Extraction method of medicinal L-isoleucine

Abstract

The invention discloses a medicinal L-isoleucine extraction method, which comprises: S1, carrying out heating inactivation on an L-isoleucine fermentation broth, and carrying out membrane filtration to obtain a membrane filtrate; s2, concentrating the membrane filtrate, cooling, crystallizing, and centrifuging to obtain an L-isoleucine crude product; s3, adding the L-isoleucine crude product into pure water, heating, dissolving, decolorizing and filtering to obtain a decolorized solution; and S4, carrying out chromatographic separation on the destaining solution to obtain an extracting solution, and carrying out concentration crystallization, filtration and drying on the extracting solution to obtain a medicinal-grade L-isoleucine finished product. The method has the following technical effects that the crude product is prepared through concentration and crystallization, residual sugar, salt and part of heteroacid in the fermentation liquor are removed, pigment is removed through decoloration, the heteroacid is further removed through chromatographic separation, the content of the heteroacid is effectively reduced, the finally extracted L-isoleucine finished product meets the medicinal grade requirement, the extraction process is stable, the yield is high, and the method is suitable for industrial production. And the finished product quality is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a method for extracting pharmaceutical-grade L-isoleucine. Background Art

[0002] L-isoleucine, also known as isoleucine, is an important component of living organisms and is an essential amino acid. In medicine, by adding L-isoleucine to nutritional infusion or drug preparation, it can promote the normal functioning of the human liver, relieve and treat liver diseases, and improve human immunity. At the same time, the compound amino acid infusion composed of three branched-chain amino acids and a large number of special amino acid drugs used in the preparation of therapeutic drugs can replace sugar to provide energy and is one of the more expensive amino acid raw materials. Pharmaceutical grade L-isoleucine has very high purity requirements, and the single impurity acid should be less than 0.2% and the total impurity acid should be less than 1%.

[0003] At present, fermentation production technology is usually used to produce L-isoleucine, and the key step to improve the purity of L-isoleucine lies in the extraction process. The existing L-isoleucine extraction method first filters the fermentation broth, then directly chromatographically separates the filtrate, and finally concentrates and crystallizes to obtain the L-isoleucine product. The L-isoleucine extracted by this extraction method not only cannot meet the pharmaceutical grade standard requirements, but also because of the presence of a large amount of residual sugar and pigment in the filtrate, it will seriously affect the service life of the chromatographic resin and increase the extraction cost. At the same time, the separation effect is also uncertain, and the quality of the finished product cannot be guaranteed. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention provides a method for extracting pharmaceutical-grade L-isoleucine, wherein a crude product is prepared by concentration and crystallization, residual sugar, salt and part of the impurity acid in the fermentation broth are removed, pigments are removed by decolorization, and impurity acid is further removed by chromatographic separation, thereby effectively reducing the impurity acid content. The L-isoleucine product finally extracted meets the pharmaceutical-grade requirements, the extraction process is stable, the quality of the finished product is guaranteed, and the extraction cost is effectively controlled.

[0005] The technical effects to be achieved by the present invention are achieved through the following technical aspects:

[0006] The present invention provides a method for extracting pharmaceutical grade L-isoleucine, comprising the following steps:

[0007] S1, L-isoleucine fermentation liquid is heated and inactivated, and then filtered through a membrane to obtain a membrane filtrate;

[0008] S2, concentrating the membrane filtrate, cooling and crystallizing, and centrifuging to obtain crude L-isoleucine;

[0009] S3, adding the crude L-isoleucine into pure water for heating and dissolving, decolorizing, and filtering to obtain a decolorized solution;

[0010] S4, the decolorized liquid is subjected to chromatographic separation to obtain an extract, and the extract is concentrated, crystallized, filtered, and dried to obtain a pharmaceutical grade L-isoleucine product.

[0011] In some embodiments, the chromatographic separation adopts continuous chromatographic separation technology, and the chromatographic resin used in the chromatographic separation adopts S3850 resin. Specifically, 6 S3850 resin continuous ion exchange columns are used for chromatographic separation.

[0012] In some embodiments, the water-to-solid ratio of the chromatographic separation is 2.50-3.50, preferably 3.10-3.20.

