CN113336663B - Method for preparing ornithine aspartate by using ornithine catalytic liquid - Google Patents
Method for preparing ornithine aspartate by using ornithine catalytic liquid Download PDFInfo
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- CN113336663B CN113336663B CN202110624190.4A CN202110624190A CN113336663B CN 113336663 B CN113336663 B CN 113336663B CN 202110624190 A CN202110624190 A CN 202110624190A CN 113336663 B CN113336663 B CN 113336663B
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/10—Citrulline; Arginine; Ornithine
Abstract
The invention discloses a method for preparing ornithine aspartate by using ornithine catalytic liquid, belonging to the field of biological medicine. The ornithine conversion solution is subjected to ultrafiltration treatment by using an ultrafiltration membrane to obtain an ultrafiltration membrane clear solution, and then the pH value is adjusted to 6.2-6.7 by using the aspartic acid, so that on one hand, the ornithine can be prepared by a one-step method, the yield is high, the total yield is 80%, on the other hand, a meta-acidic environment can be created, and the generation of related impurities is reduced. The invention adopts electrodialysis to desalt, and can avoid the generation of related substances easily caused by alkaline elution environment when the ion exchange column is used for elution.
Description
Technical Field
The invention relates to a method for preparing ornithine aspartate by using ornithine catalytic liquid, belonging to the field of biological medicine.
Background
Ornithine aspartate can directly participate in the metabolism of liver cells and activate two key enzymes in the detoxification function of the liver, so that the ornithine aspartate can assist in scavenging free radicals harmful to human bodies, enhancing the detoxification function of the liver, rapidly reducing excessive blood ammonia and promoting the repair and regeneration of the liver cells, thereby effectively improving the liver function and restoring the energy balance of the organism.
At present, the preparation method of ornithine aspartate mainly comprises two main types: the L-arginine and L-aspartic acid are used as starting materials, and the L-arginine is converted into L-ornithine by a chemical method or a biological method and then reacts with the L-aspartic acid to prepare the L-ornithine. The method has complex technical process, especially the biological method has harsh conditions and high cost, and is not suitable for industrialized mass production. The other is prepared by using L-ornithine salt and L-aspartic acid as starting materials, preparing L-ornithine free alkali by using the L-ornithine salt, and then reacting with the L-aspartic acid. L-ornithine salts employed in such processes include L-ornithine hydrochloride, L-ornithine sulfate, and L-ornithine acetate, wherein L-ornithine sulfate is not readily available and L-ornithine acetate is relatively expensive.
The method for preparing ornithine aspartate by using L-ornithine hydrochloride and L-aspartic acid as starting materials mainly comprises the following steps: dissolving L-ornithine hydrochloride in water, regulating the pH value by sodium hydroxide, desalting by adopting an electrodialysis method, and then reacting with L-aspartic acid. The electrodialysis desalting condition is harsh, the price is high, and the method is not suitable for industrial mass production. Or, L-ornithine hydrochloride is dissolved in water, desalted by cation exchange resin and then reacts with L-aspartic acid to obtain the product, the method has low yield, and is not suitable for industrial mass production, and when the product is eluted in an ion exchange column, related substances are easily generated in alkaline eluting environment, so that the product does not meet the quality requirements of raw materials.
Disclosure of Invention
[ technical problem ]
The existing method for preparing ornithine aspartate has relatively strict purification conditions, and related substances are easy to generate when an ion exchange purification method is adopted.
Technical scheme
The invention provides a method for preparing ornithine aspartate by using ornithine transformation solution, which comprises the following steps:
(1) Heating ornithine conversion solution to 55-60 ℃, preserving heat for 20-30min, and then performing ultrafiltration treatment by adopting an ultrafiltration membrane to remove protein and thalli so as to obtain an ultrafiltration membrane clear solution, wherein the working temperature of the ultrafiltration membrane is 28-32 ℃; the ornithine conversion solution is obtained by taking arginine as a substrate and converting arginine into ornithine through arginase catalysis;
(2) Regulating the pH value of the ultrafiltration membrane clear liquid obtained in the step to 6.2-6.7 by using aspartic acid;
(3) Desalting the pH-adjusted liquid by an electrodialysis method, and ending the reaction when the electric conductivity is less than or equal to 300 mu s/cm to obtain ornithine aspartate solution;
(4) Concentrating ornithine aspartate solution obtained in the step (3) to 65-75%, heating to 55-60 ℃, adding 10-15% active carbon, and decolorizing for 30-60min to obtain decolorized solution;
(5) And reversely dripping the decolorized solution into ethanol with the volume of 4-5 times, stirring at 30 ℃ to obtain crystals, and carrying out suction filtration, centrifugation and drying.
