CN114957367B - Refining method for preparing testosterone by biological method - Google Patents
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- CN114957367B CN114957367B CN202210594618.XA CN202210594618A CN114957367B CN 114957367 B CN114957367 B CN 114957367B CN 202210594618 A CN202210594618 A CN 202210594618A CN 114957367 B CN114957367 B CN 114957367B
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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 266
- 229960003604 testosterone Drugs 0.000 title claims abstract description 133
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000007670 refining Methods 0.000 title claims abstract description 40
- 238000010170 biological method Methods 0.000 title claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 63
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 60
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000012043 crude product Substances 0.000 claims abstract description 39
- 238000001914 filtration Methods 0.000 claims abstract description 27
- 238000003756 stirring Methods 0.000 claims abstract description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000047 product Substances 0.000 claims abstract description 23
- 239000012065 filter cake Substances 0.000 claims abstract description 21
- 239000000706 filtrate Substances 0.000 claims abstract description 16
- 238000002386 leaching Methods 0.000 claims abstract description 13
- 238000001816 cooling Methods 0.000 claims abstract description 12
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 239000007787 solid Substances 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000002244 precipitate Substances 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 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
- 230000010307 cell transformation Effects 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 238000011426 transformation method Methods 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 62
- 230000003804 effect on potassium Effects 0.000 abstract description 3
- 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
- 239000002904 solvent Substances 0.000 description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 239000008213 purified water Substances 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 8
- 230000014759 maintenance of location Effects 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000005909 Kieselgur Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000006243 chemical reaction Methods 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
- 230000006920 protein precipitation Effects 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000643 oven drying Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- -1 steroid compounds Chemical class 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
- 238000013019 agitation Methods 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
- 230000033228 biological regulation Effects 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
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 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
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000011499 joint compound Substances 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- CPLXHLVBOLITMK-UHFFFAOYSA-N magnesium oxide Inorganic materials [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 1
- AXZKOIWUVFPNLO-UHFFFAOYSA-N magnesium;oxygen(2-) Chemical compound [O-2].[Mg+2] AXZKOIWUVFPNLO-UHFFFAOYSA-N 0.000 description 1
- 208000037106 male hypogonadism Diseases 0.000 description 1
- 239000012046 mixed solvent Substances 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
- 239000007858 starting material Substances 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
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
Landscapes
- 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 testosterone crude product by 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 form 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 by using methanol; (3) Dropwise adding pure water, uniformly mixing, heating to above 60 ℃, concentrating and recovering 85wt% of alcohol solution; stirring and cooling to below 8 ℃ to crystallize a large amount of solids, and filtering and collecting a filter cake; sequentially eluting with ethanol and pure water, and drying the filter cake to obtain testosterone fine product. The refining method has obvious refining effect on K impurity and A impurity in the testosterone crude product prepared by a biological method, the K impurity content in the finished testosterone is less than 0.15%, the A impurity content is less than 0.05%, and the method accords with the latest EP10.1 standard; 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 in the following formula III) is a hormone medicine, belongs to steroid compounds, and is clinically used as a sex hormone medicine for treating primary and secondary male hypogonadism, maintaining male secondary characteristics, sexual functions and the like. The traditional method for synthesizing testosterone comprises the steps of oximating dehydropregnenolone acetate, performing a Bettype reaction, hydrolyzing and the like to obtain androstenedione, and then performing a two-step reaction to obtain a testosterone crude product by taking androstenedione as a raw material.
Along with the development of biocatalysis and enzyme catalysis technology, cell transformation and enzyme catalysis methods for preparing testosterone gradually replace chemical synthesis methods, androstenedione is used as a substrate by using the cell transformation or enzyme catalysis methods, 17-keto is selectively reduced to beta-hydroxyl, and 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 heteroprecursor species (formula IV) in the starting materials will also be converted to K heterospecies (formula I), and the I heteroprecursor species (formula V) will also be converted to I heterospecies (formula VI). Because the structure of K impurity (formula I) is extremely similar to that of testosterone (formula III), K impurity (formula I) and A impurity (formula II) in testosterone (formula III) prepared by a biological method are difficult to remove, a large amount of ethyl acetate and n-hexane are required to be used for crystallization for refining, the process is complex, the solvent is difficult to recycle, and the cost is high.