[0013] In some embodiments, the membrane filtration uses a ceramic membrane with a pore size of 0.05 to 1.0 μm, preferably 0.05 μm.

[0014] In some embodiments, the concentration in S2 is concentration to a solute content of 20-25%.

[0015] In some embodiments, the amount of pure water used is 30 to 40 times the quality of the crude L-isoleucine, preferably 40 times.

[0016] In some embodiments, the decolorization is performed using activated carbon, and the amount of the activated carbon used is 3-5% of the quality of the crude L-isoleucine, preferably 4%.

[0017] In some embodiments, the mother liquor of the crude L-isoleucine is combined with the residual liquid obtained by the chromatographic separation and then concentrated and crystallized to prepare feed-grade isoleucine.

[0018] In some embodiments, the mother liquor of the pharmaceutical grade L-isoleucine product is mixed with the next batch of the decolorized liquid and participates in the chromatographic separation again.

[0019] In some embodiments, the concentration in S2 uses a single-effect concentrating furnace.

[0020] In summary, compared with the prior art, the present invention has at least the following advantages:

[0021] The method for extracting pharmaceutical-grade L-isoleucine provided by the present invention comprises the following steps: firstly removing bacterial cells in a fermentation liquid by membrane filtration, then performing preliminary impurity removal by concentration and crystallization to remove most of the residual sugar, salt and part of the miscellaneous acids in the fermentation liquid, then removing the pigment by dissolving and decolorizing, removing the miscellaneous acids whose chromatographic peak time is before isoleucine by chromatographic separation, and further removing the miscellaneous acids whose chromatographic peak time is after isoleucine by concentration and crystallization of the extract, thereby effectively reducing the content of the miscellaneous acids. The L-isoleucine product finally extracted meets the pharmaceutical-grade standard requirements, the extraction process is stable, the quality of the finished product is guaranteed, and the extraction cost is effectively controlled. DETAILED DESCRIPTION

[0022] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0024] The present invention provides a method for extracting pharmaceutical grade L-isoleucine, comprising the following steps:

[0025] S1, L-isoleucine fermentation liquid is heated and inactivated, and then filtered through a membrane to obtain a membrane filtrate;

[0026] S2, concentrating the membrane filtrate, cooling and crystallizing, and centrifuging to obtain crude L-isoleucine;

[0027] S3, adding crude L-isoleucine into pure water for heating and dissolving, decolorizing, and filtering to obtain a decolorized solution;

[0028] S4. The decolorized liquid is separated by chromatography to obtain an extract, and the extract is concentrated, crystallized, filtered, and dried to obtain a pharmaceutical grade L-isoleucine product.

[0029] It should be noted that the isoleucine extracted by the fermentation broth extraction process mainly contains miscellaneous acids such as methionine, aminobutyric acid, valine, phenylalanine, and leucine. It is understandable that the sterilized fermentation broth can remove the bacteria by membrane filtration, and then concentrated and crystallized, and the membrane filtrate can be preliminarily impurized to remove most of the residual sugars, salts and some miscellaneous acids. The crude L-isoleucine obtained after centrifugation is dissolved in pure water and decolorized, and most of the pigments in the crude L-isoleucine can be removed. The miscellaneous acids such as methionine, aminobutyric acid, and valine with a chromatographic peak time in front of isoleucine can be removed by chromatographic separation, and then the extract can be concentrated and crystallized to further remove the miscellaneous acids such as phenylalanine with a chromatographic peak time behind isoleucine, thereby effectively reducing the content of miscellaneous acids, and the final L-isoleucine product obtained by extraction meets the requirements of pharmaceutical grade standards, and the extraction process is stable, and the quality of the finished product can be guaranteed. At the same time, since most of the residual sugars and pigments have been removed before chromatographic separation, the separation burden of the chromatographic resin is reduced, the service life of the chromatographic resin is extended, and the extraction cost is effectively controlled.