In one embodiment of the present invention, the ultrafiltration membrane in step (1) is operated at a temperature of 28-32 ℃.
In one embodiment of the present invention, the volumes of the dilute and concentrate compartments are the same in the electrodialysis of step (3).
In one embodiment of the present invention, the drying temperature in step (5) is 80 ℃.
[ advantageous effects ]
The ornithine conversion solution is subjected to ultrafiltration treatment by using an ultrafiltration membrane to obtain an ultrafiltration membrane clear solution, and then the pH value is adjusted to 6.2-6.7 by using the aspartic acid, so that on one hand, the ornithine can be prepared by a one-step method, the yield is high, the total yield is 80%, on the other hand, a meta-acidic environment can be created, and the generation of related impurities is reduced.
The ornithine aspartate solution is obtained by adopting the electrodialysis method, and the problem that related substances are easily generated in an alkaline elution environment when the ornithine aspartate solution is eluted in an ion exchange column can be avoided.
The ornithine aspartate prepared by the method disclosed by the invention has the impurity content meeting the requirements.
Detailed Description
The present invention will be further illustrated with reference to specific examples and comparative examples.
The main detection methods involved in the following examples are as follows:
the detection method of ornithine aspartate comprises the following steps: about 70mg of the dried sample was taken, 5mL of anhydrous formic acid and 50mL of glacial acetic acid were added and dissolved, and then, the solution was titrated with a perchloric acid titration solution (0.1 mol/L) according to a potentiometric titration method, and the result of the titration was corrected by a blank test. Each 1mL of the perchloric acid titration solution (0.1 mol/L) corresponds to 8.84mg of C 5 H 12 N 2 O 2 ·C 4 H 7 NO 4 。
The preparation method of ornithine conversion liquid comprises the following steps: in a 40L reaction system, 4kg of arginine and water are added to a constant volume of 40L, 160g of arginine converting enzyme (specific activity 1000U/g) is added, and the mixture is converted for 24 hours at 37 ℃.
Method for detecting related substances (high performance liquid chromatography):
test solution: about 0.4g of the product is taken, precisely weighed, placed in a 100ml measuring flask, added with 40ml of 0.02mol/L potassium dihydrogen phosphate buffer solution for dissolution, diluted to scale by using ethylene wax, and shaken well.
Control solution: the sample solution is precisely measured and diluted quantitatively with mobile phase to prepare solution containing ornithine aspartate in 4 mug of 1 ml.
Impurity control solutions: taking appropriate amounts of reference substances of maleic acid, fumaric acid, arginine, impurity I and impurity Il, precisely weighing, respectively adding mobile phases for dissolution, and diluting with fixed salts to prepare solutions with the concentration of about 4 mug in each 1 ml.
System applicability solution: taking appropriate amounts of ornithine aspartate, maleic acid, fumaric acid, arginine, impurities I and impurities II, placing the reference substances into a same mechanical bottle, adding an appropriate amount of mobile phase to dissolve and dilute the reference substances into a solution containing about 4mg of ornithine aspartate, and 4 mug of each of maleic acid, fumaric acid, arginine, impurities I and impurities Il in 1 ml.
Chromatographic conditions: amino-bonded silica gel is used as a filler; taking 0.02mol/L potassium dihydrogen phosphate buffer solution (taking 2.2g of potassium dihydrogen phosphate, adding 500ml of water for dissolution, adding 5ml of concentrated ammonia solution, diluting to 1000ml with water, mixing uniformly, and regulating the pH value to 5.60+/-0.05) with phosphoric acid to prepare a mobile phase; the detection wavelength is 205nm; the flow rate is 1.3ml per minute; the column temperature was 30℃and the sample volume was 20. Mu.L.
System applicability requirements: in the system applicability solution chromatogram, the separation degree between the peaks of aspartic acid, ornithine, maleic acid, fumaric acid, arginine, impurity I (namely ornithine lactam) and impurity Il is required to meet the requirement.
Assay: precisely measuring the sample solution, the control solution and the impurity control solutions, respectively injecting into a liquid chromatograph, and recording the chromatogram till 4 times of the retention time of the aspartic acid peak.
Limit: the chromatogram of the sample solution has chromatographic peaks with the same retention time as the main peak of the impurity control solution, the content of each known impurity is calculated according to the external standard method by the peak area, the content of maleic acid, fumaric acid and arginine is not more than 0.1%, the content of impurity I is not more than 0.1% (for injection) or 0.3% (for oral preparation), and the content of impurity Il is not more than 0.15%; the peak areas of other single unknown impurities are not more than the sum (0.1%) of the two main peak areas of the control solution; the total amount of impurities is less than 0.5% (for injection) or 1.0% (for oral preparation).