And testosterone refined by this method does not meet the standard of EP 10.1: k impurity is less than 0.15%, A impurity is less than 0.1%, and other single impurities are less than 0.1%.
Disclosure of Invention
The invention provides a refining method for preparing testosterone by a biological method, which aims to solve the technical problem that a testosterone refining product prepared by the biological method in the prior art does not meet the standard of EP 10.1. The refining method has better refining effect on K impurity and A impurity which are difficult to refine in testosterone, wherein the K impurity content in the finished testosterone is less than 0.15%, and the A impurity content is less than 0.05%, and meets the latest EP10.1 standard of testosterone: k impurity is less than 0.15%, A impurity is less than 0.1%. The refining process is simple, the solvent is easy to recycle, the cost is reduced, and the final product yield can reach 96% or more in the preferable 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 form flocculent denatured protein precipitate;
(2) Adding a filter aid containing activated carbon, and adsorbing flocculent denatured protein precipitate in the solution of the step (1); filtering and collecting filtrate, and leaching a small amount of residual testosterone on a filter cake by using methanol;
(3) Dropwise adding pure water into the solution, uniformly mixing, heating to above 60 ℃, concentrating and recovering 85wt% of alcohol solution; stirring and cooling to below 8 ℃ to crystallize a large amount of solids, and filtering and collecting a filter cake; sequentially eluting with ethanol and pure water, and drying the filter cake to obtain testosterone fine product.
In some specific embodiments, the alcohol comprises methanol, ethanol, ethylene glycol, isopropanol, or n-butanol, or a mixed solvent composed thereof.
In some specific embodiments, the activated carbon-containing filter aid further comprises one or more of asbestos, graphite powder, sawdust, acid clay, perlite, mud, starch, diatomaceous earth, paper making, magnesium oxide, 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 step (2), the filter aid comprising activated carbon is activated carbon and diatomaceous earth, or activated carbon and cellulose.
In some specific embodiments, in step (1), the crude testosterone is produced by a cell transformation method or an enzyme catalysis method.
In some specific embodiments, in step (1), the crude testosterone is collected by filtration.
In some specific embodiments, in step (1), the heating is performed while stirring and refluxing.
In some specific embodiments, the filtration is suction filtration. For example, the 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 adopted in each of the steps (1) and (3).
In some specific embodiments, in step (1), the volume to mass ratio of the alcohol to the crude testosterone product is: (4-12): 1, (5-8): 1 is preferably used.
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 adsorbing is performed under agitation.
In some specific embodiments, in step (2), the filter aid comprising activated carbon is used in an amount of (4-5): 15 to the crude testosterone product.
In some specific embodiments, in step (2), the diatomaceous earth and activated carbon are used in a 1:1 ratio, or the cellulose and activated carbon are used in a 3:5 ratio.
In some specific embodiments, in step (2), the volume to mass ratio of methanol to testosterone crude product is: (1-3): 3, preferably 2:3.
In some specific embodiments, in step (3), the ratio of pure water to crude testosterone is (5-10): 6, preferably 5:6 by volume/mass.
In some specific embodiments, in step (3), the pure water is added at a rate of 2 to 10ml/min; for example 5ml/min.
In some specific embodiments, in step (3), the ratio of pure water to testosterone for rinsing is (5-10) 1; preferably (6-7): 1.
In some specific embodiments, in step (3), the mixing is performed with stirring.
In some specific embodiments, in step (3), the concentrating is concentrating under reduced pressure.
In some specific embodiments, in step (3), the cooling is performed with stirring.
In some specific embodiments, in step (3), the ethanol is frozen ethanol; for example ethanol at 5-8 ℃.
In some specific embodiments, in step (3), the volume to mass ratio of ethanol to testosterone is (0.5-2) 3; preferably 1:3.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention. The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the refining method has obvious refining effect on K impurity and A impurity in the testosterone crude product prepared by a biological method, the K impurity content in the finished testosterone product is less than 0.15%, the A impurity content 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% or more, and in the preferable technical scheme, the yield of the finished product can reach 96% or more.