[0030] Further, the chromatographic separation adopts continuous chromatographic separation technology, and the chromatographic resin of the chromatographic separation adopts S3850 resin. Specifically, 6 continuous ion-exchange columns of S3850 resin are used for chromatographic separation. Since the chromatographic resin S3850 has a good effect on removing impurities of heteroacids with similar amino acid properties, it can achieve excellent separation effects when applied to the chromatographic separation of the three eutectic branched-chain amino acids of valine, isoleucine and leucine. Therefore, the use of chromatographic resin S3850 for chromatographic separation can effectively reduce the heteroacid content and improve the purity of L-isoleucine. Chromatographic separation is carried out by 6 continuous ion-exchange columns of S3850 resin, which can not only realize the automated operation of continuous feeding and continuous discharging, improve separation efficiency, but also improve resource utilization and reduce costs.

[0031] Furthermore, the water-to-material ratio of chromatographic separation is 2.50-3.50, preferably 3.10-3.20.

[0032] The membrane filtration uses a ceramic membrane with a pore size of 0.05 to 1.0 μm, preferably 0.05 μm.

[0033] Among them, the concentration in S2 is to concentrate to a solute content of 20-25%. Specifically, a single-effect concentrator is used for concentration. In some embodiments, the concentration and crystallization process in S4 can also be concentrated and crystallized using a single-effect concentrator. The use of a single-effect concentrator for concentration and crystallization can efficiently utilize energy, reduce energy consumption, and reduce concentration costs. During the operation of the single-effect concentrator, the heat released by the steam through the condenser can be recycled and supplied to the heating system, thereby reducing energy consumption. In addition, due to its small size, the single-effect concentrator can reduce the occupied area, and its simple structure makes it easy to operate and maintain.

[0034] The amount of pure water used is 30 to 40 times the quality of crude L-isoleucine, preferably 40 times.

[0035] The decolorization is carried out using activated carbon, and the amount of activated carbon used is 3-5% of the quality of the crude L-isoleucine, preferably 4%.

[0036] In some embodiments, the mother liquor of crude L-isoleucine is combined with the residual liquid obtained by chromatographic separation and then concentrated and crystallized to prepare feed-grade isoleucine. By recycling and combining the mother liquor of crude L-isoleucine and the residual liquid obtained by chromatographic separation and reprocessing, isoleucine that meets the requirements of feed-grade standards can be obtained, which can be put into the production of another product, effectively improving the utilization rate of production resources, improving production efficiency, and avoiding resource waste, which has certain environmental value. By recycling and reusing the mother liquor of crude L-isoleucine and the residual liquid obtained by chromatographic separation, the comprehensive yield of the prepared isoleucine reaches 90%.

[0037] In some embodiments, the mother liquor of the finished pharmaceutical-grade L-isoleucine product is mixed with the next batch of decolorizing liquid and is again subjected to chromatographic separation. By recycling and reusing the finished product mother liquor, not only can the use of new solvents be reduced, costs can be reduced, and waste of resources can be avoided; but also the residual L-isoleucine in the finished product mother liquor can be separated, thereby improving the purity and yield of the finished L-isoleucine product.

[0038] Example 1

[0039] This embodiment provides a pharmaceutical grade L-isoleucine, which is extracted by the following extraction method, which specifically includes the following steps:

[0040] S1. After 5000L fermentation, 3500L of L-isoleucine fermentation liquid was obtained, and the L-isoleucine content was 4.0%. After heating and inactivation, it was directly filtered through a 0.2μm ceramic membrane, and the membrane filtrate was collected, with a yield of 96%;

[0041] S2, the collected membrane filtrate is concentrated to 10-20% of the original volume and 20% content by a single-effect concentrator, then placed in a furnace, cooled and crystallized, and centrifuged to obtain crude L-isoleucine, the weight of the crude product after purification is 105kg, and the yield of the fermentation broth is 75%;

[0042] S3, adding crude L-isoleucine to 3675L pure water, heating to dissolve, adding 4.2kg activated carbon for decolorization, and filtering to obtain a decolorized solution;

[0043] S4. The obtained decolorized liquid is placed on 6 continuous ion exchange columns filled with S3850 resin. After adjusting the parameters, the water-to-solid ratio is controlled to be 3.16. The extract and the residual liquid are collected separately. The extract is concentrated, crystallized, filtered, and dried to obtain a pharmaceutical grade L-isoleucine product with a weight of 76 kg.

