step (2), the centrifuged supernatant was adsorbed by T81 macroporous adsorption resin and then eluted with pure acetone. The component-containing eluate was collected.
[0079] Step (3), after the eluate was concentrated and dried, ethanol was added to dissolve to at least 200000 μg / ml. Purification was performed on a silica gel column, and elution was performed at 2 BV / h under normal pressure with 10:90 (methanol: ethyl acetate) 2 BV and 20:80 (methanol: ethyl acetate) 2 BV, respectively. The component-containing eluate was collected, and the eluate was concentrated under vacuum at -0.08 MPa and 25°C to obtain a concentrate.
[0080] Step (4), the concentrate was dissolved in an appropriate amount of ethanol to not less than 200000 μg / ml. After clarification, the solution was filtered through a 0.45 μm filter membrane to obtain a preparation column loading solution. The C18 high-pressure preparation was performed, and the high-pressure preparation eluate was 20% methanol aqueous solution with a flow rate of 50 ml / min. The intermediate product with a purity greater than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0081] Step (5), 95% purity intermediate product was combined, and the solvent was concentrated under vacuum at not less than -0.08 MPa and 25°C. Pure methanol was added to the concentrated solvent in a volume: weight of the material before crystallization = 8:1 (ml: g) until complete dissolution. The mixture was stirred overnight at 4°C,
[0082] filtered, and the crystals were dried in a vacuum at not less than -0.08 MPa and 30°C to obtain the finished product 0.26 g with a purity greater than 99% and a yield of 17.12%.
[0083] It can be known from the comparison between this comparative example and the previous examples that the temperature of the fermentation broth treatment (such as the temperature during solid-liquid separation) has an effect on the yield. A too high temperature can reduce the yield of the product.
[0084] Comparative Example 2:
[0085] Step (1), 0.1% other denaturant (sodium dodecyl sulfate) was added before the 41 L fermentation broth was discharged, the pH was adjusted to about 8, and the supernatant was collected by tube centrifugation at 12000 rpm and 4°C, 36 L, and the titer was 49 μg / ml.
[0086] Step (2), the centrifugal supernatant was adsorbed by T81 macroporous adsorption resin and then resolved with pure acetone, and the component-containing eluate was collected.
[0087] Step (3), after the resolution liquid was concentrated and dried, ethanol was added to dissolve to at least 200000 μg / ml, and the silica gel column was purified, and the component-containing eluate was collected by elution with 15:85 (methanol: ethyl acetate) 2BV, 20:80 (methanol: ethyl acetate) 2BV, and 2BV / h under normal pressure, and the eluate was concentrated and dried under vacuum-0.08 MPa and 25°C to obtain a concentrate.
[0088] Step (4), a proper amount of ethanol was added to the concentrate to dissolve to not less than 200000 μg / ml, and after the solution was clarified, it was filtered through a 0.45 μm filter membrane to obtain a preparation column liquid, which was loaded onto a C 18 High pressure preparation, the high pressure preparation eluent was 20% methanol aqueous solution, the flow rate was 50 ml / min, and the intermediate product with a purity of more than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0089] Step (5), the 95% pure intermediate product was combined, and the solvent was concentrated and dried under vacuum not less than-0.08 MPa and 25°C, pure methanol was added, the volume: the weight of the material before crystallization = 8:1 (ml: g), and the solution was stirred at 4°C overnight,
[0090] Filtration, the crystal was dried under vacuum not less than-0.08 MPa and 30°C to obtain the finished product 0.3 g, the purity of the finished product was more than 99%, and the yield was 19.84%.
[0091] By comparing the examples and the previous examples, it can be seen that different alkane chain lengths of the same type of denaturant can also affect the yield of the product.
[0092] Comparative Example 3:
[0093] Step (1), 0.1% sodium dodecyl sulfate was added before the 41 L fermentation broth was discharged, the pH was adjusted to about 8, and the supernatant was collected by tube centrifugation at 12000 rpm and 4°C, 36 L, and the titer was 49 μg / ml.
[0094] Step (2), the centrifugal supernatant was adsorbed by T81 macroporous adsorption resin and then resolved with pure acetone, and the component-containing eluate was collected.
[0095] Step (3), after the concentrated dry of the eluate, add ethanol to at least 200000 μg / ml, and purify by silica gel column, elute successively with 5:95 (methanol: ethyl acetate) 2BV, 15:85 (methanol: ethyl acetate) 2BV, at 2BV / h under normal pressure, collect the eluate containing the component, and concentrate the solvent of the eluate under vacuum-0.08 MPa, 25°C to obtain a concentrate.
