CN114957367A - Refining method for preparing testosterone by biological method - Google Patents
Refining method for preparing testosterone by biological method Download PDFInfo
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- CN114957367A CN114957367A CN202210594618.XA CN202210594618A CN114957367A CN 114957367 A CN114957367 A CN 114957367A CN 202210594618 A CN202210594618 A CN 202210594618A CN 114957367 A CN114957367 A CN 114957367A
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- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 title claims abstract description 256
- 229960003604 testosterone Drugs 0.000 title claims abstract description 128
- 238000007670 refining Methods 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000010170 biological method Methods 0.000 title claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 60
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 60
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000012043 crude product Substances 0.000 claims abstract description 37
- 238000001914 filtration Methods 0.000 claims abstract description 31
- 239000000047 product Substances 0.000 claims abstract description 31
- 238000003756 stirring Methods 0.000 claims abstract description 27
- 239000012065 filter cake Substances 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000010438 heat treatment Methods 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 239000000706 filtrate Substances 0.000 claims abstract description 14
- 238000001816 cooling Methods 0.000 claims abstract description 11
- 239000002244 precipitate Substances 0.000 claims abstract description 11
- 239000007787 solid Substances 0.000 claims abstract description 11
- 238000002386 leaching Methods 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 238000000967 suction filtration Methods 0.000 claims description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 230000010307 cell transformation Effects 0.000 claims description 9
- 239000001913 cellulose Substances 0.000 claims description 9
- 229920002678 cellulose Polymers 0.000 claims description 9
- 238000006555 catalytic reaction Methods 0.000 claims description 8
- 238000001291 vacuum drying Methods 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 238000011426 transformation method Methods 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 39
- 230000000694 effects Effects 0.000 abstract description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 32
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 24
- 125000005842 heteroatom Chemical group 0.000 description 13
- 239000008213 purified water Substances 0.000 description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- 230000014759 maintenance of location Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 8
- 230000002255 enzymatic effect Effects 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- -1 steroid compound Chemical class 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 description 4
- 229960005471 androstenedione Drugs 0.000 description 4
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 description 4
- 238000002425 crystallisation Methods 0.000 description 4
- 230000008025 crystallization Effects 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000011877 solvent mixture Substances 0.000 description 3
- 239000005909 Kieselgur Substances 0.000 description 2
- CPLXHLVBOLITMK-UHFFFAOYSA-N Magnesium oxide Chemical compound [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
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- 239000007858 starting material Substances 0.000 description 2
- MZWRIOUCMXPLKV-RFOVXIPZSA-N 16-Dehydropregnenolone acetate Chemical compound C([C@@H]12)C[C@]3(C)C(C(C)=O)=CC[C@H]3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC(=O)C)C1 MZWRIOUCMXPLKV-RFOVXIPZSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 239000010425 asbestos Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003804 effect on potassium Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 201000003585 eunuchism Diseases 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 239000010440 gypsum Substances 0.000 description 1
- 229910052602 gypsum Inorganic materials 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000011499 joint compound Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- 208000037106 male hypogonadism Diseases 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000006146 oximation reaction Methods 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052895 riebeckite Inorganic materials 0.000 description 1
- 230000036299 sexual function Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
- C07J1/0003—Androstane derivatives
- C07J1/0018—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa
- C07J1/0022—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa the substituent being an OH group free esterified or etherified
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a refining method of a testosterone crude product prepared by a biological method, which comprises the following steps: (1) uniformly mixing the dried testosterone crude product with alcohol, and heating to above 60 ℃ to completely dissolve the testosterone crude product and generate flocculent denatured protein precipitate; (2) adding a filter aid comprising activated carbon to adsorb the flocculent denatured protein precipitate; filtering and collecting filtrate, and leaching a filter cake with methanol; (3) dropwise adding pure water, uniformly mixing, heating to above 60 ℃, concentrating and recovering 85 wt% of alcohol solution; stirring and cooling to below 8 ℃ to crystallize out a large amount of solid, and filtering and collecting a filter cake; and leaching the mixture by using ethanol and pure water in sequence, and drying a filter cake to obtain a testosterone refined product. The refining method has obvious refining effect on the K impurity and the A impurity in the testosterone crude product prepared by a biological method, the content of the K impurity in the finished testosterone product is less than 0.15 percent, the content of the A impurity is less than 0.05 percent, and the latest EP10.1 standard is met; the yield of the finished product can reach 93 percent or more.