Drawings
FIG. 1 is an HPLC plot of testosterone obtained by the cell transformation method; wherein A is testosterone crude product; b is refined testosterone essence which is filtered by activated carbon and diatomite.
FIG. 2 is an HPLC plot of testosterone obtained by an enzyme-catalyzed process; wherein A is testosterone crude product; b is refined testosterone essence which is filtered by activated carbon and diatomite.
FIG. 3 is an HPLC plot of ethanol-refined testosterone; wherein A is testosterone crude product; b is refined testosterone extract refined by ethanol.
Fig. 4 is an HPLC profile of activated carbon and cellulose-assisted filtration refined testosterone; wherein A is testosterone crude product; b is refined testosterone essence which is filtered and refined by the aid of active carbon and cellulose.
Fig. 5 is an HPLC profile of crude testosterone for chemical refining.
Figure 6 is an HPLC chart of refined testosterone extract crystallized from ethyl acetate and n-hexane.
Fig. 7 is an HPLC profile of refined testosterone extract from ethyl acetate crystallization.
Figure 8 is an HPLC profile of acetone-refined testosterone extract.
Figure 9 is an HPLC profile of refined testosterone from isopropanol.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1 purification of crude testosterone product obtained by cell transformation method
The first step: the testosterone-containing 1L cell fermentation broth is filtered to collect about 30g of testosterone crude product, and after the testosterone crude product is dried in vacuum, 240mL of methanol is added, and the mixture is heated to 70 ℃ with stirring and refluxed for 1 hour until the material is completely dissolved and flocculent denatured protein precipitation occurs.
And a second step of: to the above solution, 5g of diatomaceous earth and 5g of activated carbon were added, and the mixture was stirred at a constant temperature to adsorb insoluble matters (i.e., flocculent denatured protein precipitate) by a filter aid, and the filtrate was collected by filtration. A small amount of residual testosterone on the filter cake was rinsed with 20mL methanol.
And a third step of: and (3) dropwise adding 25mL of purified water into the solution at a rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain a methanol solution with 85wt% and recycling, stirring, cooling to below 8 ℃ to crystallize a large amount of solids, filtering, collecting a filter cake, leaching with 10mL of frozen ethanol, leaching with 200mL of purified water, and vacuum drying the filter cake to obtain testosterone fine product.
The yield is more than 96%, the purity of HPLC inspection is more than 99.8%, and the K impurity and A impurity limit accords with the EP10.1 standard. The HPLC profile of crude testosterone is shown in A and Table 1 of figure 1, and the HPLC profile of fine testosterone is shown in B and Table 2 of figure 1.
TABLE 1 cell-converted crude testosterone HPLC
| Peak number | Retention time | Relative retention time | Area of | Area percent | Impurity species | Impurity content |
| 1 | 2.685 | 0.290 | 26269 | 0.528 | Solvent peak | / |
| 2 | 7.953 | 0.858 | 20785 | 0.418 | K impurity | 0.418％ |
| 3 | 8.400 | 0.906 | 4952 | 0.100 | Unknown impurities | 0.1％ |
| 4 | 9.269 | 1.000 | 4872808 | 97.997 | Testosterone (Testosterone) | |
| 5 | 13.402 | 1.446 | 47569 | 0.957 | A impurity | 0.857％ |
TABLE 2 cell-converted testosterone top-quality HPLC
Example 2 purification of crude testosterone product by enzymatic catalysis
The first step: 300mL of enzyme catalytic conversion solution containing 30g of testosterone crude product is filtered, the testosterone crude product is collected, 240mL of methanol is added after the testosterone crude product is dried in vacuum, and the mixture is heated to 70 ℃ under stirring and refluxed for 1h until the material is completely dissolved and flocculent denatured protein precipitation occurs.
And a second step of: to the above solution, 5g of diatomaceous earth and 5g of activated carbon were added, and the mixture was stirred at a constant temperature to adsorb insoluble matters by a filter aid, and the filtrate was filtered to obtain a filtrate, and a small amount of testosterone remaining on the filter cake was rinsed with 20mL of methanol.
