CN114957367B

CN114957367B - A kind of refining method for preparing testosterone by biological method

Info

Publication number: CN114957367B
Application number: CN202210594618.XA
Authority: CN
Inventors: 刘明欣; 何鑫; 陈海林; 刘林; 杨艳青
Original assignee: Huanggang Renfu Pharmaceutical Co ltd
Current assignee: Huanggang Renfu Pharmaceutical Co ltd
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2023-12-29
Anticipated expiration: 2042-05-27
Also published as: CN114957367A

Abstract

本发明公开了一种生物法制备的睾酮粗品的精制方法，其包括以下步骤：(1)将干燥的睾酮粗品与醇混匀，升温至60℃以上，使所述睾酮粗品完全溶解并出现絮状变性蛋白沉淀；(2)加入包含活性炭的助滤剂以吸附所述絮状变性蛋白沉淀；过滤收集滤液，滤饼用甲醇淋洗；(3)滴加纯水，混匀后升温至60℃以上，浓缩并回收85wt％的醇溶液；搅拌降温至8℃以下析晶出大量固体，过滤收集滤饼；依次用乙醇和纯水淋洗，滤饼干燥后得睾酮精品。本发明的精制方法对生物法制备的睾酮粗品中K杂及A杂精制效果明显，成品睾酮中K杂含量＜0.15％，A杂含量＜0.05％，符合最新EP10.1标准；成品收率能够达到93％及以上。The invention discloses a method for refining crude testosterone prepared by biological methods, which includes the following steps: (1) Mix the dried crude testosterone with alcohol, and raise the temperature to above 60°C to completely dissolve the crude testosterone and cause flocculation. The flocculent denatured protein precipitates; (2) Add a filter aid containing activated carbon to adsorb the flocculent denatured protein precipitate; collect the filtrate by filtration, and rinse the filter cake with methanol; (3) Add pure water dropwise, mix and raise the temperature to 60 ℃ or above, concentrate and recover 85wt% alcohol solution; stir and cool down to below 8 ℃ to crystallize a large amount of solid, filter and collect the filter cake; sequentially rinse with ethanol and pure water, and dry the filter cake to obtain testosterone fine product. The refining method of the present invention has an obvious effect on refining K impurities and A impurities in crude testosterone prepared by biological methods. The content of K impurities in the finished testosterone is <0.15%, and the content of A impurities is <0.05%, which conforms to the latest EP10.1 standard; the yield of the finished product can Achieve 93% and above.

Description

Refining method for preparing testosterone by biological method

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

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:

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.

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

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.