[0044] Example 2

[0045] This embodiment provides a pharmaceutical grade L-isoleucine, which is extracted by the following extraction method, which specifically includes the following steps:

[0046] S1. After 5000L fermentation, 3500L of L-isoleucine fermentation liquid was obtained, and the L-isoleucine content was 4.0%. After heating and inactivation, it was directly filtered through a 0.05μm ceramic membrane, and the membrane filtrate was collected, with a yield of 96%;

[0047] S2, the collected membrane filtrate is concentrated to 10-20% of the original volume and 25% in a single-effect concentrator, then placed in a furnace, cooled and crystallized, and centrifuged to obtain crude L-isoleucine, the weight of the crude product after purification is 105kg, and the yield of the fermentation broth is 75%;

[0048] S3, adding crude L-isoleucine to 4200 L pure water, heating to dissolve, adding 4.2 kg activated carbon for decolorization, and filtering to obtain a decolorized solution;

[0049] S4. The obtained decolorized liquid is placed on 6 continuous ion exchange columns filled with S3850 resin. After adjusting the parameters, the water-to-solid ratio is controlled to be 3.16. The extract and the residual liquid are collected separately. The extract is concentrated, crystallized, filtered, and dried to obtain a pharmaceutical grade L-isoleucine product with a weight of 78 kg.

[0050] Example 3

[0051] This embodiment provides a pharmaceutical grade L-isoleucine, which is extracted by the following extraction method, which specifically includes the following steps:

[0052] S1. After 5000L fermentation, 3500L of L-isoleucine fermentation liquid was obtained, and the L-isoleucine content was 4.0%. After heating and inactivation, it was directly filtered through a 1.0μm ceramic membrane, and the membrane filtrate was collected, with a yield of 96%;

[0053] S2, the collected membrane filtrate is concentrated to 10-20% of the original volume and 20% content by a single-effect concentrator, then placed in a furnace, cooled and crystallized, and centrifuged to obtain crude L-isoleucine, the weight of the crude product after purification is 105kg, and the yield of the fermentation broth is 75%;

[0054] S3, adding crude L-isoleucine to 3675L pure water, heating to dissolve, adding 3.15kg activated carbon for decolorization, and filtering to obtain a decolorized solution;

[0055] S4. The obtained decolorized liquid is placed on 6 continuous ion exchange columns filled with S3850 resin. After adjusting the parameters, the water-to-solid ratio is controlled to be 2.67. The extract and the residual liquid are collected separately. The extract is concentrated, crystallized, filtered, and dried to obtain a pharmaceutical grade L-isoleucine product with a finished product weight of 78 kg.

[0056] Comparative Example 1

[0057] This comparative example provides an L-isoleucine, which is extracted by the following extraction method, which specifically comprises the following steps:

[0058] S1. After 5000L fermentation, 3500L of L-isoleucine fermentation liquid was obtained, and the L-isoleucine content was 4.0%. After heating and inactivation, it was directly filtered through a 0.2μm ceramic membrane, and the membrane filtrate was collected, with a yield of 96%;

[0059] S2, the collected membrane filtrate is concentrated to 10-20% of the original volume and 20% content by a single-effect concentrator, then placed in a furnace, cooled and crystallized, and centrifuged to obtain crude L-isoleucine, the weight of the crude product after purification is 105kg, and the yield of the fermentation broth is 75%;

[0060] S3, adding crude L-isoleucine to 3675L pure water, heating to dissolve, adding 4.2kg activated carbon for decolorization, and filtering to obtain a decolorized solution;

[0061] S4, the obtained decolorized liquid is first applied to the positive column of 732 resin, and then to the negative column of 717 resin, the upper column liquid is collected, concentrated and crystallized, filtered, and dried to obtain the L-isoleucine finished product, the finished product weight is 60kg.