Description
Technical Field
The invention relates to the technical field of steroid medicine preparation, in particular to a refining method for preparing testosterone by a biological method.
Background
Testosterone (shown as the following formula III) is a hormone drug, belongs to a steroid compound, and is clinically used as a sex hormone drug for treating primary and secondary male hypogonadism and maintaining the second characteristics and the sexual function of males. The traditional method for synthesizing testosterone comprises the steps of oximation of dehydropregnenolone acetate, beta reaction, hydrolysis and the like to obtain androstenedione, and then the androstenedione is used as a raw material to obtain a testosterone crude product through two-step reaction.
With the development of biocatalysis and enzyme catalysis technologies, a chemical synthesis method is gradually replaced by the cell transformation and enzyme catalysis method for preparing testosterone, androstenedione is used as a substrate by using the cell transformation or enzyme catalysis method, 17-keto is selectively reduced to form beta-hydroxy, and the androstenedione is converted into testosterone in one step (route a), so that the production process is simplified, and the production cost is saved.
However, in this reaction, the K hetero-precursor species (formula IV) in the starting material is also converted to K hetero-precursor species (formula I) and the I hetero-precursor species (formula V) is also converted to I hetero-precursor species (formula VI). The structure of the K hetero (formula I) is extremely similar to that of the testosterone (formula III), so that the K hetero (formula I) and the A hetero (formula II) in the testosterone (formula III) prepared by a biological method are difficult to remove, a large amount of ethyl acetate and n-hexane are required for crystallization and refining, the process is complex, the solvent is difficult to recover, and the cost is high.
And the testosterone refined by the method does not meet the standard of EP 10.1: k is less than 0.15%, A is less than 0.1%, and other single impurity is less than 0.1%.
Disclosure of Invention
In order to solve the technical problem that the refined product of testosterone prepared by a biological method in the prior art does not meet the standard of EP10.1, the invention provides a refining method for preparing testosterone by a biological method. The refining method has a good refining effect on K impurities and A impurities which are difficult to refine in testosterone, the content of the K impurities in the finished testosterone is less than 0.15%, the content of the A impurities is less than 0.05%, and the latest EP10.1 standard of testosterone is met: the impurity K is less than 0.15 percent, and the impurity A is less than 0.1 percent. And the refining process is simple, the solvent is easy to recover, the cost is reduced, and the yield of the final product can reach 96% or more in the preferred technical scheme.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
(1) uniformly mixing the dried testosterone crude product with alcohol, and heating to above 60 ℃ to completely dissolve the testosterone crude product and generate flocculent denatured protein precipitate;
(2) adding a filter aid containing activated carbon, and adsorbing flocculent denatured protein precipitate in the solution in the step (1); filtering and collecting filtrate, and leaching a small amount of residual testosterone on a filter cake with methanol;
(3) dropwise adding pure water into the solution, uniformly mixing, heating to above 60 ℃, concentrating and recovering 85 wt% of alcohol solution; stirring and cooling to below 8 ℃ to crystallize out a large amount of solid, and filtering and collecting a filter cake; and leaching the mixture by using ethanol and pure water in sequence, and drying a filter cake to obtain a testosterone refined product.
In some specific embodiments, the alcohol includes methanol, ethanol, ethylene glycol, isopropanol, or n-butanol, or a mixed solvent composed thereof.
In some specific embodiments, the filter aid comprising activated carbon further comprises one or more of asbestos, graphite powder, sawdust, acid clay, perlite, mud, starch, diatomaceous earth, paper, magnesia, gypsum, cellulose, and aluminum hydroxide.