And a third step of: and (3) dropwise adding 25mL of purified water into the solution at a rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain a methanol solution with 85wt% and recycling, stirring, cooling to below 8 ℃ to crystallize to obtain a large amount of solids, filtering, collecting a filter cake, leaching with 10mL of frozen ethanol, leaching with 200mL of purified water, vacuum drying the filter cake to obtain testosterone fine product, wherein the yield is more than 96%, the purity of HPLC (high performance liquid chromatography) is higher than 99.8%, and the K impurity and A impurity limit accords with the EP10.1 standard. The HPLC profile of crude testosterone is shown in A and Table 3 of figure 2, and the HPLC profile of fine testosterone is shown in B and Table 4 of figure 2.
TABLE 3 enzyme-catalyzed crude testosterone HPLC
| Peak number | Retention time | Relative retention time | Area of | Area percent | Impurity species | Impurity content |
| 1 | 2.693 | 0.290 | 3201 | 0.030 | Solvent impurity | / |
| 2 | 7.965 | 0.857 | 25489 | 0.242 | K impurity | 0.242％ |
| 3 | 8.412 | 0.906 | 6911 | 0.066 | Unknown impurities | 0.066％ |
| 4 | 9.289 | 1.000 | 10407259 | 98.991 | Testosterone (Testosterone) | |
| 5 | 13.438 | 1.447 | 70500 | 0.671 | A impurity | 0.671％ |
Table 4 enzyme catalyzed testosterone top-quality HPLC
EXAMPLE 3 refining of crude ethanol of testosterone
The first step: 300mL of enzyme catalytic conversion solution containing 30g of testosterone crude product is filtered, testosterone crude product is collected, after the testosterone crude product is dried in vacuum, 240mL of ethanol solution (ethanol > 95%) is added, and the temperature is raised to 60 ℃ under stirring to reflux for 1h until the material is completely dissolved and flocculent denatured protein precipitation occurs.
And a second step of: to the above solution, 5g of diatomaceous earth and 5g of activated carbon were added, and the mixture was stirred at a constant temperature to allow the insoluble matter to be adsorbed by a filter aid, and the filtrate was collected by filtration, and a small amount of testosterone remaining on the filter cake was rinsed with 20mL of methanol.
And a third step of: and (3) dropwise adding 25mL of purified water into the solution at a rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain an ethanol solution with 85wt% and recycling, stirring, cooling to below 8 ℃ to crystallize to obtain a large amount of solids, filtering, collecting a filter cake, leaching with 10mL of frozen ethanol, leaching with 200mL of purified water, vacuum drying the filter cake to obtain a testosterone fine product, wherein the yield is more than 93%, the purity of HPLC (high performance liquid chromatography) delivery inspection is higher than 99.8%, and the K impurity and A impurity limit accords with the EP10.1 standard. The HPLC profile of crude testosterone is shown in A of figure 3 and Table 5, and the HPLC profile of fine testosterone is shown in B of figure 3 and Table 6.
TABLE 5 enzyme catalyzed crude testosterone HPLC
| Peak number | When reservedInterval (C) | Relative retention time | Area of | Area percent | Impurity species | Impurity content |
| 1 | 2.693 | 0.290 | 3201 | 0.030 | Solvent impurity | / |
| 2 | 7.965 | 0.857 | 25489 | 0.242 | K impurity | 0.242％ |
| 3 | 8.412 | 0.906 | 6911 | 0.066 | Unknown impurities | 0.066％ |
| 4 | 9.289 | 1.000 | 10407259 | 98.991 | Testosterone (Testosterone) | |
| 5 | 13.438 | 1.447 | 70500 | 0.671 | A impurity | 0.671％ |
Table 6 enzyme catalyzed testosterone top-quality HPLC
EXAMPLE 4 purification of crude testosterone active carbon and cellulose by filtration
The first step: 300mL of enzyme catalytic conversion solution containing 30g of testosterone crude product is filtered, the testosterone crude product is collected, 240mL of methanol is added after the testosterone crude product is dried in vacuum, and the mixture is heated to 70 ℃ under stirring and refluxed for 1h until the material is completely dissolved and flocculent denatured protein precipitation occurs.
And a second step of: 3g of cellulose and 5g of activated carbon are added into the solution, the solution is stirred at a constant temperature to enable insoluble substances to be adsorbed by a filter aid, the filtrate is filtered to obtain filtrate, and a small amount of residual testosterone on the filter cake is leached by 20mL of methanol.