[0062] Comparative Example 2

[0063] This comparative example provides an L-isoleucine, which is extracted by the following extraction method, which specifically comprises the following steps:

[0064] S1. After 5000L fermentation, 3500L of L-isoleucine fermentation liquid was obtained, and the L-isoleucine content was 4.0%. After heating and inactivation, it was directly filtered through a 0.2μm ceramic membrane, and the membrane filtrate was collected, with a yield of 96%;

[0065] S2. The obtained membrane filtrate is first applied to the positive column of 732 resin and then to the negative column of 717 resin. The upper column liquid is collected for concentration, crystallization and filtration to obtain crude L-isoleucine. The crude product is refined to obtain the finished L-isoleucine.

[0066] Test Example 1

[0067] In this test example, the contents of L-isoleucine and various miscellaneous acids were measured on the L-isoleucine products of Examples 1 to 3 and Comparative Examples 1 to 2. The results are shown in Table 1.

[0068] Table 1 Determination results of the contents of L-isoleucine and various miscellaneous acids in the finished L-isoleucine products of Examples 1 to 3 and Comparative Examples 1 to 2

[0069]

[0070]

[0071] By comparing the L-isoleucine content and the content of each impurity acid in the L-isoleucine finished products obtained in Examples 1 to 3 and Comparative Examples 1 to 2 in Table 1, it can be seen that in the pharmaceutical-grade L-isoleucine finished products obtained by the extraction method of the pharmaceutical-grade L-isoleucine provided by the present invention in Examples 1 to 3, the L-isoleucine content is higher than 99.0%, the content of each impurity acid is lower than 0.2%, and the total impurity acid content is lower than 1%, which meets the standard requirements of the pharmaceutical-grade L-isoleucine in the European Pharmacopoeia EP 10.0. However, Comparative Example 1 does not use 6 continuous ion exchange columns filled with S3850 resin for continuous chromatographic separation, and the L-isoleucine finished product extracted by it cannot meet the pharmaceutical-grade standard requirements; Comparative Example 2 directly performs chromatographic separation on the membrane filtrate, and the L-isoleucine finished product extracted by it cannot meet the pharmaceutical-grade standard requirements. The extraction method of the pharmaceutical-grade L-isoleucine provided by the present invention can not only produce L-isoleucine that meets the pharmaceutical-grade standard requirements, but also has a stable extraction process, can ensure the quality of the finished product, and can effectively control the extraction cost.

[0072] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims

1. A method for extracting pharmaceutical grade L-isoleucine, characterized in that: The following steps are involved: S1, L-isoleucine fermentation broth is heated and inactivated, and then filtered through a membrane to obtain a membrane filtrate; S2, concentrating the membrane filtrate, cooling and crystallizing, and centrifuging to obtain crude L-isoleucine; S3, adding the crude L-isoleucine into pure water for heating and dissolving, decolorizing, and filtering to obtain a decolorized solution; S4, the decolorized liquid is separated by chromatography to obtain an extract, and the extract is concentrated, crystallized, filtered, and dried to obtain a pharmaceutical grade L-isoleucine product.

2. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The chromatographic separation adopts continuous chromatographic separation technology, and the chromatographic resin of the chromatographic separation adopts S3850 resin.

3. The method for extracting pharmaceutical grade L-isoleucine according to claim 2, characterized in that: The water-to-material ratio of the chromatographic separation is 2.50-3.

50.

4. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The membrane filtration uses a ceramic membrane with a pore size of 0.05 to 1.0 μm.

5. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The concentration in S2 is to concentrate until the solute content is 20-25%.

6. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The amount of the pure water used is 30 to 40 times the quality of the crude L-isoleucine.

7. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The decolorization adopts activated carbon decolorization, and the amount of the activated carbon used is 3-5% of the quality of the crude L-isoleucine.

8. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The mother liquor of the crude L-isoleucine is combined with the residual liquid obtained by the chromatographic separation and then concentrated and crystallized to prepare feed-grade isoleucine.

9. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The mother liquor of the pharmaceutical grade L-isoleucine finished product is mixed with the next batch of the decolorizing solution and participates in the chromatographic separation again.

10. The method for extracting pharmaceutical grade L-isoleucine according to claim 1, characterized in that: The concentration in S2 adopts a single-effect concentration furnace.

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