In some specific embodiments, the conditions of the refining process are selected from the group consisting of:
i) in the step (1), the alcohol is methanol or ethanol;
ii) in the step (2), the filter aid containing the activated carbon is activated carbon and diatomite or activated carbon and cellulose.
In some embodiments, in step (1), the crude testosterone is prepared by a cell transformation method or an enzymatic method.
In some specific embodiments, in step (1), the crude testosterone is collected by filtration.
In some specific embodiments, in step (1), the temperature is increased while stirring and refluxing are performed.
In some specific embodiments, the filtration is suction filtration. For example, filtration in steps (1), (2) and (3) is carried out by suction filtration.
In some specific embodiments, the drying is vacuum drying. For example, vacuum drying is used in both steps (1) and (3).
In some specific embodiments, in step (1), the volume-to-mass ratio of the alcohol to the crude testosterone is: (4-12): 1, preferably (5-8): 1.
In some specific embodiments, in step (1), when the alcohol is methanol, the temperature is raised to above 70 ℃.
In some specific embodiments, in step (2), the adsorption is performed under agitation.
In some specific embodiments, in the step (2), the dosage ratio of the filter aid containing the activated carbon to the crude testosterone is (4-5): 15.
In some specific embodiments, in step (2), the dosage ratio of the diatomite to the activated carbon is 1:1, or the dosage ratio of the cellulose to the activated carbon is 3: 5.
In some specific embodiments, in the step (2), the volume-to-mass ratio of the methanol to the crude testosterone is: (1-3) and preferably 2: 3.
In some specific embodiments, in the step (3), the volume-to-mass ratio of the pure water to the crude testosterone is (5-10): 6, and preferably 5: 6.
In some specific embodiments, in the step (3), the adding speed of the pure water is 2-10 ml/min; for example 5 ml/min.
In some specific embodiments, in the step (3), the volume-to-mass ratio of the pure water to the testosterone for leaching is (5-10): 1; preferably (6-7): 1.
In some specific embodiments, in step (3), the blending is performed under agitation.
In some specific embodiments, in step (3), the concentration is concentration under reduced pressure.
In some specific embodiments, in step (3), the reducing temperature is performed under stirring.
In some specific embodiments, in step (3), the ethanol is frozen ethanol; such as 5-8 deg.C ethanol.
In some specific embodiments, in the step (3), the volume-to-mass ratio of the ethanol to the testosterone is (0.5-2): 3; preferably 1: 3.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention. The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the refining method has obvious refining effect on the K impurity and the A impurity in the testosterone crude product prepared by a biological method, the content of the K impurity in the finished testosterone product is less than 0.15%, the content of the A impurity in the finished testosterone product is less than 0.05%, and the refined finished product meets the latest EP10.1 standard. The yield of the finished product can reach 93 percent or more, and in the preferred technical scheme, the yield of the finished product can reach 96 percent or more.
Drawings
FIG. 1 is an HPLC profile of testosterone obtained by a cell transformation method; wherein A is a testosterone crude product; and B is testosterone refined product refined by activated carbon and diatomite assisted filtration.
FIG. 2 is an HPLC profile of testosterone using an enzymatic method; wherein A is a testosterone crude product; and B is testosterone refined product refined by activated carbon and diatomite assisted filtration.
FIG. 3 is an HPLC profile of ethanol refined testosterone; wherein A is a testosterone crude product; b is testosterone refined by ethanol.
FIG. 4 is an HPLC chromatogram of activated carbon and cellulose assisted refined testosterone; wherein A is a testosterone crude product; b is testosterone refined by active carbon and cellulose for filter aid refining.
FIG. 5 is an HPLC profile of crude testosterone used for chemical refining.
FIG. 6 is HPLC chromatogram of refined testosterone product refined by crystallization of ethyl acetate and n-hexane.
FIG. 7 is an HPLC chromatogram of a refined testosterone product refined by ethyl acetate crystallization.
FIG. 8 is an HPLC chromatogram of acetone-refined testosterone fine product.