Enzymatic conversion of L-arginine to L-ornithine conversion solution
(1) Conversion conditions:
volume: 5000L; substrate concentration: l-arginine 100g/L; l-arginase concentration: 1000U/L, temperature: 37 ℃, rotation speed: 100rpm, pH 9-10, the pH is about 10 after the substrate L-arginine is dissolved, so that the pH does not need to be adjusted.
(2) The preparation process comprises the following steps:
a. accurately weighing 500kg of substrate L-arginine, adding into 7000L jacketed reaction tank containing 3000L of water, fixing volume to 4000L for the first time, setting the temperature to 37 ℃ and the rotating speed to 100rpm, stirring for dissolving and heating substrate solution.
b. The L-arginase solution was added when the temperature in the reaction tank reached 37℃in a volume of 5000L in accordance with an enzyme concentration of 1000U/L, and then the volume was fixed to 5000L for the second time. And controlling steam and circulating water quantity of the reaction kettle, keeping the temperature in the reaction kettle at 37+/-1 ℃, uniformly stirring, starting the reaction, timing, reacting for 24 hours, heating to deactivate enzyme, and ending the reaction.
Example 1
(1) Heating ornithine conversion solution to 60 ℃, preserving heat for 30min, and then adopting an ultrafiltration membrane to carry out ultrafiltration treatment to remove protein and thalli, wherein the working temperature of the ultrafiltration membrane is 30 ℃, and the membrane inlet pressure is 0.1Mpa, so as to obtain an ultrafiltration membrane clear solution;
(2) Regulating the pH value of the clear solution of the ultrafiltration membrane obtained in the step to 6.2 by using aspartic acid;
(3) Desalting the pH-adjusted liquid by an electrodialysis method, placing 10L of the pH-adjusted liquid in a dilute chamber, adding 10L of pure water in a concentrated chamber, applying a voltage of 50v, carrying out electrodialysis, and ending the reaction when the conductivity is 300 mu s/cm to obtain ornithine aspartate solution;
(4) Concentrating the ornithine aspartate solution obtained in the step (3) to 70% of Baume degree, heating to 60 ℃, adding 15% active carbon for decolorization for 30min for one time to obtain decolorized solution;
(5) 200mL of decolorized solution is reversely dripped into 800mL of ethanol, stirred for 5 hours at 30 ℃ to obtain crystals, filtered, centrifuged and dried for 16 hours at 80 ℃. The impurity content of the obtained product meets the requirements.
The technology is a one-step method for preparing ornithine aspartate with high yield and total yield of 80%. In the prior art, ornithine is mostly prepared into ornithine hydrochloride, after ornithine hydrochloride is desalted, aspartic acid is used for regulating pH value to prepare the ornithine, the steps are multiple, the yield is low, and the total yield only reaches 50%.
Example 2
(1) Heating ornithine conversion solution to 55 ℃, preserving heat for 30min, and then performing ultrafiltration treatment by adopting an ultrafiltration membrane to remove protein and thalli to obtain an ultrafiltration membrane clear solution, wherein the working temperature of the ultrafiltration membrane is 32 ℃;
(2) Regulating the pH value of the clear solution of the ultrafiltration membrane obtained in the step to 6.5 by using aspartic acid;
(3) Desalting the pH-adjusted liquid by an electrodialysis method, placing the pH-adjusted liquid in a dilute chamber, adding pure water with the same volume into a concentrated chamber, adding voltage of 50v, carrying out electrodialysis, and ending the reaction when the electric conductivity is less than or equal to 300 mu s/cm to obtain ornithine aspartate solution;
(4) Concentrating ornithine aspartate solution obtained in the step (3) to 75%, heating to 60 ℃, and adding 10% active carbon for decolorization for 50min to obtain decolorized solution;
(5) And reversely dripping the decolorized solution into ethanol with the volume of 4 times, stirring at 30 ℃ to obtain crystals, carrying out suction filtration, centrifuging, and drying at 80 ℃ for 16 hours. The impurity content of the obtained product meets the requirements, the content of maleic acid, fumaric acid and arginine is lower than 0.1%, the impurity I is lower than 0.1%, and the impurity Il is lower than 0.15%.