And a third step of: and (3) dropwise adding 25mL of purified water into the solution at a rate of 5mL/min, uniformly stirring, heating to 60 ℃, concentrating under reduced pressure to obtain a methanol solution with 85wt% and recycling, stirring, cooling to below 8 ℃ to crystallize to obtain a large amount of solids, filtering, collecting a filter cake, leaching with 10mL of frozen ethanol, leaching with 200mL of purified water, vacuum drying the filter cake to obtain a testosterone fine product, wherein the yield is more than 93%, the purity of HPLC (high performance liquid chromatography) is higher than 99.8%, and the K impurity and A impurity limit accords with the EP10.1 standard. The HPLC profile of crude testosterone is shown in A and Table 7 of FIG. 4, and the HPLC profile of fine testosterone is shown in B and Table 8 of FIG. 4.
TABLE 7 enzyme catalyzed crude testosterone HPLC
| Peak number | Retention time | Relative retention time | Area of | Area percent | Impurity species | Impurity content |
| 1 | 2.742 | 0.290 | 15654 | 0.030 | Solvent impurity | / |
| 2 | 8.245 | 0.856 | 22231 | 0.242 | K impurity | 0.242％ |
| 3 | 8.713 | 0.905 | 7418 | 0.066 | Unknown impurities | 0.066％ |
| 4 | 9.632 | 1.000 | 7145895 | 98.991 | Testosterone (Testosterone) | |
| 5 | 13.926 | 1.446 | 36157 | 0.671 | A impurity | 0.671％ |
Table 8 enzyme catalyzed testosterone top-quality HPLC
Example 5 comparative experiments
The crude testosterone product used in the refining method described below is not limited to the crude testosterone product obtained by a chemical method, a cell transformation method or an enzyme catalysis method, unless otherwise specified.
5.1 crystallization and refining of ethyl acetate and n-hexane:
10g of testosterone crude product (figure 5) is taken, 5-8 times volume of ethyl acetate (50-80 mL) is added, and the temperature is raised to 70 ℃ under stirring until the material is completely dissolved.
Filtering to remove denatured protein, collecting filtrate, preserving heat for 1h, slowly dripping 3-5 times of n-hexane (30-50 mL) into the solution, slowly cooling to about 10 ℃, and precipitating a large amount of solids.
Filtering to collect testosterone fine product, and oven drying at 60deg.C to constant weight.
The yield is 80-85%, and the refining effect is as follows: k impurity content changes: 0.223% -0.200%; a, the impurity content is changed: 0.534% -0.101%; the remaining single impurities were > 0.1% (FIG. 6). The testosterone refined by the method does not meet the EP10.1 requirement, and the yield is lower.
5.2 crystallization and refining of ethyl acetate:
10g of testosterone crude product (figure 5) is taken, 5-8 times volume of ethyl acetate (50-80 mL) is added, and the temperature is raised to 70 ℃ under stirring until the material is completely dissolved.
Filtering to remove denatured protein, collecting filtrate, concentrating the filtrate in vacuum until the volume of ethyl acetate is 1-2 times of the volume of the solvent, and precipitating a large amount of solids.
Cooling, crystallizing, filtering, collecting testosterone fine product, and oven drying at 60deg.C to constant weight.
The yield is 80-85%, and the refining effect is as follows: k impurity content changes: 0.212% → 0.187%; a, the impurity content is changed: 0.534% → 0.052%; the remaining single impurities were > 0.1% (FIG. 7). The testosterone refined by the method does not meet the EP10.1 requirement, and the yield is lower. Experiments show that the method has no refining effect on the testosterone synthesized by a biological method, and also does not meet the limit of K impurity regulation in EP 0.1.
5.3 refining acetone:
10g of testosterone crude product (figure 5) is taken, 6-9 times of acetone (60-90 mL) is added, and the temperature is raised to 50 ℃ under stirring until the material is completely dissolved.
The denatured proteins were removed by filtration and the filtrate was collected, and the filtrate was concentrated in vacuo to a residual 1-fold volume of acetone, and a large amount of solids precipitated.
Cooling, crystallizing, filtering, collecting testosterone fine product, and oven drying at 60deg.C to constant weight.