FIG. 9 is an HPLC chromatogram of a testosterone fine product refined with isopropanol.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. 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.
EXAMPLE 1 purification of crude Testosterone from the cell transformation method
The first step is as follows: collecting testosterone crude product with about 30g by filtering 1L cell fermentation liquid containing testosterone, vacuum drying testosterone crude product, adding 240mL methanol, heating to 70 deg.C under stirring, refluxing for 1h until the material is completely dissolved and flocculent denatured protein precipitate appears.
The second step is that: to the solution, 5g of diatomaceous earth and 5g of activated carbon were added, and the mixture was stirred at a constant temperature to adsorb insoluble matter (i.e., flocculent denatured protein precipitate) by a filter aid, followed by filtration to collect the filtrate. The small amount of residual testosterone on the filter cake was rinsed with 20mL methanol.
The third step: and (2) dropwise adding 25mL of purified water into the solution at the rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain 85 wt% methanol solution, recovering, stirring, cooling to below 8 ℃, crystallizing to obtain a large amount of solid, performing suction filtration to collect a filter cake, performing suction filtration by using 10mL of frozen ethanol, performing drip filtration by using 200mL of purified water, and performing vacuum drying on the filter cake to obtain a testosterone refined product.
The yield is more than 96 percent, the HPLC submission purity is more than 99.8 percent, and the limits of the K impurity and the A impurity meet the EP10.1 standard. The HPLC chromatogram of the testosterone crude product is shown in A and Table 1 of figure 1, and the HPLC chromatogram of the testosterone refined product is shown in B and Table 2 of figure 1.
TABLE 1 cell transformation of crude Testosterone HPLC
| Peak number | Retention time | Relative retention time | Area of | Area% | Species of impurities | Content of |
| 1 | 2.685 | 0.290 | 26269 | 0.528 | Peak of solvent | / |
| 2 | 7.953 | 0.858 | 20785 | 0.418 | K hetero | 0.418％ |
| 3 | 8.400 | 0.906 | 4952 | 0.100 | Unknown impurity | 0.1％ |
| 4 | 9.269 | 1.000 | 4872808 | 97.997 | |
|
| 5 | 13.402 | 1.446 | 47569 | 0.957 | A hetero compound | 0.857％ |
TABLE 2 cell transformation of Testosterone essence HPLC
EXAMPLE 2 purification of crude Testosterone by enzymatic method
The first step is as follows: 300mL of the enzyme catalysis conversion solution containing 30g of testosterone crude product is filtered, the testosterone crude product is collected, after the testosterone crude product is dried in vacuum, 240mL of methanol is added, the temperature is raised to 70 ℃ under stirring, and the mixture is refluxed for 1h until the material is completely dissolved and flocculent denatured protein precipitates appear.
The second step is that: adding 5g of diatomite and 5g of activated carbon into the solution, stirring the solution at a constant temperature to enable insoluble substances to be adsorbed by a filter aid, filtering the filtrate with the yield, and leaching a small amount of residual testosterone on a filter cake by using 20mL of methanol.
The third step: dropwise adding 25mL of purified water into the solution at the rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain 85 wt% methanol solution, recovering, stirring, cooling to below 8 ℃, crystallizing to obtain a large amount of solid, performing suction filtration to collect a filter cake, performing suction filtration by using 10mL of frozen ethanol, performing drip filtration by using 200mL of purified water, and performing vacuum drying on the filter cake to obtain a testosterone refined product, wherein the yield is more than 96%, the HPLC (high performance liquid chromatography) detection purity is higher than 99.8%, and the limitation of K impurity and A impurity meets the EP10.1 standard. The HPLC pattern of the testosterone crude product is shown in A and table 3 of figure 2, and the HPLC pattern of the testosterone refined product is shown in B and table 4 of figure 2.