[0096] Step (4), add an appropriate amount of ethanol to the concentrate to not less than 200000 μg / ml, filter through a 0.45 μm filter membrane after dissolving, and obtain a preparation column liquid, and purify by C18 high pressure preparation, elute the high pressure preparation eluate with 20% methanol aqueous solution at a flow rate of 50 ml / min, and collect the intermediate product with a purity of more than 95% at a wavelength of 290 nm of an ultraviolet spectrophotometer detector.
[0097] Step (5), combine the 95% pure intermediate product, concentrate the solvent under vacuum not less than-0.08 MPa, 25°C, add pure ethanol to the volume: weight of the material before crystallization = 2:1 (ml: g), until completely dissolved, stir overnight at 4°C, filter, and dry the crystals under vacuum not less than-0.08 MPa, 30°C to obtain the finished product 0.7 g, with a purity of 97%-98% and a yield of 40%.
[0098] It can be known by comparing the example and the previous examples that when the crystallization solvent is ethanol, the crystallization effect is poor, which affects the purity of the final product.
[0099] Comparative Example 4:
[0100] Step (1), add 0.1% sodium dodecyl sulfate to 38 L of fermentation liquid before tanking, adjust the pH to about 8, and centrifuge by a tubular centrifuge at 12000 rpm and 4°C to collect 35 L of supernatant with a titer of 38 μg / ml.
[0101] Step (2), adsorb the centrifuged supernatant by T81 macroporous adsorption resin, and elute with pure acetone to collect the eluate containing the component.
[0102] Step (3), after the concentrated dry of the eluate, add ethanol to at least 200000 μg / ml, and purify by silica gel column, elute successively with 5:95 (methanol: ethyl acetate) 2BV, 15:85 (methanol: ethyl acetate) 2BV, at 2BV / h under normal pressure, collect the eluate containing the component, and concentrate the solvent of the eluate under vacuum-0.08 MPa, 25°C to obtain a concentrate.
[0103] Step (4), the concentrate was dissolved in proper amount of ethanol to a concentration of no less than 200000 μg / ml, and the solution was filtered through a 0.45 μm filter to obtain a preparation column liquid. The preparation column liquid was subjected to high pressure preparation on a C18 column, and the high pressure preparation eluent was 30% methanol aqueous solution at a flow rate of 50 ml / min. The intermediate product with a purity of more than 95% was collected at a wavelength of 290 nm under ultraviolet spectrophotometric detection.
[0104] Step (5), the 95% purity intermediate product was combined, and the solvent was concentrated under vacuum at a pressure of no less than -0.08 MPa and a temperature of 25°C. Pure methanol was added to the concentrated product at a volume to weight ratio of 8:1 (ml:g) of the crystallized product, and the mixture was stirred at 4°C overnight until the product was completely dissolved. The product was filtered, and the crystals were dried under vacuum at a pressure of no less than -0.08 MPa and a temperature of 30°C to obtain the finished product 0.54 g with a purity of 97%-98% and a yield of 40.6%.
[0105] It can be known from the comparison between the present example and the previous examples that when the elution concentration of chromatography is too high, the separation of some impurities is not conducive, and the purity of the final product is low.
Claims
1. A method for purifying mitomycin C, characterized in that: Includes the following steps: Step 1: After adding surfactant and / or guanidine hydrochloride to the fermentation broth, perform solid-liquid separation; Step 2: Separate the solution containing mitomycin C by chromatographic column chromatography; Step 3: Obtain mitomycin C solid from mitomycin C solution.
2. The method according to claim 1, characterized in that: Step 2 further includes steps 2-1 and 2-2; Step 2-1: After solid-liquid separation, the supernatant is adsorbed using macroporous adsorption resin and then eluted with elution buffer 1 to obtain elution product 1. After removing the solvent from elution product 1, it is prepared into a column loading solution and adsorbed onto a silica gel column, and then eluted with elution buffer 2 to obtain elution product 2. Step 2-2: After removing the solvent from elution product 2, dissolve it in a solvent and then pass it through C... 18 After adsorption, elution with eluent 3 yielded a solution containing mitomycin C.
3. The method according to claim 2, characterized in that: Eluent 1 is selected from acetone, ethanol, and methanol.
4. The method according to claim 2, characterized in that: Eluent 2 is a mixture of methanol and ethyl acetate.
5. The method according to claim 2, characterized in that: Eluent 3 is a methanol solution.
6. The method according to claim 2, characterized in that: After removing the solvent from elution product 1, prepare the column loading solution with ethanol.
7. The method according to claim 2, characterized in that: Eluent 3 is a methanol solution, and the concentration of methanol is no higher than 30% by volume.
8. The method according to claim 1, characterized in that: The surfactant is sodium dodecyl sulfate.
9. The method according to claim 1, characterized in that: In step 1, the temperature of the fermentation broth should not exceed room temperature.
10. The method according to claim 2, characterized in that: In step 3, after concentrating the mitomycin C solution, methanol is added to dissolve it, and then crystallization is performed to obtain solid mitomycin C.