Example 3
(1) Heating ornithine conversion solution to 60 ℃, preserving heat for 30min, and then performing ultrafiltration treatment by adopting an ultrafiltration membrane to remove protein and thalli to obtain an ultrafiltration membrane clear solution, wherein the working temperature of the ultrafiltration membrane is 32 ℃;
(2) Regulating the pH value of the clear solution of the ultrafiltration membrane obtained in the step to 6.7 by using aspartic acid;
(3) Desalting the pH-adjusted liquid by an electrodialysis method, placing the pH-adjusted liquid in a dilute chamber, adding pure water with the same volume in a concentrated chamber, adding voltage of 50v, carrying out electrodialysis, and ending the reaction when the electric conductivity is less than or equal to 300us/cm to obtain ornithine aspartate solution;
(4) Concentrating ornithine aspartate solution obtained in the step (3) to 75%, heating to 60 ℃, adding 15% active carbon, and decolorizing for 60min to obtain decolorized solution;
(5) And reversely dripping the decolorized solution into ethanol with the volume of 4 times, stirring at 30 ℃ to obtain crystals, carrying out suction filtration, centrifuging, and drying at 80 ℃ for 16 hours. The impurity content of the obtained product meets the requirements, the content of maleic acid, fumaric acid and arginine is lower than 0.1%, the impurity I is lower than 0.1%, and the impurity Il is lower than 0.15%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A method for preparing ornithine aspartate, which is characterized by preparing ornithine aspartate by ornithine transformation solution, comprising the following steps:
(1) Heating ornithine conversion solution to 55-60 ℃, preserving heat for 20-30min, and then adopting an ultrafiltration membrane to carry out ultrafiltration treatment to remove protein and thalli so as to obtain an ultrafiltration membrane clear solution; the ornithine conversion solution is obtained by taking arginine as a substrate and converting arginine into ornithine through arginase catalysis;
(2) Regulating the pH value of the ultrafiltration membrane clear liquid obtained in the step (1) to 6.2-6.7 by using aspartic acid;
(3) Desalting the pH-adjusted liquid by an electrodialysis method, and ending the reaction when the electric conductivity is less than or equal to 300 mu s/cm to obtain ornithine aspartate solution;
(4) Concentrating ornithine aspartate solution obtained in the step (3) to 65-75%, heating to 55-60 ℃, adding 10-15% active carbon, and decolorizing for 30-60min to obtain decolorized solution;
(5) And reversely dripping the decolorized solution into ethanol with the volume of 4-5 times, stirring at 30 ℃ to obtain crystals, and carrying out suction filtration, centrifugation and drying.
2. The process for preparing ornithine aspartate according to claim 1, wherein the ultrafiltration membrane in step (1) has an operating temperature of 28-32 ℃.
3. The method for preparing ornithine aspartate according to claim 1, wherein the volumes of the dilute chamber and the concentrated chamber are the same in the electrodialysis of the step (3).
4. The process for preparing ornithine aspartate according to claim 1, wherein the drying temperature in step (5) is 80 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104058981A (en) * | 2013-03-18 | 2014-09-24 | 辽宁科泰生物基因制药股份有限公司 | Preparation method of ornithine aspartate |
| CN104193632A (en) * | 2014-08-12 | 2014-12-10 | 无锡晶海氨基酸有限公司 | Method for separating L-ornithine from L-ornithine conversion solution prepared by using enzyme biotechnology and forming L-ornithine hydrochloride |
| CN105154499A (en) * | 2015-09-30 | 2015-12-16 | 精晶药业股份有限公司 | Preparation method of L-aspartate-L-ornithine |
| WO2021104432A1 (en) * | 2019-11-28 | 2021-06-03 | 武汉远大弘元股份有限公司 | L-ornithine composite salt and preparation method therefor and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104058981A (en) * | 2013-03-18 | 2014-09-24 | 辽宁科泰生物基因制药股份有限公司 | Preparation method of ornithine aspartate |
| CN104193632A (en) * | 2014-08-12 | 2014-12-10 | 无锡晶海氨基酸有限公司 | Method for separating L-ornithine from L-ornithine conversion solution prepared by using enzyme biotechnology and forming L-ornithine hydrochloride |
| CN105154499A (en) * | 2015-09-30 | 2015-12-16 | 精晶药业股份有限公司 | Preparation method of L-aspartate-L-ornithine |
| WO2021104432A1 (en) * | 2019-11-28 | 2021-06-03 | 武汉远大弘元股份有限公司 | L-ornithine composite salt and preparation method therefor and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 基于电渗析技术的复合氨基酸脱盐与分离;王艳君 等;《安徽工业大学学报》;第36卷(第2期);第132-135页 * |
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