TABLE 3 enzymatic catalysis of crude Testosterone HPLC
| Peak number | Retention time | Relative retention time | Area of | Area% | Species of impurities | Content of |
| 1 | 2.693 | 0.290 | 3201 | 0.030 | Solvent mixture | / |
| 2 | 7.965 | 0.857 | 25489 | 0.242 | K hetero | 0.242％ |
| 3 | 8.412 | 0.906 | 6911 | 0.066 | Unknown impurity | 0.066％ |
| 4 | 9.289 | 1.000 | 10407259 | 98.991 | |
|
| 5 | 13.438 | 1.447 | 70500 | 0.671 | A hetero compound | 0.671％ |
TABLE 4 enzymatic Testosterone boutique HPLC
EXAMPLE 3 purification of crude Testosterone EtOH
The first step is as follows: 300mL of the enzymatic conversion solution containing 30g of testosterone crude product is filtered, the testosterone crude product is collected, after the testosterone crude product is dried in vacuum, 240mL of ethanol solution (ethanol is more than 95 percent) is added, the temperature is raised to 60 ℃ under stirring, and the mixture is refluxed for 1 hour until the material is completely dissolved and flocculent denatured protein precipitates.
The second step is that: 5g of diatomite and 5g of activated carbon are added into the solution, the solution is kept in a constant temperature state and stirred to enable insoluble substances to be adsorbed by a filter aid, filtrate is collected through filtration, and a small amount of residual testosterone on a filter cake is rinsed by 20mL of methanol.
The third step: dropwise adding 25mL of purified water into the solution at the rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain 85 wt% ethanol solution, recovering, stirring, cooling to below 8 ℃, crystallizing to obtain a large amount of solid, performing suction filtration to collect a filter cake, performing suction filtration by using 10mL of frozen ethanol, performing drip filtration by using 200mL of purified water, and performing vacuum drying on the filter cake to obtain a testosterone refined product, wherein the yield is more than 93%, the HPLC (high performance liquid chromatography) detection purity is higher than 99.8%, and the limitation of K impurity and A impurity meets the EP10.1 standard. The HPLC profile of testosterone crude product is shown in A and Table 5 of FIG. 3, and the HPLC profile of testosterone fine product is shown in B and Table 6 of FIG. 3.
TABLE 5 enzymatic Testosterone crude HPLC
| Number of peak | Retention time | Relative retention time | Area of | Area% | Species of impurities | Content of |
| 1 | 2.693 | 0.290 | 3201 | 0.030 | Solvent mixture | / |
| 2 | 7.965 | 0.857 | 25489 | 0.242 | K hetero | 0.242％ |
| 3 | 8.412 | 0.906 | 6911 | 0.066 | Unknown impurity | 0.066％ |
| 4 | 9.289 | 1.000 | 10407259 | 98.991 | |
|
| 5 | 13.438 | 1.447 | 70500 | 0.671 | A hetero compound | 0.671％ |
TABLE 6 enzymatic Testosterone boutique HPLC
EXAMPLE 4 Testosterone crude activated carbon and cellulose aid filtration refining
The first step is as follows: 300mL of the enzyme catalysis conversion solution containing 30g of testosterone crude product is filtered, the testosterone crude product is collected, after the testosterone crude product is dried in vacuum, 240mL of methanol is added, the temperature is raised to 70 ℃ under stirring, and the mixture is refluxed for 1h until the material is completely dissolved and flocculent denatured protein precipitates appear.
The second step: adding 3g of cellulose and 5g of activated carbon into the solution, stirring the solution at a constant temperature to enable insoluble substances to be adsorbed by a filter aid, filtering the filtrate with the yield, and leaching a small amount of residual testosterone on a filter cake by using 20mL of methanol.
The third step: dropwise adding 25mL of purified water into the solution at the rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain 85 wt% methanol solution, recovering, stirring, cooling to below 8 ℃, crystallizing to obtain a large amount of solid, performing suction filtration to collect a filter cake, performing suction filtration by using 10mL of frozen ethanol, performing drip filtration by using 200mL of purified water, and performing vacuum drying on the filter cake to obtain a testosterone refined product, wherein the yield is more than 93%, the HPLC (high performance liquid chromatography) detection purity is higher than 99.8%, and the limitation of K impurity and A impurity meets the EP10.1 standard. The HPLC profile of testosterone crude product is shown in A and Table 7 of FIG. 4, and the HPLC profile of testosterone fine product is shown in B and Table 8 of FIG. 4.