The yield is 80%, and the refining effect is as follows: k impurity content changes: 0.235%. Fwdarw.0.112%, a impurity content varies: 0.534% → 0.252% (fig. 8). The testosterone refined by the method does not meet the EP10.1 requirement, and the yield is lower.
5.4 refining isopropyl alcohol:
10g of testosterone crude product synthesized by an enzyme catalysis method (figure 5) is taken, 5-8 times of isopropanol (50-80 mL) is added, and the temperature is raised to 60 ℃ under stirring until the material is completely dissolved. The denatured protein is removed by filtration, the filtrate is collected, 50 to 70 percent of purified water (5 to 7 mL) is added to the filtrate according to the testosterone mass, and the solvent is concentrated under reduced pressure until the solvent is 2 times the volume of the solvent.
Cooling and crystallizing until a large amount of solids are separated out, filtering and collecting testosterone fine products, and drying at 60 ℃ until the weight is constant.
Yield 90%, refining effect: k impurity content changes: 0.235% -0.114%; a, the impurity content is changed: 0.534% → 0.057% (fig. 9). The testosterone refined by the method meets the EP10.1 requirement.
Claims (11)
1. The refining method of the crude testosterone product by the biological method is characterized by comprising the following steps of:
(1) Uniformly mixing the dried testosterone crude product with alcohol, heating to above 60 ℃, and stirring and refluxing while heating to completely dissolve the testosterone crude product and form flocculent denatured protein precipitate; the alcohol is methanol or ethanol; the testosterone crude product is prepared by a cell transformation method or an enzyme catalysis method and is collected by filtration; the volume mass ratio of the alcohol to the testosterone crude product is as follows: (5-8) 1;
(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 by using methanol; the filter aid containing the activated carbon is activated carbon and diatomite or activated carbon and cellulose; the dosage ratio of the diatomite to the activated carbon is 1:1, and the dosage ratio of the cellulose to the activated carbon is 3:5; the dosage ratio of the filter aid containing activated carbon to the testosterone crude product is (4-5) 15;
(3) Dropwise adding pure water, uniformly mixing, heating to above 60 ℃, concentrating and recovering 85wt% of alcohol solution, wherein the volume-mass ratio of the pure water to the testosterone crude product is (5-10): 6; stirring and cooling to below 8 ℃ to crystallize a large amount of solids, and filtering and collecting a filter cake; sequentially eluting with ethanol and pure water, and drying the filter cake to obtain testosterone essence; the volume-mass ratio of the pure water used for leaching and the testosterone crude product is (5-10): 1.
2. The refining process of claim 1, wherein the refining process conditions are selected from one or more of the following:
i) In the steps (1) - (3), the filtering is suction filtration;
ii) in steps (1) and (3), the drying is vacuum drying.
3. The refining method according to claim 1, wherein in the step (1), when the alcohol is methanol, the temperature is raised to 70℃or higher.
4. The refining method according to claim 1, wherein the adsorption is performed by stirring at a constant temperature.
5. The refining method according to claim 1, wherein in the step (2), the volume/mass ratio of the methanol to the testosterone crude product is (1-3): 3.
6. The refining method according to claim 5, wherein in the step (2), the volume/mass ratio of the methanol to the testosterone crude product is 2:3.
7. The method according to any one of claims 1 to 6, wherein in the step (3), the conditions of the method are selected from the group consisting of:
i) The volume-mass ratio of the pure water to the testosterone crude product is 5:6;
ii) the adding speed of the pure water is 2-10 ml/min;
iii) The volume-mass ratio of the pure water used for leaching and the testosterone crude product is (6-7) 1.
8. The refining method as claimed in claim 7, wherein the pure water is added at a rate of 5ml/min.
9. The refining method according to claim 8, wherein in the step (3), the conditions of the refining method are selected from the group consisting of:
i) The mixing is carried out under stirring;
ii) said concentrating to reduced pressure concentrating;
iii) The temperature reduction is carried out under stirring.
10. The refining method according to claim 1, wherein in the step (3), the conditions of the refining method 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) 3.
11. The refining method according to claim 10, wherein in the step (3), the volume to mass ratio of the ethanol to the testosterone is 1:3.
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