TABLE 7 enzymatic Testosterone crude HPLC
| Peak number | Retention time | Relative retention time | Area of | Area% | Species of impurities | Content of |
| 1 | 2.742 | 0.290 | 15654 | 0.030 | Solvent mixture | / |
| 2 | 8.245 | 0.856 | 22231 | 0.242 | K hetero | 0.242％ |
| 3 | 8.713 | 0.905 | 7418 | 0.066 | Unknown impurity | 0.066％ |
| 4 | 9.632 | 1.000 | 7145895 | 98.991 | |
|
| 5 | 13.926 | 1.446 | 36157 | 0.671 | A hetero compound | 0.671％ |
TABLE 8 enzymatic Testosterone boutique HPLC
Example 5 comparative experiment
Unless otherwise specified, the following purification methods use crude testosterone that is not limited to chemical, cell transformation, or enzymatic methods.
5.1, crystallizing and refining ethyl acetate and n-hexane:
taking 10g of testosterone crude product (figure 5), adding ethyl acetate (50-80 mL) with the volume 5-8 times that of the testosterone crude product, and heating to 70 ℃ under stirring until the material is completely dissolved.
Filtering to remove denatured protein, collecting filtrate, keeping the temperature for 1h, slowly dripping n-hexane (30-50 mL) with the volume of 3-5 times into the solution, slowly cooling to about 10 ℃, and separating out a large amount of solids.
Filtering and collecting testosterone essence, and drying at 60 ℃ to constant weight.
The yield is 80-85%, and the refining effect is as follows: variation in the content of K hetero: 0.223% → 0.200%; change of impurity content: 0.534% → 0.101%; the remaining single hetero > 0.1% (FIG. 6). The method can obtain refined testosterone with low yield, which does not meet EP10.1 requirement.
5.2 ethyl acetate crystallization refining:
taking 10g of the testosterone crude product (shown in figure 5), adding 5-8 times of ethyl acetate (50-80 mL), and heating to 70 ℃ under stirring until the material is completely dissolved.
And (3) filtering to remove denatured protein, collecting filtrate, and concentrating the filtrate in vacuum until 1-2 times volume of ethyl acetate remains and a large amount of solid is separated out.
Cooling, crystallizing, filtering, collecting refined testosterone product, and oven drying at 60 deg.C to constant weight.
The yield is 80-85%, and the refining effect is as follows: variation in the content of K hetero: 0.212% → 0.187%; change of impurity content: 0.534% → 0.052%; the remaining single hetero > 0.1% (FIG. 7). The method can obtain refined testosterone with low yield, which does not meet EP10.1 requirement. Moreover, it was found that this method does not have a refining effect on biologically synthesized testosterone and does not meet the limits specified in EP 0.1.
5.3 acetone refining:
taking 10g of testosterone crude product (figure 5), adding acetone (60-90 mL) with the volume 6-9 times of the testosterone crude product, and heating to 50 ℃ under stirring until the materials are completely dissolved.
Filtering to remove denatured protein, collecting filtrate, and vacuum concentrating the filtrate until 1 volume of acetone remains, and a large amount of solid is separated out.
Cooling, crystallizing, filtering, collecting refined testosterone product, and oven drying at 60 deg.C to constant weight.
The yield is 80%, and the refining effect is as follows: variation in the content of K impurities: 0.235% → 0.112%, a hetero content variation: 0.534% → 0.252% (fig. 8). The method can obtain refined testosterone with low yield, which does not meet EP10.1 requirement.
5.4 refining isopropanol:
taking 10g of testosterone crude product (shown in figure 5) synthesized by an enzyme catalysis method, adding 5-8 times of isopropanol (50-80 mL), and heating to 60 ℃ under stirring until the material is completely dissolved. Filtering to remove denatured protein, collecting filtrate, adding 50-70% of purified water (5-7 mL) according to the mass of testosterone into the filtrate, and concentrating the solvent under reduced pressure until 2 times of the volume of the solvent remains.
Cooling and crystallizing until a large amount of solid is separated out, filtering and collecting testosterone refined products, and drying at 60 ℃ to constant weight.
The yield is 90%, and the refining effect is as follows: variation in the content of K hetero: 0.235% → 0.114%; change of impurity content: 0.534% → 0.057% (fig. 9). The testosterone refined by the method meets the requirements of EP 10.1.
Claims (10)
1. A refining method of testosterone crude products prepared by a biological method is characterized by comprising the following steps:
(1) uniformly mixing the dried testosterone crude product with alcohol, and heating to above 60 ℃ to completely dissolve the testosterone crude product and generate flocculent denatured protein precipitate;
(2) adding a filter aid comprising activated carbon to adsorb the flocculent denatured protein precipitate; filtering and collecting filtrate, and leaching a small amount of residual testosterone on a filter cake with methanol;
(3) dropwise adding pure water, uniformly mixing, heating to above 60 ℃, concentrating and recovering 85 wt% of alcohol solution; stirring and cooling to below 8 ℃ to crystallize out a large amount of solid, and filtering and collecting a filter cake; and leaching the mixture by using ethanol and pure water in sequence, and drying a filter cake to obtain a testosterone refined product.
2. The refining process of claim 1, wherein the refining process conditions are selected from the group consisting of:
i) in the step (1), the alcohol is methanol or ethanol;
ii) in the step (2), the filter aid containing the activated carbon is activated carbon and diatomite or activated carbon and cellulose.
3. The refining process of claim 1, wherein in step (1), the refining process conditions are selected from one or more of the following:
i) the testosterone crude product is prepared by a cell transformation method or an enzyme catalysis method;
ii) the crude testosterone is collected by filtration;
iii) stirring and refluxing while raising the temperature.
4. The refining process of claim 3, wherein the refining process conditions are selected from one or more of the following:
i) in the steps (1) - (3), the filtration is suction filtration;
ii) in steps (1) and (3), the drying is vacuum drying.
5. The refining process of claim 1, wherein in step (1), the refining process conditions are selected from the group consisting of:
i) the volume-mass ratio of the alcohol to the crude testosterone product is as follows: (4-12) 1, preferably (5-8) 1;
ii) when the alcohol is methanol, heating to above 70 ℃.
6. The refining process of claim 1, wherein in step (2), the refining process conditions are selected from the group consisting of:
i) the adsorption is carried out at a constant temperature by stirring;
ii) the using amount ratio of the filter aid containing the activated carbon to the crude testosterone product is (4-5): 15.
7. The refining process of claim 5, wherein in step (2), the refining process conditions are selected from the group consisting of:
i) the dosage ratio of the diatomite to the active carbon is 1:1, or the dosage ratio of the cellulose to the active carbon is 3: 5;
ii) the volume mass ratio of the methanol to the crude testosterone product is (1-3) to 3, preferably 2 to 3.
8. The refining method according to claim 1 to 7, wherein in the step (3), the conditions of the refining method are selected from the group consisting of:
i) the volume-mass ratio of the pure water to the crude testosterone is (5-10) to 6, preferably 5 to 6;
ii) the adding speed of the pure water is 2-10 ml/min; for example 5 ml/min;
iii) the volume-to-mass ratio of the pure water for leaching to the crude testosterone is (5-10) to 1; preferably (6-7): 1.
9. The refining process of claim 8, wherein in step (3), the refining process conditions are selected from the group consisting of:
i) the uniform mixing is carried out under stirring;
ii) said concentrating is concentrating under reduced pressure;
iii) said lowering of the temperature is carried out under stirring.
10. The refining process of claim 1, wherein in step (3), the refining process conditions are selected from the group consisting of:
i) the ethanol is frozen ethanol;
ii) the volume mass ratio of the ethanol to the testosterone is (0.5-2) to 3; preferably 1: 3.
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